Establishment of a depression model using dexamethasone-treated three-dimensional cultured rat cortical cells

Establishment of a depression model using dexamethasone-treated three-dimensional cultured rat cortical cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Establishment of a depression model using dexamethasone-treated three-dimensional cultured rat cortical cells Mi Kyoung Seo, Sehoon Jeong, Woo Seok Cheon, Dong Yun Lee, Sumin Lee, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5413832/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In vitro models are useful for exploring the molecular mechanisms underlying impaired neuroplasticity in depression. In this study, we developed a three-dimensional spheroid model using the synthetic glucocorticoid dexamethasone to investigate its effects on key pathways involved in neuroplasticity, specifically focusing on BDNF, sirtuin 1, and mTORC1 signaling. A micro-spheroid device was fabricated using photolithography and soft lithography, and cortical spheroids were generated from primary rat cortical cells. These spheroids were treated with varying concentrations of dexamethasone, and their structure included neurons, astrocytes, microglia, and oligodendrocytes. Dexamethasone treatment (100, 200, and 300 µM) resulted in a dose-dependent reduction in cell viability, BDNF mRNA expression, and neurite outgrowth. At 100 µM, dexamethasone reduced the expression of BDNF and sirtuin 1 and decreased phosphorylation of ERK1/2. It also lowered the phosphorylation levels of mTORC1, 4E-BP1, and p70S6K, as well as synaptic proteins such as PSD-95 and GluA1. Dexamethasone treatment inhibited pathways related to neuroplasticity. While the dexamethasone-treated spheroids may serve as a basis for developing an in vitro model of depression, further validation is required to confirm its broader applicability. Depression Dexamethasone Micro-spheroid device mTORC1 signaling Neuroplasticity Spheroid Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Depression is a prevalent mental health disorder affecting approximately 3.8% of the global population, with a higher incidence among adults (5.0%) and those over 60 years old (5.7%) (Institute of Health Metrics and Evaluation 2019 ). It manifests through various symptoms such as anhedonia, reduced motivation, hopelessness, sleep disturbances, appetite changes, and decreased physical activity (Otte et al. 2016 ), often accompanied by an increased risk of suicide (Bachmann 2018 ). As projected by the World Health Organization, depression could become the second leading cause of global disability by 2030, presenting a significant burden on both individuals and society (Li et al. 2021 ). Although selective serotonin reuptake inhibitors (SSRIs) are widely used and effective antidepressants, their therapeutic benefits often take weeks to manifest, and many patients do not respond adequately, leading to treatment-resistant depression (Commons and Linnros 2019 ; Akil et al. 2018 ). This highlights the need for new therapeutic approaches that can provide faster and more reliable effects. Understanding the underlying biological mechanisms of depression is key to advancing treatment options. While the exact pathophysiology of depression remains unclear, growing evidence points to impaired neuroplasticity as a central factor (Duman and Aghajanian 2012 ; Malykhin and Coupland 2015 ; McEwen et al. 2012 ; Price and Drevets 2010 ). Brain-derived neurotrophic factor (BDNF) plays a critical role in supporting neurogenesis and synaptic plasticity, and it is closely associated with antidepressant effects (Autry and Monteggia 2012 ; Castrén and Kojima 2017 ; Lindholm and Castrén 2014 ). Reduced BDNF levels have been observed in individuals with depression, and restoring BDNF activity is linked to antidepressant outcomes (Bocchio-Chiavetto et al. 2010 ; Duman and Monteggia 2006 ; Pittenger and Duman 2008 ). Signaling through BDNF and its receptor, tropomyosin receptor kinase B (TrkB), activates pathways such as the phosphatidyl inositol-3 kinase (PI3K)/protein kinase B (Akt) and mitogen‑activated protein kinase (MEK)/extracellular signal‑regulated kinase 1/2 (ERK1/2) signaling cascades, promoting neuronal growth and plasticity (Duman et al. 2016 ). The mechanistic target of rapamycin complex I (mTORC1) signaling pathway, which modulates synaptic protein synthesis, is another critical downstream target of BDNF/TrkB signaling, and its dysfunction has been implicated in stress-induced neuroplastic deficits (Chao and Hempstead 1995 ; Duman et al. 2016 ; Saarelainen et al. 2003 ). mTORC1 signaling enhances mRNA translation via its downstream substrates p70S6 kinase (p70S6K) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), leading to increased expression of synaptic proteins, such as postsynaptic density protein-95 (PSD-95), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit glutamate receptor 1 (GluA1), and synapsin 1. Chronic stress downregulates the synthesis of BDNF and synaptic proteins via the mTORC1 signaling pathway and ultimately impairs neuroplasticity (Duman et al. 2016 ). Moreover, sirtuin 1, a protein that influences neuroplasticity, has been linked to depression through genetic studies (CONVERGE consortium 2015 ; Kishi et al. 2010 ; Kovanen et al. 2015 ) and found to have decreased expression in depressed patients (Kishi et al. 2010 ). While animal models of depression have been extensively used to study these molecular pathways, in vitro models offer a complementary approach to investigate depression-related neuroplasticity. Traditionally, in vitro studies have relied on two-dimensional (2D) cultures, where cells grow in a flat, planar manner. However, the brain’s complex three-dimensional (3D) structure, composed of interconnected neurons, astrocytes, and glial cells, is not well represented by 2D cultures. Thus, developing 3D models that better mimic the brain’s microenvironment is essential for understanding depression at the cellular and molecular levels. The importance of refining in vitro models has become even more relevant given recent shifts in regulatory policies, such as the US FDA’s decision to no longer require animal testing prior to human trials (Wadman 2023 ). Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, which governs the stress response, is a well-documented feature of depression (Troubat et al. 2021 ). This system regulates the secretion of glucocorticoids in response to stress, with elevated glucocorticoid levels contributing to hippocampal and prefrontal cortex dysfunction in depressed individuals (Hill and Spencer-Segal 2021 ). Antidepressant treatments have been shown to normalize HPA axis activity, supporting its relevance in depression recovery (Troubat et al. 2021 ). In this study, we aimed to develop a 3D spheroid model that more accurately replicates the neurobiological environment associated with depression. Using a micro-spheroid device, we generated 3D neural spheroids from primary rat cortical cells. To simulate the glucocorticoid hypersecretion seen in depression, we treated the spheroids with varying concentrations of dexamethasone (DEX), a synthetic glucocorticoid. We then explored the effects of DEX on key molecular pathways implicated in neuroplasticity, including BDNF, sirtuin 1, and mTORC1 signaling. Method Fabrication of the micro-spheroid device for 3D neural spheroids The micro-spheroid device was fabricated from polydimethylsiloxane (PDMS) prepolymer (10:1 mixture Sylgard 184 DOW CORNING, USA) using one-step photolithography and soft lithography techniques. Initially, an array of microwells was made by PDMS soft lithography from an SU-8™ patterned Si master mold with inverted replication (pillar structure) of microwell arrays (diameter: 200 µm, depth: 200 µm). To create the master mold (Fig. 1 A, a), a photosensitive epoxy layer (SU-8™; MicroChem, USA) with 200 µm thicknesses was patterned onto a 4-inch diameter silicon substrate. The photosensitive epoxy layer, which forms the microwell arrays, is patterned by spin-coating SU-8 2075 at 1200 rpm to make a thickness of 200 µm, and first soft-baked at 65°C for 24 h and then at 95°C for 40 min. It was then exposed to UV light through a photomask, followed by a postexposure bake in two steps, first at 65°C for 5 min and then at 95°C for 15 min. The master mold was then coated with (tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilane (United Chemical Technologies, USA) to facilitate PDMS release from the master mold. A PDMS prepolymer (10:1 mixture) was poured into the master mold and cured at 80°C for 2 h to replicate the PDMS device from the master mold (Fig. 1 A, b). A 10-mm diameter punch bit (Syneo, USA) mounted on a drill press was used to punch out reservoirs to hold the cell culture media (Fig. 1 A, c). Second, PDMS replicas can be cut into 10 mm diameter discs by circular punch bits (Fig. 1 A, d), and each PDMS micro-spheroid device can be placed in each well of a 48-well cell culture plate. The micro-spheroid devices were then treated with oxygen plasma, immersed in deionized water and subsequently autoclaved. After sterilization, the micro-spheroid device was placed in a 48-well cell culture plate (Fig. 1 B). 3D neural spheroids were prepared using the microwell arrays in the micro-spheroid device (Fig. 1 C). Dissociated primary cortical cells were added at a concentration of 10 million cells per a micro-spheroid device. Finally, the cells were cultured for 3 days in a temperature and humidity controlled 5% CO 2 incubator, and then the 200 µm sized 3D neural spheroids formed in Fig. 1 D were collected by rapidly stirring the PDMS micro-spheroid device in the medium bath. Isolation of primary cortical cells Primary cortical tissues were isolated from the brains of Sprague–Dawley rat (Hana Biotech, Pyeongtaek-si, Gyeonggi-do, Korea) fetuses (embryonic day 19). The method of cortical cell isolation was slightly modified from that developed by Pacifici and Pruzzi (2012). Briefly, the tissues were cut into small sections and triturated by pipetting 30 times in neurobasal media (Gibco; ThermoFisher Scientific, Waltham, MA, USA) containing 0.25% trypsin–ethylene-diamine-tetraacetic acid (Gibco). The cell solution was placed at room temperature for 20 min and then suspended in growth medium (neurobasal medium containing 1% fetal bovine serum, 1% horse serum, 2% serum-free B27 growth medium, 250 mM glutamine, and 1% penicillin–streptomycin; all from Gibco). Debris was removed by passing the solution through a 100 µm cell strainer (SPL Life Sciences, Gyeonggi-do, Korea). The cell solution was centrifuged at 180 g for 10 min, and the supernatant was removed. The cell pellet was suspended in growth medium and carefully pipetted several times. Cortical cells were seeded at a density of 2 × 10 7 /micro-spheroid device (see 3D neural spheroid culture). The procedures were performed according to the guidelines of the Institutional Animal Care and Use Committee (IACUC; Inje University, Republic of Korea) and the ARRIVE (Kilkenny et al. 2010 ). The animal experiments were approved by the IACUC of the College of Medicine of Inje University (approval no.: 2021–010). Three-dimensional neural spheroid culture Cortical cell spheroids were prepared using the micro-spheroid device, which is microwells for producing uniformly sized spheroids with a diameter of 200 µm (Fig. 2 ). A micro-spheroid device was placed in a 48-well plate, and cortical cells were seeded at a density of 1 × 10 7 /micro-spheroid device. Cortical cells were allowed to settle into microwells for 1 h at 37°C, while the cells attached to the outside of the microwells were washed using a growth medium. Cells were aggregated in the microwells for 3 days (Fig. 2 ). At 3 days in vitro (DIV), aggregates were suspended in a 3 mL growth medium and transferred to poly-D-lysine (Sigma-Aldrich, St. Louis, MO, USA) and Matrigel (BD Biosciences, Franklin Lakes, NJ, USA)-coated plates. For the cell viability assay, 10–15 spheroids (100 µL) were plated in a 48-well plate. For quantitative real-time polymerase chain reaction (qRT-PCR), 20–30 spheroids (200 µL) were plated in a 12-well plate. For Western blotting, 40–60 spheroids (400 µL) were plated in a 6-well plate. For immunofluorescence, 20–30 spheroids (200 µL) were plated in a 1-well cell culture slide (SPL Life Sciences). On DIV 10, the spheroids were exposed to DEX (water-soluble; Sigma-Aldrich) at 100–300 µM for 5 days. On DIV 15, the spheroids were harvested for further experiments. Whole spheroids were maintained at 37°C, 5% CO 2 , and 95% humidity. The growth medium was changed every 2–3 days. The results from at least three independent spheroid cultures were recorded. Immunofluorescence To detect the presence of neurons and glia in cortical spheroids, the spheroids were collected on DIV 3, 4, and 10. Additionally, spheroids were harvested after 5 days of exposure to 100 µM DEX to determine whether DEX altered synaptic protein expression. The spheroids were washed with phosphate-buffered saline (PBS; GenDEPOT) and fixed in 4% paraformaldehyde (Biosesang, Seongnam-si, Gyeonggi-do, Korea) for 20 min at room temperature. The spheroids were permeabilized in 0.1% Triton X-100 (GenDEPOT) for 3 min, blocked with 4% bovine serum albumin (GenDEPOT) for 1 h at room temperature, and incubated with the primary antibodies (Table 1 ) overnight at 4°C. After incubation, the spheroids were washed with PBS and incubated with the secondary antibodies for 1 h (Table 1 ). Subsequently, the spheroids were washed with PBS, and the nuclei were stained with Hoechst 33258 (1:10,000; Invitrogen, Carlsbad, CA, USA). After washing with PBS, the spheroids were mounted on a 1-well cell culture slide using Biomeda Gel/Mount (Electron Microscopy Sciences, Foster City, CA, USA). The images were captured at a resolution of 1024 × 1024 pixels using the A1 + confocal laser scanning microscope (Nikon, Tokyo, Japan). Table 1 List of antibodies used in this study. Antigen Manufacturer (cat. no., RRID) Application IF WB β-III-tubulin BioLegend (801202, RRID:AB_2313773) 1:1000 MAP-2 Millipore (MAB3418, RRID:AB_94856) 1:1000 Nestin Millipore (MAB353, RRID:AB_94911) 1:1000 GFAP Santa Cruz Biotechnology (sc-33673, RRID:AB_627673) 1:1000 O1 Millipore (MAB344, RRID:AB_94860) 1:1000 CD11b Millipore (CBL1512, RRID:AB_93253) 1:1000 Hoechest 33258 Invitrogen (H21491) 1:10000 BDNF Abcam (ab108319, RRID:AB_10862052) 1:1000 Sirtuin1 Millipore (07-131, RRID:AB_10067921) 1:1000 p-Thr 202 /Tyr 204 -ERK1/2 Cell Signaling Technology (9101, RRID:AB_331646) 1:1000 ERK1/2 Cell signaling Technology (4695, RRID:AB_390779) 1:1000 p-Ser 2448 -mTORC1 Cell Signaling Technology (2971, RRID:AB_330970) 1:1000 mTORC1 Cell Signaling Technology (2972, RRID: AB_330978) 1:1000 p-Thr 37/46 -4E-BP1 Cell Signaling Technology (2855, RRID:AB_560835) 1:1000 4E-BP1 Cell Signaling Technology (9452, RRID:AB_331692) 1:1000 p-Thr 389 -p70S6K Cell Signaling Technology (9205, RRID:AB_330944) 1:1000 p70S6K Cell Signaling Technology (9202, RRID:AB_331676) 1:1000 PSD-95 Cell Signaling Technology (3450, RRID: AB_2292883) 1:1000 1:1000 GluA1 Cell Signaling Technology (13185, RRID:AB_2732897) 1:1000 1:1000 α-tubulin Sigma-Aldrich (T9026, RRID:AB_477593) 1:2000 Alexa Fluor 594 goat anti-mouse IgG (H + L) Thermo Fisher Scientific (A-11032, RRID:AB_2534091) 1:1000 Alexa Fluor 488 goat anti-rabbit IgG (H + L) Thermo Fisher Scientific (A-11008, RRID:AB_143165) 1:1000 Mouse anti-rabbit IgG-HRP Santa Cruz Biotechnology (sc-2357, RRID:AB_628497) 1:2000 Anti-mouse IgG-HRP Cell signaling Technology (7076, RRID:AB_330924) 1:5000 IF, Immunofluorescence; WB, Western blotting Cell viability The cytotoxic effect of DEX on neural spheroids was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Amresco, Solon, OH, USA) assay. Briefly, after exposure of spheroids in 48-well plates to 0, 100, 200, or 300 µM DEX for 5 days, 0.5 mg/mL MTT was added to the wells. The plates were incubated at 37°C for 4 h. After incubation, the medium was removed, and 400 µL dimethyl sulfoxide (GenDEPOT, Katy, TX, USA) was added to dissolve the formazan. Subsequently, 100 µL of the dissolved solution from each well was transferred to 96-well plates and analyzed using a SpectraMax M2e Microplate Readers (Molecular Devices, San Jose, CA, USA) at a wavelength of 570 nm. Measurement of BDNF mRNA levels by qRT-PCR Total RNA was extracted from spheroids after 5 days of exposure to 0, 100, 200, or 300 µM DEX using Tri-RNA reagent (Favogen, Kaohsiung, Taiwan) according to the manufacturer’s instructions. RNA quantity and purity were determined using the NanoDrop™ ND-1000 UV-Vis Spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). Single-strand cDNA was synthesized from 1 µg RNA using amfiRivertII™ cDNA Synthesis Master Mix (GenDEPOT) according to the manufacturer’s instructions, and 100 ng of the cDNA was subjected to qRT-PCR using TOPreal™ qPCR 2× PreMIX (SYBR Green with low ROX; Enzynomics, Daejeon, Korea). The final primer concentration was 10 pmol. The reaction parameters were 95°C for 10 min, followed by 45 cycles of heating at 95°C for 35 s, annealing at 55°C for 35 s, and extension at 72°C for 35 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene. The following primer sequences were used: BDNF: forward 5'-CCAACGAAGAAAACCATAAG-3', reverse 5'-AATACTGTCACACAC GCTCA-3'; GAPDH: forward 5'-AACAGCAACTCCCATTCTT C-3', reverse 5'-TGGTCCA GGGTTTCTTACTC-3'. The relative BDNF mRNA level was calculated using the 2 −△△CT method (Livak and Schmittgen 2001 ). The qRT-PCR was repeated two times per group for each from the three independent cultures. Neurite outgrowth analysis After 5 days of exposure to 0, 100, 200, or 300 µM DEX, the spheroids were fixed in 4% paraformaldehyde and immunostained with anti-microtubule-associated protein-2 (MAP-2; see Immunofluorescence). Photographs of each spheroid were captured as 20 images cropped from top to bottom using the A1 + confocal laser scanning microscope; the middle (i.e., 10th) image of the spheroid was analyzed using Sholl ImageJ software ( https://imagej.net/Sholl_Analysis ). The number of intersections/distance from the spheroid center was plotted (Harris et al. 2018 ; Zhong et al. 2020 ). The Area Under the Curve of each group was calculated from each spheroid and then averaged. Intersections were counted after a 400 µm diameter (i.e., the average size of the spheroids on DIV 15). Western blotting Total proteins were extracted from spheroids after 5 days of exposure to 100 µM DEX using lysis buffer containing RIPA buffer (Elpisbiotech, Daejeon, Korea), 1× protease inhibitor cocktail (GenDEPOT), and 1× phosphatase inhibitor (GenDEPOT). The pellets were removed by centrifugation (13,000 g, 20 min, 4°C), and the supernatant was collected. Protein concentrations were measured using the Bradford assay (Bradford Protein Assay Plus Reagents; GenDEPOT). Total protein lysates (20 µg) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (GenDEPOT) for 2 h using a 100-V power supply. The membranes were blocked in Tris-buffered saline with Tween-20 (TBS-T; GenDEPOT) and 5% skim milk for 1 h and then incubated with the primary antibodies (Table 1 ) overnight at 4°C. The membranes were washed with TBS-T for 10 min and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Table 1 ) at room temperature for 1 h. The Western blot bands were detected using West-Q Pico Dura ECL Solution (GenDEPOT) and visualized using the Amersham™ Imager 600 (GE Healthcare, Chicago, IL, USA). The protein bands were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The Western blot analyses were repeated two times per group for each from the four independent cultures. Statistical analysis Statistical analyses were performed using GraphPad Prism software (version 9.4.1; GraphPad software, Inc., San Diego, CA, USA; RRID:SCR_002798). BDNF mRNA level and neurite outgrowth values were analyzed by one-way analysis of variance followed by Tukey ’s post hoc tests. The unpaired t -test was used to compare the two groups. P < 0.05 was considered statistically significant. Results Self-assembled 3D spheroids in cortical cells Primary cortical cells dissociated from embryonic day 19 cortical tissues from rat fetuses were seeded in a micro-spheroid device to facilitate cellular self-assembly into 3D spheroids (Fig. 3 ). The cortical cells were aggregated in the micro-spheroid device from DIV 1 to 3. On DIV 3, spheroids of equal size (200 µm diameter) were generated. From DIV 15, the diameter remained constant at almost 400 µm. To assess the formation of intercellular networks within 3D cortical spheroids, we conducted a comprehensive investigation of various cell types generated in primary culture, including neurons, astrocytes, oligodendrocytes, neural progenitor cells, and microglia. This analysis involved the use of immunostaining techniques using specific markers for each cell type (Fig. 3 ). On DIV 3, 4, and 10, cortical spheroids expressed markers of neurons (β-III-tubulin and MAP-2), astrocytes (glial fibrillary acidic protein), microglia (CD11b), oligodendrocytes (O1), and neural progenitor cells (nestin). Spheroids on DIV 3 exhibited globular patterns. Neurite and glial processes extending outward from the spheroid were observed at DIV 4. At DIV 10, the neurites were longer and formed a more complex network, and the neural cells continued to extend the process outward from the spheroid. Changes in cell viability, BDNF mRNA level, and neurite outgrowth in neural spheroids after DEX exposure To evaluate the dose-dependent effect of DEX on cytotoxicity, neural spheroids on DIV 10 were exposed to different doses of DEX (100, 200, and 300 µM) for 5 days, and an MTT assay was performed on DIV 15. Neural spheroids exposed to DEX showed a dose-dependent decrease in cell viability ( F [3,76] = 11.500, p < 0.001; 100 µM, p = 0.004, 82.89% of control; 200 µM, p < 0.001, 79.28% of control; 300 µM, p < 0.001, 72.92% of control, Fig. 4 A). We also investigated whether 100–300 µM DEX decreases the BDNF level and neuronal complexity. DEX concentrations of 100, 200, and 300 µM significantly reduced the BDNF mRNA level ( F [3,12] = 29.600, p < 0.001; 100 µM, p < 0.001; 200 µM, p < 0.001; 300 µM, p < 0.001, Fig. 4 B). Neurite outgrowth was visualized by immuno-fluorescence using an anti-MAP-2 antibody and quantified using Sholl ImageJ software (Fig. 4 C). DEX doses of 100, 200, and 300 µM produced dose-dependent reductions in the number of intersections at 300 µm from the spheroid center (100 µM, 126.38 ± 9.86 intersections; 200 µM, 129.50 ± 9.80 intersections; 300 µM, 86.13 ± 10.41 intersections) and at 400 µm from the spheroid center (100 µM, 100.50 ± 7.53 intersections; 200 µM, 95.13 ± 10.30 intersections; 300 µM, 65.13 ± 6.98 intersections, Fig. 4 C, b). Additionally, exposure to 100, 200, and 300 µM DEX decreased the total number of intersections to 21.08%, 20.68%, and 21.08%, respectively. The Area Under the Curve was significantly reduced after 100, 200, and 300 µM DEX treatment compared with the controls ( F [3,23] = 11.100, p < 0.001; 100 µM, p = 0.019; 200 µM, p = 0.017; 300 µM, p < 0.001, Fig. 4 C, c). Changes in cell viability, BDNF mRNA level, and neurite outgrowth in neural spheroids after DEX exposure The preliminary experiments showed that co-administration of 200 or 300 µM DEX with antidepressants disrupted the morphology of neural spheroids due to cytotoxic effects (data not shown). Therefore, neural spheroids were exposed to 100 µM DEX for 5 days. As expected, treatment with 100 µM DEX significantly reduced the protein levels of BDNF ( t = 7.480, p < 0.001, Fig. 5 A), sirtuin 1 ( t = 9.130, p < 0.001, Fig. 5 B), and phospho-ERK1/2, a potential downstream target of sirtuin 1 ( t = 2.810, p = 0.031, Fig. 5 C). Changes in the phosphorylation of mTORC1-mediated proteins and the expression of synaptic proteins in neural spheroids after DEX exposure To investigate the effects of DEX on mTORC1 signaling in neural spheroids, the phosphorylation levels of mTORC1, 4E-BP1, and p70S6K, as well as the expression levels of the synaptic proteins PSD-95 and GluA1 were determined by Western blotting. The exposure of spheroids to 100 µM DEX significantly reduced the phosphorylation levels of mTORC1 ( t = 3.710, p = 0.010, Fig. 6 A, a), 4E-BP1 ( t = 5.070, p = 0.002, Fig. 6 A, b), and p70S6K ( t = 5.030, p = 0.002, Fig. 6 A, c), as well as the expression levels of PSD-95 ( t = 3.330, p = 0.016, Fig. 6 B, a) and GluA1 ( t = 3.670, p = 0.010, Fig. 6 B, b). Neurites in cortical spheroids showed immunoreactivity for each of the synaptic markers PSD-95 and GluA1 as shown by immunofluorescence (Fig. 6 B). Discussion In this study, we utilized a 3D neural spheroid culture system derived from primary rat cortical cells. The use of 3D spheroid models presents a potential alternative to traditional 2D cultures and animal models for investigating depression and other neurological conditions. These models offer an environment that more closely mimics the in vivo brain structure, maintaining key cellular interactions and the extracellular matrix in a 3D space (Dingle et al. 2015 ). Treatment of the 3D neural spheroids with DEX, a synthetic glucocorticoid, resulted in reduced cell viability, BDNF mRNA levels, and neurite outgrowth, all in a dose-dependent manner. Notably, exposure to 100 µM DEX led to a decrease in the protein levels of BDNF, sirtuin 1, and components of the mTORC1 signaling pathway, such as phospho-ERK1/2, phospho-mTORC1, and synaptic proteins (PSD-95 and GluA1). These findings indicate that this 3D spheroid model may serve as a useful in vitro system for studying the molecular mechanisms that contribute to impaired neuroplasticity in depression. The method employed in this study allowed for the production of uniform 3D cortical spheroids, approximately 200 µm in diameter, using a micro-spheroid device. This setup ensured consistent size and morphology, avoiding the central necrosis that can occur in larger spheroids due to insufficient nutrient and oxygen supply (Pamies et al. 2017 ). Importantly, the cultures were scaffold-free, meaning that the cells naturally produced their own extracellular matrix, such as laminin (Dingle et al. 2015 ), which has been shown to support neurogenesis and synapse formation (Barros et al. 2011 ; Relucio et al. 2012 ). In the present study, we established a scaffold-free, self-assembled 3D neural culture system that forms complex 3D structures of neurons and glia in a relatively short time of 15 days. This system offers a relatively simple and reproducible way to create 3D cultures that may be applied to studying various aspects of central nervous system function. Compared to traditional 2D cultures and animal models, 3D in vitro models may provide a more physiologically relevant platform for examining complex processes like neuroplasticity. For instance, similar 3D systems have been used to model Alzheimer's disease and perform neurotoxicity screenings using human-induced pluripotent stem cells (iPSCs)-derived spheroids (Choi et al. 2014 ; Kobolak et al. 2020 ; Zhong et al. 2020 ). In our study, we used this model to simulate HPA axis dysregulation associated with glucocorticoid hypersecretion by treating the spheroids with DEX, and we observed downregulation of key neuroplasticity-related proteins and pathways. DEX has been widely used in rodent models to induce depression-like behaviors, including reductions in reward-seeking and increased immobility (Wang et al. 2021 ; Wu et al. 2021 ). In previous studies, chronic DEX administration has been linked to structural changes in the brain, such as neuronal atrophy, decreased dendritic outgrowth, and impaired synaptic plasticity (Wang et al. 2021 ). In fact, in our previous studies of 2D cultures, DEX treatment reduced total dendritic length and spine formation in primary hippocampal cells; this plasticity was associated with decreased expression of synaptic proteins (PSD-95, synapsin 1, and GluA1) via downregulation of mTORC1 signaling (Park et al. 2018 ; Seo et al. 2020 ). Our results align with these findings, as DEX exposure in our 3D model similarly affected synaptic proteins and neuroplasticity pathways, notably the mTORC1 signaling pathway, which is critical for synaptic function and plasticity (Duman and Monteggia 2006 ). The doses of DEX used in this study (100–300 µM) were selected based on preliminary experiments, with 100 µM chosen for subsequent analyses due to its significant impact on cell viability and neuroplasticity markers without causing spheroid disintegration. The DEX concentrations used in this study are higher than those used in other 2D or 3D culture models. Cerebral organoids derived from human iPSCs were treated with 10, 100, 1000 nM and 100 µM DEX (Cruceanu et al. 2022 ). Mice hippocampal slice cultures were treated with 1-100 nM (Saito et al. 2016 ). In 2D cultures using neuronal cell lines, DEX was applied at various concentrations (0.01–100 µM) (Bassil et al. 2023 ). It is important to note that DEX concentrations and responses may vary across different models, depending on the cell type, experimental conditions, and culture medium (Bassil et al. 2023 ). The mTORC1 signaling pathway plays an important role in mediating the rapid antidepressant effect of ketamine by improving synaptic plasticity (Duman and Voleti 2012 ; Li et al. 2010 ). Although ketamine has a rapid onset of action, it has numerous side effects, including psychotic and dissociative symptoms, and potential for abuse. Thus, drugs with antidepressant effects similar to those of ketamine but without side effects are urgently needed. We propose that the DEX-induced 3D neural spheroid model can be used to replace or complement animal models of depression. Future studies could use this 3D in vitro model to evaluate ketamine-like antidepressant candidates. Accumulating evidence suggests that the function of sirtuin 1 may be abnormal in depression (Abe-Higuchi et al. 2016 ; CONVERGE consortium 2015 ; Kishi et al. 2010 ; Kovanen, et al. 2015 ). Chronic stress reduced sirtuin 1 expression and activity in the hippocampus (Abe-Higuchi et al. 2016 ; Shen et al. 2018 ). Moreover, the sirtuin 1 inhibitor sirtinol promotes depression-like behavior, while the sirtuin 1 activator resveratrol induces stress resilience. Inhibition of sirtuin 1 was associated with dendritic atrophy induced by chronic stress. Importantly, activation of the hippocampal sirtuin 1 increased ERK1/2 phosphorylation under chronic stress, and activation and inhibition of ERK1/2 led to antidepressant-like and depression-like behaviors, respectively (Abe-Higuchi et al. 2016 ; Ferland et al. 2013 ), highlighting a role of ERK1/2, a potential downstream target of sirtuin 1, in depression (Duman et al. 2007 ; Iñiguez et al. 2010 ; Li et al. 2008 ). Taken together, we propose that sirtuin 1/EKR1/2 signaling converges on the mTORC1 signaling pathway. To confirm this, further studies using the DEX-induced 3D neural spheroid model with pharmacological (inhibitor or activator) or genetic (siRNA or vial-mediated gene transfer) tools to modulate the sirtuin 1 function are needed. This study provides preliminary insights into the potential of 3D neural spheroid models as a tool for investigating the cellular mechanisms underlying depression. Further research is needed to validate this model using various antidepressant treatments, including SSRIs and ketamine, to confirm its relevance for studying drug effects in vitro . Additionally, examining the electrophysiological properties of the spheroids, such as synaptic activity and neuronal communication, would enhance our understanding of their functionality and potential as a model system. Overall, while this 3D model offers advantages over conventional 2D systems, its application as a model for depression still requires further exploration. Future studies could use this system to investigate the molecular actions of rapid-acting antidepressants, such as ketamine, which modulates the mTORC1 pathway to improve synaptic plasticity. The search for alternative antidepressants with fewer side effects remains a critical area of investigation, and the DEX-induced 3D neural spheroid model may contribute to this effort. In conclusion, we developed a 3D in vitro model of depression using primary cortical cell-based spheroid cultures. DEX treatment led to a reduction in neuroplasticity-related markers, including downregulation of the BDNF/ERK1/2/mTORC1 signaling pathway (Fig. 7 ). While this 3D model reflects certain aspects of the brain microenvironment, further studies are needed to fully assess its utility in exploring the molecular mechanisms underlying neuroplasticity. The system offers a potential tool for investigating antidepressant mechanisms, although additional validation is required. Overall, the findings from this study may contribute to a better understanding of depression's pathophysiology and could inform future research into therapeutic strategies. Declarations Competing interest The authors declare no competing financial interests. Ethical approval All experimental procedures were performed in accordance with the guidelines for the care and use of laboratory animals for scientific purposes with protocols approved by the Committee for Animal Experimentation and the Institutional Animal Laboratory Review Board of Inje Medical College (approval no. 2021-010). Statement on ARRIVE Guidelines We declared that this study was carried out in compliance with the ARRIVE guidelines. Funding This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government Ministry of Science and ICT (MSIT) (NRF-2023R1A2C2005016 to S.W. Park and NRF-2021R1A2C1003591 to J.G. Lee). And, this research was supported by Basic Science Research Program through the NRF funded by the Ministry of Education (NRF-2021R1I1A3061265 to S. Jeong). Author Contribution Conceptualization: Mi Kyoung Seo, Sehoon Jeong, Jung Goo Lee, and Sung Woo Park; Methodology: Mi Kyoung Seo, Woo Seok Cheon, Dong Yun Lee, Sumin Lee, Gyu-Hui Lee, Deok-Gyeong Kang, and Dae-Hyun Seog; Formal analysis and investigation: Mi Kyoung Seo, Woo Seok Cheon, and Dae-Hyun Seog; Writing – original draft preparation: Sehoon Jeong, Jung Goo Lee, and Sung Woo Park; Writing – review and editing: Mi Kyoung Seo, Woo Seok Cheon, Dae-Hyun Seog, and Seong-Ho Kim; Funding acquisition: Sehoon Jeong, Jung Goo Lee, and Sung Woo Park; Resources: Sehoon Jeong and Sumin Lee; Supervision: Jung Goo Lee and Sung Woo Park. All authors read and approved the final manuscript. Data Availability The data presented in this study are available on request from the corresponding author. References Abe-Higuchi N, Uchida S, Yamagata H, Higuchi F, Hobara T, Hara K, Kobayashi A, Watanabe Y (2016) Hippocampal Sirtuin 1 Signaling Mediates Depression-like Behavior. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5413832","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":376301053,"identity":"4891493b-46f4-4b3a-b0c2-5dedf96c9be0","order_by":0,"name":"Mi Kyoung Seo","email":"","orcid":"","institution":"Inje University","correspondingAuthor":false,"prefix":"","firstName":"Mi","middleName":"Kyoung","lastName":"Seo","suffix":""},{"id":376301054,"identity":"4e78cf1b-1f76-44b3-8bca-26be67f5acc2","order_by":1,"name":"Sehoon Jeong","email":"","orcid":"","institution":"Sejong University","correspondingAuthor":false,"prefix":"","firstName":"Sehoon","middleName":"","lastName":"Jeong","suffix":""},{"id":376301055,"identity":"006cf82b-abc5-4810-b508-08c20b32deb4","order_by":2,"name":"Woo Seok Cheon","email":"","orcid":"","institution":"Inje University","correspondingAuthor":false,"prefix":"","firstName":"Woo","middleName":"Seok","lastName":"Cheon","suffix":""},{"id":376301056,"identity":"4dbf7263-5359-42f5-a646-c73b5b69d818","order_by":3,"name":"Dong Yun Lee","email":"","orcid":"","institution":"Inje University","correspondingAuthor":false,"prefix":"","firstName":"Dong","middleName":"Yun","lastName":"Lee","suffix":""},{"id":376301057,"identity":"762e6715-9cca-452b-8f20-d0187258c743","order_by":4,"name":"Sumin Lee","email":"","orcid":"","institution":"Inje University","correspondingAuthor":false,"prefix":"","firstName":"Sumin","middleName":"","lastName":"Lee","suffix":""},{"id":376301058,"identity":"305c3607-9539-436e-b830-d41f7f9f520a","order_by":5,"name":"Gyu-Hui Lee","email":"","orcid":"","institution":"Inje University","correspondingAuthor":false,"prefix":"","firstName":"Gyu-Hui","middleName":"","lastName":"Lee","suffix":""},{"id":376301059,"identity":"e4fa9772-b3cf-4b96-9cbe-3d60649b3caa","order_by":6,"name":"Deok-Gyeong Kang","email":"","orcid":"","institution":"Inje University","correspondingAuthor":false,"prefix":"","firstName":"Deok-Gyeong","middleName":"","lastName":"Kang","suffix":""},{"id":376301060,"identity":"5c933ffd-dc64-416f-ab5a-bfacf547e556","order_by":7,"name":"Dae-Hyun Seog","email":"","orcid":"","institution":"Inje University","correspondingAuthor":false,"prefix":"","firstName":"Dae-Hyun","middleName":"","lastName":"Seog","suffix":""},{"id":376301061,"identity":"bcd2a602-5219-4cb9-bfa3-a2b3c0a7cb83","order_by":8,"name":"Seong-Ho Kim","email":"","orcid":"","institution":"Inje University","correspondingAuthor":false,"prefix":"","firstName":"Seong-Ho","middleName":"","lastName":"Kim","suffix":""},{"id":376301062,"identity":"4b534d9e-43a8-4706-ab87-132e68530c47","order_by":9,"name":"Jung Goo Lee","email":"","orcid":"","institution":"Inje University","correspondingAuthor":false,"prefix":"","firstName":"Jung","middleName":"Goo","lastName":"Lee","suffix":""},{"id":376301063,"identity":"81464dc2-5881-499c-bb08-8ba56314bbee","order_by":10,"name":"Sung Woo Park","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYNCCAzY8QALMNCBWSxrpWg7DmYS1yM/IPfi54sx5GXPGwwcYftQwGJs3ENBicCMvWfLMjds8lg3HEhh7jjGYyRwgpEU6x0Cy4cNtHoMDZwwYeBsYbCQIOmx2jvHPhg/ngFrOf2D8S4wWhts5ZpINNw6AbGFgBtpiRlCLwf13aZYNZ5KBWo4ZHJY5JmFM2GE9Zw/fbDhmZ29w4/DDh29qbAxnEHQYAw+UljgAikzCPkHSwt9AjOpRMApGwSgYiQAAOqZCsTud+jwAAAAASUVORK5CYII=","orcid":"","institution":"Inje University","correspondingAuthor":true,"prefix":"","firstName":"Sung","middleName":"Woo","lastName":"Park","suffix":""}],"badges":[],"createdAt":"2024-11-08 05:53:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5413832/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5413832/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70030675,"identity":"e5110ff0-2b15-492b-b1a0-b16c785e795a","added_by":"auto","created_at":"2024-11-27 16:22:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":503427,"visible":true,"origin":"","legend":"\u003cp\u003eMicro-spheroid device fabrication and layout.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic of the manufacturing process for the micro-spheroid device. (\u003cstrong\u003eB\u003c/strong\u003e) Each micro-spheroid device fits into 1 well of a conventional 48-well polystyrene culture plate. (\u003cstrong\u003eC\u003c/strong\u003e) Schematic illustration of the Micro-spheroid device. 3D and cross-sectional view showing the microwell arrays; each reservoir: 200 μm diameter and 200 μm height. (\u003cstrong\u003eD\u003c/strong\u003e) An illustration showing the generation of size-controlled neural aggregates using microwell arrays.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5413832/v1/7d18161f1ae047d7459dfaaf.png"},{"id":70030673,"identity":"68159395-2b5a-4ad9-a4f0-b7b6af10debd","added_by":"auto","created_at":"2024-11-27 16:22:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":274394,"visible":true,"origin":"","legend":"\u003cp\u003e3D cortical spheroid fabrication.\u003c/p\u003e\n\u003cp\u003eA micro-spheroid device with a diameter of 1 cm was used to fabricate spheroids with a diameter of 200 μm. Primary cortical cells were seeded into the micro-spheroid device. Monodispersed cortical cells were allowed to aggregate for 3 days. On DIV 3, self-assembled spheroids were transferred to various well plates for further experiments. Images were obtained using an inverted microscope.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5413832/v1/0d36ec95ea328a6915442285.png"},{"id":70030674,"identity":"2bce34c1-ad85-4f66-8321-27a572fa719a","added_by":"auto","created_at":"2024-11-27 16:22:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":506811,"visible":true,"origin":"","legend":"\u003cp\u003eCortical spheroids with neural cell types.\u003c/p\u003e\n\u003cp\u003eOn DIV 3, 4, and 10, spheroids were immunostained using antibodies specific for neural cell types. Neurons (β-III-tubulin, red), dendrites (MAP-2, purple), astrocytes (glial fibrillary acidic protein, green), microglia (CD11b, sky blue), oligodendrocytes (O1, brown), and neural stem cells (nestin, yellow) were identified in spheroids by confocal microscopy. Nuclei were stained with DAPI (blue). Scale bar: 200 μm.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5413832/v1/92b4b2895b957f2dbdcaa53d.png"},{"id":70031568,"identity":"39cc1a11-c965-46d3-8bd4-c71663278274","added_by":"auto","created_at":"2024-11-27 16:30:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":660184,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in cell viability, BDNF mRNA level, and neurite outgrowth after dexamethasone (DEX) exposure.\u003c/p\u003e\n\u003cp\u003eOn DIV 10, neural spheroids were exposed to 100, 200, or 300 μM DEX for 5 days. The experiments were performed on DIV 15. (\u003cstrong\u003eA\u003c/strong\u003e) Cell viability was evaluated by MTT assay. (\u003cstrong\u003eB\u003c/strong\u003e) In two different wells per group for each from three independent cultures (n=6), the BDNF mRNA level was evaluated by qRT-PCR. (\u003cstrong\u003eC\u003c/strong\u003e) Neurite outgrowth from 11 spheroids was analyzed using ImageJ Sholl software. (\u003cstrong\u003ea\u003c/strong\u003e) Neural spheroids were immunostained with a MAP-2 antibody. Representative images are shown. Scale bar: 200 µm. (\u003cstrong\u003eb\u003c/strong\u003e) Sholl analysis was used to calculate the number of neurite intersections at different distances from the spheroid center. The x-axis represents the radius from the center, and the y-axis represents the number of intersections with the concentric circles generated by the Sholl ImageJ software. (\u003cstrong\u003ec\u003c/strong\u003e) The Area Under the Curve was calculated for each group shown in (b). Data represent means ± SEM from three independent experiments. \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 or \u003csup\u003e\u003cem\u003e***\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. controls (0 µM DEX).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5413832/v1/4b6260127f89e5e8d22af8fc.png"},{"id":70030679,"identity":"520d3b37-ae42-45cf-97d5-48df152fcbf8","added_by":"auto","created_at":"2024-11-27 16:22:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":182075,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in the levels of BDNF and sirtuin 1 proteins and ERK1/2 phosphorylation after dexamethasone (DEX) exposure.\u003c/p\u003e\n\u003cp\u003eOn DIV 10, neural spheroids were exposed to 100 µM DEX for 5 days. In two different wells per group for each from four independent cultures (n=8), the protein levels were analyzed by SDS-PAGE and Western blotting using each of the primary antibodies. Western blotting revealed the levels of BDNF (\u003cstrong\u003eA\u003c/strong\u003e), sirtuin 1 (\u003cstrong\u003eB\u003c/strong\u003e), and phospho-Thr\u003csup\u003e202\u003c/sup\u003e/Tyr\u003csup\u003e204\u003c/sup\u003e-ERK1/2 (\u003cstrong\u003eC\u003c/strong\u003e). Quantitative analyses were normalized to the α-tubulin level for BDNF and sirtuin 1 and the total ERK level for phospho-ERK1/2. Data represent means ± SEM from three independent experiments. \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 or \u003csup\u003e\u003cem\u003e***\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. controls (CON; 0 µM DEX).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5413832/v1/76aff0d6d45b4389741e5da8.png"},{"id":70030677,"identity":"e9b64cca-5dcc-4267-a1a6-bf8b3f9e017d","added_by":"auto","created_at":"2024-11-27 16:22:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1101551,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in the phosphorylation of mTORC1-mediated proteins and the expression of synaptic proteins after dexamethasone (DEX) exposure.\u003c/p\u003e\n\u003cp\u003eOn DIV 10, neural spheroids were exposed to 100 µM DEX for 5 days. In two different wells per group for each from four independent cultures (n=8), Western blotting and immunofluorescence were performed using each primary antibody. (\u003cstrong\u003eA\u003c/strong\u003e) Western blotting revealed the levels of phospho-Ser\u003csup\u003e2448\u003c/sup\u003e-mTORC1 (\u003cstrong\u003ea\u003c/strong\u003e), phospho-Thr\u003csup\u003e37/46\u003c/sup\u003e-4E-BP1 (\u003cstrong\u003eb\u003c/strong\u003e), and phospho-Thr\u003csup\u003e389\u003c/sup\u003e-p70S6K (\u003cstrong\u003ec\u003c/strong\u003e). (\u003cstrong\u003eB\u003c/strong\u003e) Western blot analysis and representative images of PSD-95 (\u003cstrong\u003ea\u003c/strong\u003e) and GluA1 (\u003cstrong\u003eb\u003c/strong\u003e) are shown. Synaptic markers: PSD-95 (green) co-stained with neuronal marker MAP-2 (red); GluA1 (green) co-stained with MAP-2 (red). Scale bar: 100 µm. Data are presented as means ± SEM from three independent experiments. \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 or \u003csup\u003e\u003cem\u003e***\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs. controls (CON; 0 µM DEX).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5413832/v1/30b2b8d0bc796fe12da9fd58.png"},{"id":70030678,"identity":"8e484b25-9ee6-403f-9661-8dc3aee6ee66","added_by":"auto","created_at":"2024-11-27 16:22:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":227346,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of the molecular mechanisms underlying DEX-induced impaired neuroplasticity.\u003c/p\u003e\n\u003cp\u003eExposure to DEX downregulates the signaling pathways that affect neuroplasticity, including BDNF, sirtuin 1, and mTORC1 signaling. Activation of the mTORC1 signaling pathway induces the synthesis of synaptic proteins as well as BDNF. Secreted BDNF interacts with its receptor TrkB, further activating mTORC1 signaling via PI3K/Akt and MEK/EKR1/2. Therefore, the synaptic plasticity is enhanced. Sirtuin 1 is also involved in the regulation of neuroplasticity. Akt, protein kinase B; AMPA, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; BDNF, brain-derived neurotrophic factor; ERK1/2, extracellular signal-regulated kinase 1/2; GluA1, AMPA receptor subunit glutamate receptor1; MEK, mitogen‑activated protein kinase; mTORC1, mechanistic target of rapamycin complex I; p70S6K, p70S6 kinase; PI3K, phosphatidyl inositol-3 kinase; PSD-95, postsynaptic density protein-95; TrKB, tropomyosin receptor kinase B; 4E-BP1, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1. Original illustration created using BioRender (biorender.com).\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5413832/v1/2dd12e42ab7f1c3872412b99.png"},{"id":72263991,"identity":"80e45005-4a75-4c41-a4fa-7f4a8c49b068","added_by":"auto","created_at":"2024-12-24 11:23:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4204040,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5413832/v1/05e9c9e0-f904-4f0d-b620-7e1389dd48f1.pdf"},{"id":70030680,"identity":"bc909370-0a81-41b1-a4c6-ce0a9f3fb1ec","added_by":"auto","created_at":"2024-11-27 16:22:34","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":415055,"visible":true,"origin":"","legend":"","description":"","filename":"Supplemantaryinformation241108.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5413832/v1/84f6142fd4565dc0421460a7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Establishment of a depression model using dexamethasone-treated three-dimensional cultured rat cortical cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDepression is a prevalent mental health disorder affecting approximately 3.8% of the global population, with a higher incidence among adults (5.0%) and those over 60 years old (5.7%) (Institute of Health Metrics and Evaluation \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It manifests through various symptoms such as anhedonia, reduced motivation, hopelessness, sleep disturbances, appetite changes, and decreased physical activity (Otte et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), often accompanied by an increased risk of suicide (Bachmann \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). As projected by the World Health Organization, depression could become the second leading cause of global disability by 2030, presenting a significant burden on both individuals and society (Li et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although selective serotonin reuptake inhibitors (SSRIs) are widely used and effective antidepressants, their therapeutic benefits often take weeks to manifest, and many patients do not respond adequately, leading to treatment-resistant depression (Commons and Linnros \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Akil et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This highlights the need for new therapeutic approaches that can provide faster and more reliable effects.\u003c/p\u003e \u003cp\u003eUnderstanding the underlying biological mechanisms of depression is key to advancing treatment options. While the exact pathophysiology of depression remains unclear, growing evidence points to impaired neuroplasticity as a central factor (Duman and Aghajanian \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Malykhin and Coupland \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; McEwen et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Price and Drevets \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Brain-derived neurotrophic factor (BDNF) plays a critical role in supporting neurogenesis and synaptic plasticity, and it is closely associated with antidepressant effects (Autry and Monteggia \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Castr\u0026eacute;n and Kojima \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Lindholm and Castr\u0026eacute;n \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Reduced BDNF levels have been observed in individuals with depression, and restoring BDNF activity is linked to antidepressant outcomes (Bocchio-Chiavetto et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Duman and Monteggia \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Pittenger and Duman \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Signaling through BDNF and its receptor, tropomyosin receptor kinase B (TrkB), activates pathways such as the phosphatidyl inositol-3 kinase (PI3K)/protein kinase B (Akt) and mitogen‑activated protein kinase (MEK)/extracellular signal‑regulated kinase 1/2 (ERK1/2) signaling cascades, promoting neuronal growth and plasticity (Duman et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The mechanistic target of rapamycin complex I (mTORC1) signaling pathway, which modulates synaptic protein synthesis, is another critical downstream target of BDNF/TrkB signaling, and its dysfunction has been implicated in stress-induced neuroplastic deficits (Chao and Hempstead \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Duman et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Saarelainen et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). mTORC1 signaling enhances mRNA translation via its downstream substrates p70S6 kinase (p70S6K) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), leading to increased expression of synaptic proteins, such as postsynaptic density protein-95 (PSD-95), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit glutamate receptor 1 (GluA1), and synapsin 1. Chronic stress downregulates the synthesis of BDNF and synaptic proteins via the mTORC1 signaling pathway and ultimately impairs neuroplasticity (Duman et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Moreover, sirtuin 1, a protein that influences neuroplasticity, has been linked to depression through genetic studies (CONVERGE consortium \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kishi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Kovanen et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and found to have decreased expression in depressed patients (Kishi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile animal models of depression have been extensively used to study these molecular pathways, \u003cem\u003ein vitro\u003c/em\u003e models offer a complementary approach to investigate depression-related neuroplasticity. Traditionally, \u003cem\u003ein vitro\u003c/em\u003e studies have relied on two-dimensional (2D) cultures, where cells grow in a flat, planar manner. However, the brain\u0026rsquo;s complex three-dimensional (3D) structure, composed of interconnected neurons, astrocytes, and glial cells, is not well represented by 2D cultures. Thus, developing 3D models that better mimic the brain\u0026rsquo;s microenvironment is essential for understanding depression at the cellular and molecular levels. The importance of refining \u003cem\u003ein vitro\u003c/em\u003e models has become even more relevant given recent shifts in regulatory policies, such as the US FDA\u0026rsquo;s decision to no longer require animal testing prior to human trials (Wadman \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, which governs the stress response, is a well-documented feature of depression (Troubat et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This system regulates the secretion of glucocorticoids in response to stress, with elevated glucocorticoid levels contributing to hippocampal and prefrontal cortex dysfunction in depressed individuals (Hill and Spencer-Segal \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Antidepressant treatments have been shown to normalize HPA axis activity, supporting its relevance in depression recovery (Troubat et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we aimed to develop a 3D spheroid model that more accurately replicates the neurobiological environment associated with depression. Using a micro-spheroid device, we generated 3D neural spheroids from primary rat cortical cells. To simulate the glucocorticoid hypersecretion seen in depression, we treated the spheroids with varying concentrations of dexamethasone (DEX), a synthetic glucocorticoid. We then explored the effects of DEX on key molecular pathways implicated in neuroplasticity, including BDNF, sirtuin 1, and mTORC1 signaling.\u003c/p\u003e"},{"header":"Method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFabrication of the micro-spheroid device for 3D neural spheroids\u003c/h2\u003e \u003cp\u003eThe micro-spheroid device was fabricated from polydimethylsiloxane (PDMS) prepolymer (10:1 mixture Sylgard 184 DOW CORNING, USA) using one-step photolithography and soft lithography techniques. Initially, an array of microwells was made by PDMS soft lithography from an SU-8\u0026trade; patterned Si master mold with inverted replication (pillar structure) of microwell arrays (diameter: 200 \u0026micro;m, depth: 200 \u0026micro;m). To create the master mold (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, a), a photosensitive epoxy layer (SU-8\u0026trade;; MicroChem, USA) with 200 \u0026micro;m thicknesses was patterned onto a 4-inch diameter silicon substrate. The photosensitive epoxy layer, which forms the microwell arrays, is patterned by spin-coating SU-8 2075 at 1200 rpm to make a thickness of 200 \u0026micro;m, and first soft-baked at 65\u0026deg;C for 24 h and then at 95\u0026deg;C for 40 min. It was then exposed to UV light through a photomask, followed by a postexposure bake in two steps, first at 65\u0026deg;C for 5 min and then at 95\u0026deg;C for 15 min. The master mold was then coated with (tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilane (United Chemical Technologies, USA) to facilitate PDMS release from the master mold. A PDMS prepolymer (10:1 mixture) was poured into the master mold and cured at 80\u0026deg;C for 2 h to replicate the PDMS device from the master mold (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, b). A 10-mm diameter punch bit (Syneo, USA) mounted on a drill press was used to punch out reservoirs to hold the cell culture media (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, c). Second, PDMS replicas can be cut into 10 mm diameter discs by circular punch bits (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, d), and each PDMS micro-spheroid device can be placed in each well of a 48-well cell culture plate. The micro-spheroid devices were then treated with oxygen plasma, immersed in deionized water and subsequently autoclaved. After sterilization, the micro-spheroid device was placed in a 48-well cell culture plate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). 3D neural spheroids were prepared using the microwell arrays in the micro-spheroid device (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Dissociated primary cortical cells were added at a concentration of 10\u0026nbsp;million cells per a micro-spheroid device. Finally, the cells were cultured for 3 days in a temperature and humidity controlled 5% CO\u003csub\u003e2\u003c/sub\u003e incubator, and then the 200 \u0026micro;m sized 3D neural spheroids formed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD were collected by rapidly stirring the PDMS micro-spheroid device in the medium bath.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIsolation of primary cortical cells\u003c/h3\u003e\n\u003cp\u003ePrimary cortical tissues were isolated from the brains of Sprague\u0026ndash;Dawley rat (Hana Biotech, Pyeongtaek-si, Gyeonggi-do, Korea) fetuses (embryonic day 19). The method of cortical cell isolation was slightly modified from that developed by Pacifici and Pruzzi (2012). Briefly, the tissues were cut into small sections and triturated by pipetting 30 times in neurobasal media (Gibco; ThermoFisher Scientific, Waltham, MA, USA) containing 0.25% trypsin\u0026ndash;ethylene-diamine-tetraacetic acid (Gibco). The cell solution was placed at room temperature for 20 min and then suspended in growth medium (neurobasal medium containing 1% fetal bovine serum, 1% horse serum, 2% serum-free B27 growth medium, 250 mM glutamine, and 1% penicillin\u0026ndash;streptomycin; all from Gibco). Debris was removed by passing the solution through a 100 \u0026micro;m cell strainer (SPL Life Sciences, Gyeonggi-do, Korea). The cell solution was centrifuged at 180 g for 10 min, and the supernatant was removed. The cell pellet was suspended in growth medium and carefully pipetted several times. Cortical cells were seeded at a density of 2 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e /micro-spheroid device (see 3D neural spheroid culture). The procedures were performed according to the guidelines of the Institutional Animal Care and Use Committee (IACUC; Inje University, Republic of Korea) and the ARRIVE (Kilkenny et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The animal experiments were approved by the IACUC of the College of Medicine of Inje University (approval no.: 2021\u0026ndash;010).\u003c/p\u003e\n\u003ch3\u003eThree-dimensional neural spheroid culture\u003c/h3\u003e\n\u003cp\u003eCortical cell spheroids were prepared using the micro-spheroid device, which is microwells for producing uniformly sized spheroids with a diameter of 200 \u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A micro-spheroid device was placed in a 48-well plate, and cortical cells were seeded at a density of 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e/micro-spheroid device. Cortical cells were allowed to settle into microwells for 1 h at 37\u0026deg;C, while the cells attached to the outside of the microwells were washed using a growth medium. Cells were aggregated in the microwells for 3 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). At 3 days \u003cem\u003ein vitro\u003c/em\u003e (DIV), aggregates were suspended in a 3 mL growth medium and transferred to poly-D-lysine (Sigma-Aldrich, St. Louis, MO, USA) and Matrigel (BD Biosciences, Franklin Lakes, NJ, USA)-coated plates. For the cell viability assay, 10\u0026ndash;15 spheroids (100 \u0026micro;L) were plated in a 48-well plate. For quantitative real-time polymerase chain reaction (qRT-PCR), 20\u0026ndash;30 spheroids (200 \u0026micro;L) were plated in a 12-well plate. For Western blotting, 40\u0026ndash;60 spheroids (400 \u0026micro;L) were plated in a 6-well plate. For immunofluorescence, 20\u0026ndash;30 spheroids (200 \u0026micro;L) were plated in a 1-well cell culture slide (SPL Life Sciences). On DIV 10, the spheroids were exposed to DEX (water-soluble; Sigma-Aldrich) at 100\u0026ndash;300 \u0026micro;M for 5 days. On DIV 15, the spheroids were harvested for further experiments. Whole spheroids were maintained at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e, and 95% humidity. The growth medium was changed every 2\u0026ndash;3 days. The results from at least three independent spheroid cultures were recorded.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eTo detect the presence of neurons and glia in cortical spheroids, the spheroids were collected on DIV 3, 4, and 10. Additionally, spheroids were harvested after 5 days of exposure to 100 \u0026micro;M DEX to determine whether DEX altered synaptic protein expression. The spheroids were washed with phosphate-buffered saline (PBS; GenDEPOT) and fixed in 4% paraformaldehyde (Biosesang, Seongnam-si, Gyeonggi-do, Korea) for 20 min at room temperature. The spheroids were permeabilized in 0.1% Triton X-100 (GenDEPOT) for 3 min, blocked with 4% bovine serum albumin (GenDEPOT) for 1 h at room temperature, and incubated with the primary antibodies (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) overnight at 4\u0026deg;C. After incubation, the spheroids were washed with PBS and incubated with the secondary antibodies for 1 h (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Subsequently, the spheroids were washed with PBS, and the nuclei were stained with Hoechst 33258 (1:10,000; Invitrogen, Carlsbad, CA, USA). After washing with PBS, the spheroids were mounted on a 1-well cell culture slide using Biomeda Gel/Mount (Electron Microscopy Sciences, Foster City, CA, USA). The images were captured at a resolution of 1024 \u0026times; 1024 pixels using the A1\u003csup\u003e+\u003c/sup\u003e confocal laser scanning microscope (Nikon, Tokyo, Japan).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of antibodies used in this study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAntigen\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eManufacturer\u003c/p\u003e \u003cp\u003e(cat. no., RRID)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eApplication\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eIF\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eWB\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-III-tubulin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBioLegend\u003c/p\u003e \u003cp\u003e(801202,\u003c/p\u003e \u003cp\u003eRRID:AB_2313773)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMAP-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMillipore\u003c/p\u003e \u003cp\u003e(MAB3418,\u003c/p\u003e \u003cp\u003eRRID:AB_94856)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNestin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMillipore\u003c/p\u003e \u003cp\u003e(MAB353,\u003c/p\u003e \u003cp\u003eRRID:AB_94911)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGFAP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSanta Cruz Biotechnology\u003c/p\u003e \u003cp\u003e(sc-33673,\u003c/p\u003e \u003cp\u003eRRID:AB_627673)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eO1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMillipore\u003c/p\u003e \u003cp\u003e(MAB344,\u003c/p\u003e \u003cp\u003eRRID:AB_94860)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD11b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMillipore\u003c/p\u003e \u003cp\u003e(CBL1512,\u003c/p\u003e \u003cp\u003eRRID:AB_93253)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHoechest 33258\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInvitrogen\u003c/p\u003e \u003cp\u003e(H21491)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:10000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBDNF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003cp\u003e(ab108319,\u003c/p\u003e \u003cp\u003eRRID:AB_10862052)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSirtuin1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMillipore\u003c/p\u003e \u003cp\u003e(07-131,\u003c/p\u003e \u003cp\u003eRRID:AB_10067921)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep-Thr\u003csup\u003e202\u003c/sup\u003e/Tyr\u003csup\u003e204\u003c/sup\u003e-ERK1/2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003cp\u003e(9101,\u003c/p\u003e \u003cp\u003eRRID:AB_331646)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eERK1/2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell signaling Technology\u003c/p\u003e \u003cp\u003e(4695,\u003c/p\u003e \u003cp\u003eRRID:AB_390779)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep-Ser\u003csup\u003e2448\u003c/sup\u003e-mTORC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003cp\u003e(2971,\u003c/p\u003e \u003cp\u003eRRID:AB_330970)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emTORC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003cp\u003e(2972,\u003c/p\u003e \u003cp\u003eRRID: AB_330978)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep-Thr\u003csup\u003e37/46\u003c/sup\u003e-4E-BP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003cp\u003e(2855,\u003c/p\u003e \u003cp\u003eRRID:AB_560835)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4E-BP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003cp\u003e(9452,\u003c/p\u003e \u003cp\u003eRRID:AB_331692)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep-Thr\u003csup\u003e389\u003c/sup\u003e-p70S6K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003cp\u003e(9205,\u003c/p\u003e \u003cp\u003eRRID:AB_330944)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep70S6K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003cp\u003e(9202,\u003c/p\u003e \u003cp\u003eRRID:AB_331676)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePSD-95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003cp\u003e(3450,\u003c/p\u003e \u003cp\u003eRRID: AB_2292883)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGluA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003cp\u003e(13185,\u003c/p\u003e \u003cp\u003eRRID:AB_2732897)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eα-tubulin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSigma-Aldrich\u003c/p\u003e \u003cp\u003e(T9026,\u003c/p\u003e \u003cp\u003eRRID:AB_477593)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:2000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlexa Fluor 594 goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003cp\u003e(A-11032, RRID:AB_2534091)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlexa Fluor 488 goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e \u003cp\u003e(A-11008, RRID:AB_143165)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMouse anti-rabbit IgG-HRP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSanta Cruz Biotechnology\u003c/p\u003e \u003cp\u003e(sc-2357, RRID:AB_628497)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:2000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-mouse IgG-HRP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell signaling Technology\u003c/p\u003e \u003cp\u003e(7076,\u003c/p\u003e \u003cp\u003eRRID:AB_330924)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:5000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eIF, Immunofluorescence; WB, Western blotting\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eCell viability\u003c/h3\u003e\n\u003cp\u003eThe cytotoxic effect of DEX on neural spheroids was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Amresco, Solon, OH, USA) assay. Briefly, after exposure of spheroids in 48-well plates to 0, 100, 200, or 300 \u0026micro;M DEX for 5 days, 0.5 mg/mL MTT was added to the wells. The plates were incubated at 37\u0026deg;C for 4 h. After incubation, the medium was removed, and 400 \u0026micro;L dimethyl sulfoxide (GenDEPOT, Katy, TX, USA) was added to dissolve the formazan. Subsequently, 100 \u0026micro;L of the dissolved solution from each well was transferred to 96-well plates and analyzed using a SpectraMax M2e Microplate Readers (Molecular Devices, San Jose, CA, USA) at a wavelength of 570 nm.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of BDNF mRNA levels by qRT-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from spheroids after 5 days of exposure to 0, 100, 200, or 300 \u0026micro;M DEX using Tri-RNA reagent (Favogen, Kaohsiung, Taiwan) according to the manufacturer\u0026rsquo;s instructions. RNA quantity and purity were determined using the NanoDrop\u0026trade; ND-1000 UV-Vis Spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). Single-strand cDNA was synthesized from 1 \u0026micro;g RNA using amfiRivertII\u0026trade; cDNA Synthesis Master Mix (GenDEPOT) according to the manufacturer\u0026rsquo;s instructions, and 100 ng of the cDNA was subjected to qRT-PCR using TOPreal\u0026trade; qPCR 2\u0026times; PreMIX (SYBR Green with low ROX; Enzynomics, Daejeon, Korea). The final primer concentration was 10 pmol. The reaction parameters were 95\u0026deg;C for 10 min, followed by 45 cycles of heating at 95\u0026deg;C for 35 s, annealing at 55\u0026deg;C for 35 s, and extension at 72\u0026deg;C for 35 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene. The following primer sequences were used: BDNF: forward 5'-CCAACGAAGAAAACCATAAG-3', reverse 5'-AATACTGTCACACAC GCTCA-3'; GAPDH: forward 5'-AACAGCAACTCCCATTCTT C-3', reverse 5'-TGGTCCA GGGTTTCTTACTC-3'. The relative BDNF mRNA level was calculated using the 2\u003csup\u003e\u0026minus;△△CT\u003c/sup\u003e method (Livak and Schmittgen \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The qRT-PCR was repeated two times per group for each from the three independent cultures.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNeurite outgrowth analysis\u003c/h3\u003e\n\u003cp\u003eAfter 5 days of exposure to 0, 100, 200, or 300 \u0026micro;M DEX, the spheroids were fixed in 4% paraformaldehyde and immunostained with anti-microtubule-associated protein-2 (MAP-2; see Immunofluorescence). Photographs of each spheroid were captured as 20 images cropped from top to bottom using the A1\u003csup\u003e+\u003c/sup\u003e confocal laser scanning microscope; the middle (i.e., 10th) image of the spheroid was analyzed using Sholl ImageJ software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.net/Sholl_Analysis\u003c/span\u003e\u003cspan address=\"https://imagej.net/Sholl_Analysis\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The number of intersections/distance from the spheroid center was plotted (Harris et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhong et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The Area Under the Curve of each group was calculated from each spheroid and then averaged. Intersections were counted after a 400 \u0026micro;m diameter (i.e., the average size of the spheroids on DIV 15).\u003c/p\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cp\u003eTotal proteins were extracted from spheroids after 5 days of exposure to 100 \u0026micro;M DEX using lysis buffer containing RIPA buffer (Elpisbiotech, Daejeon, Korea), 1\u0026times; protease inhibitor cocktail (GenDEPOT), and 1\u0026times; phosphatase inhibitor (GenDEPOT). The pellets were removed by centrifugation (13,000 g, 20 min, 4\u0026deg;C), and the supernatant was collected. Protein concentrations were measured using the Bradford assay (Bradford Protein Assay Plus Reagents; GenDEPOT). Total protein lysates (20 \u0026micro;g) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (GenDEPOT) for 2 h using a 100-V power supply. The membranes were blocked in Tris-buffered saline with Tween-20 (TBS-T; GenDEPOT) and 5% skim milk for 1 h and then incubated with the primary antibodies (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) overnight at 4\u0026deg;C. The membranes were washed with TBS-T for 10 min and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) at room temperature for 1 h. The Western blot bands were detected using West-Q Pico Dura ECL Solution (GenDEPOT) and visualized using the Amersham\u0026trade; Imager 600 (GE Healthcare, Chicago, IL, USA). The protein bands were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The Western blot analyses were repeated two times per group for each from the four independent cultures.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism software (version 9.4.1; GraphPad software, Inc., San Diego, CA, USA; RRID:SCR_002798). BDNF mRNA level and neurite outgrowth values were analyzed by one-way analysis of variance followed by \u003cem\u003eTukey\u003c/em\u003e\u0026rsquo;s post hoc tests. The unpaired \u003cem\u003et\u003c/em\u003e-test was used to compare the two groups. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSelf-assembled 3D spheroids in cortical cells\u003c/h2\u003e \u003cp\u003ePrimary cortical cells dissociated from embryonic day 19 cortical tissues from rat fetuses were seeded in a micro-spheroid device to facilitate cellular self-assembly into 3D spheroids (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The cortical cells were aggregated in the micro-spheroid device from DIV 1 to 3. On DIV 3, spheroids of equal size (200 \u0026micro;m diameter) were generated. From DIV 15, the diameter remained constant at almost 400 \u0026micro;m.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess the formation of intercellular networks within 3D cortical spheroids, we conducted a comprehensive investigation of various cell types generated in primary culture, including neurons, astrocytes, oligodendrocytes, neural progenitor cells, and microglia. This analysis involved the use of immunostaining techniques using specific markers for each cell type (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). On DIV 3, 4, and 10, cortical spheroids expressed markers of neurons (β-III-tubulin and MAP-2), astrocytes (glial fibrillary acidic protein), microglia (CD11b), oligodendrocytes (O1), and neural progenitor cells (nestin). Spheroids on DIV 3 exhibited globular patterns. Neurite and glial processes extending outward from the spheroid were observed at DIV 4. At DIV 10, the neurites were longer and formed a more complex network, and the neural cells continued to extend the process outward from the spheroid.\u003c/p\u003e \u003cp\u003e \u003cb\u003eChanges in cell viability, BDNF mRNA level, and neurite outgrowth in neural spheroids after DEX exposure\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the dose-dependent effect of DEX on cytotoxicity, neural spheroids on DIV 10 were exposed to different doses of DEX (100, 200, and 300 \u0026micro;M) for 5 days, and an MTT assay was performed on DIV 15. Neural spheroids exposed to DEX showed a dose-dependent decrease in cell viability (\u003cem\u003eF\u003c/em\u003e \u003csub\u003e[3,76]\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;11.500, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 100 \u0026micro;M, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004, 82.89% of control; 200 \u0026micro;M, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, 79.28% of control; 300 \u0026micro;M, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, 72.92% of control, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also investigated whether 100\u0026ndash;300 \u0026micro;M DEX decreases the BDNF level and neuronal complexity. DEX concentrations of 100, 200, and 300 \u0026micro;M significantly reduced the BDNF mRNA level (\u003cem\u003eF\u003c/em\u003e \u003csub\u003e[3,12]\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;29.600, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 100 \u0026micro;M, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 200 \u0026micro;M, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 300 \u0026micro;M, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Neurite outgrowth was visualized by immuno-fluorescence using an anti-MAP-2 antibody and quantified using Sholl ImageJ software (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). DEX doses of 100, 200, and 300 \u0026micro;M produced dose-dependent reductions in the number of intersections at 300 \u0026micro;m from the spheroid center (100 \u0026micro;M, 126.38\u0026thinsp;\u0026plusmn;\u0026thinsp;9.86 intersections; 200 \u0026micro;M, 129.50\u0026thinsp;\u0026plusmn;\u0026thinsp;9.80 intersections; 300 \u0026micro;M, 86.13\u0026thinsp;\u0026plusmn;\u0026thinsp;10.41 intersections) and at 400 \u0026micro;m from the spheroid center (100 \u0026micro;M, 100.50\u0026thinsp;\u0026plusmn;\u0026thinsp;7.53 intersections; 200 \u0026micro;M, 95.13\u0026thinsp;\u0026plusmn;\u0026thinsp;10.30 intersections; 300 \u0026micro;M, 65.13\u0026thinsp;\u0026plusmn;\u0026thinsp;6.98 intersections, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, b). Additionally, exposure to 100, 200, and 300 \u0026micro;M DEX decreased the total number of intersections to 21.08%, 20.68%, and 21.08%, respectively. The Area Under the Curve was significantly reduced after 100, 200, and 300 \u0026micro;M DEX treatment compared with the controls (\u003cem\u003eF\u003c/em\u003e \u003csub\u003e[3,23]\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;11.100, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 100 \u0026micro;M, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.019; 200 \u0026micro;M, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.017; 300 \u0026micro;M, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, c).\u003c/p\u003e \u003cp\u003e \u003cb\u003eChanges in cell viability, BDNF mRNA level, and neurite outgrowth in neural spheroids after DEX exposure\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe preliminary experiments showed that co-administration of 200 or 300 \u0026micro;M DEX with antidepressants disrupted the morphology of neural spheroids due to cytotoxic effects (data not shown). Therefore, neural spheroids were exposed to 100 \u0026micro;M DEX for 5 days.\u003c/p\u003e \u003cp\u003eAs expected, treatment with 100 \u0026micro;M DEX significantly reduced the protein levels of BDNF (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.480, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), sirtuin 1 (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.130, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), and phospho-ERK1/2, a potential downstream target of sirtuin 1 (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.810, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.031, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eChanges in the phosphorylation of mTORC1-mediated proteins and the expression of synaptic proteins in neural spheroids after DEX exposure\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the effects of DEX on mTORC1 signaling in neural spheroids, the phosphorylation levels of mTORC1, 4E-BP1, and p70S6K, as well as the expression levels of the synaptic proteins PSD-95 and GluA1 were determined by Western blotting. The exposure of spheroids to 100 \u0026micro;M DEX significantly reduced the phosphorylation levels of mTORC1 (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.710, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.010, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, a), 4E-BP1 (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.070, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, b), and p70S6K (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.030, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, c), as well as the expression levels of PSD-95 (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.330, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.016, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, a) and GluA1 (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.670, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.010, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, b). Neurites in cortical spheroids showed immunoreactivity for each of the synaptic markers PSD-95 and GluA1 as shown by immunofluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we utilized a 3D neural spheroid culture system derived from primary rat cortical cells. The use of 3D spheroid models presents a potential alternative to traditional 2D cultures and animal models for investigating depression and other neurological conditions. These models offer an environment that more closely mimics the \u003cem\u003ein vivo\u003c/em\u003e brain structure, maintaining key cellular interactions and the extracellular matrix in a 3D space (Dingle et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTreatment of the 3D neural spheroids with DEX, a synthetic glucocorticoid, resulted in reduced cell viability, BDNF mRNA levels, and neurite outgrowth, all in a dose-dependent manner. Notably, exposure to 100 \u0026micro;M DEX led to a decrease in the protein levels of BDNF, sirtuin 1, and components of the mTORC1 signaling pathway, such as phospho-ERK1/2, phospho-mTORC1, and synaptic proteins (PSD-95 and GluA1). These findings indicate that this 3D spheroid model may serve as a useful \u003cem\u003ein vitro\u003c/em\u003e system for studying the molecular mechanisms that contribute to impaired neuroplasticity in depression.\u003c/p\u003e \u003cp\u003eThe method employed in this study allowed for the production of uniform 3D cortical spheroids, approximately 200 \u0026micro;m in diameter, using a micro-spheroid device. This setup ensured consistent size and morphology, avoiding the central necrosis that can occur in larger spheroids due to insufficient nutrient and oxygen supply (Pamies et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Importantly, the cultures were scaffold-free, meaning that the cells naturally produced their own extracellular matrix, such as laminin (Dingle et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), which has been shown to support neurogenesis and synapse formation (Barros et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Relucio et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In the present study, we established a scaffold-free, self-assembled 3D neural culture system that forms complex 3D structures of neurons and glia in a relatively short time of 15 days. This system offers a relatively simple and reproducible way to create 3D cultures that may be applied to studying various aspects of central nervous system function.\u003c/p\u003e \u003cp\u003eCompared to traditional 2D cultures and animal models, 3D \u003cem\u003ein vitro\u003c/em\u003e models may provide a more physiologically relevant platform for examining complex processes like neuroplasticity. For instance, similar 3D systems have been used to model Alzheimer's disease and perform neurotoxicity screenings using human-induced pluripotent stem cells (iPSCs)-derived spheroids (Choi et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kobolak et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhong et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In our study, we used this model to simulate HPA axis dysregulation associated with glucocorticoid hypersecretion by treating the spheroids with DEX, and we observed downregulation of key neuroplasticity-related proteins and pathways.\u003c/p\u003e \u003cp\u003eDEX has been widely used in rodent models to induce depression-like behaviors, including reductions in reward-seeking and increased immobility (Wang et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In previous studies, chronic DEX administration has been linked to structural changes in the brain, such as neuronal atrophy, decreased dendritic outgrowth, and impaired synaptic plasticity (Wang et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In fact, in our previous studies of 2D cultures, DEX treatment reduced total dendritic length and spine formation in primary hippocampal cells; this plasticity was associated with decreased expression of synaptic proteins (PSD-95, synapsin 1, and GluA1) via downregulation of mTORC1 signaling (Park et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Seo et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Our results align with these findings, as DEX exposure in our 3D model similarly affected synaptic proteins and neuroplasticity pathways, notably the mTORC1 signaling pathway, which is critical for synaptic function and plasticity (Duman and Monteggia \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The doses of DEX used in this study (100\u0026ndash;300 \u0026micro;M) were selected based on preliminary experiments, with 100 \u0026micro;M chosen for subsequent analyses due to its significant impact on cell viability and neuroplasticity markers without causing spheroid disintegration. The DEX concentrations used in this study are higher than those used in other 2D or 3D culture models. Cerebral organoids derived from human iPSCs were treated with 10, 100, 1000 nM and 100 \u0026micro;M DEX (Cruceanu et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Mice hippocampal slice cultures were treated with 1-100 nM (Saito et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In 2D cultures using neuronal cell lines, DEX was applied at various concentrations (0.01\u0026ndash;100 \u0026micro;M) (Bassil et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It is important to note that DEX concentrations and responses may vary across different models, depending on the cell type, experimental conditions, and culture medium (Bassil et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe mTORC1 signaling pathway plays an important role in mediating the rapid antidepressant effect of ketamine by improving synaptic plasticity (Duman and Voleti \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Although ketamine has a rapid onset of action, it has numerous side effects, including psychotic and dissociative symptoms, and potential for abuse. Thus, drugs with antidepressant effects similar to those of ketamine but without side effects are urgently needed. We propose that the DEX-induced 3D neural spheroid model can be used to replace or complement animal models of depression. Future studies could use this 3D \u003cem\u003ein vitro\u003c/em\u003e model to evaluate ketamine-like antidepressant candidates.\u003c/p\u003e \u003cp\u003eAccumulating evidence suggests that the function of sirtuin 1 may be abnormal in depression (Abe-Higuchi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; CONVERGE consortium \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kishi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Kovanen, et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Chronic stress reduced sirtuin 1 expression and activity in the hippocampus (Abe-Higuchi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Shen et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Moreover, the sirtuin 1 inhibitor sirtinol promotes depression-like behavior, while the sirtuin 1 activator resveratrol induces stress resilience. Inhibition of sirtuin 1 was associated with dendritic atrophy induced by chronic stress. Importantly, activation of the hippocampal sirtuin 1 increased ERK1/2 phosphorylation under chronic stress, and activation and inhibition of ERK1/2 led to antidepressant-like and depression-like behaviors, respectively (Abe-Higuchi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ferland et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), highlighting a role of ERK1/2, a potential downstream target of sirtuin 1, in depression (Duman et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; I\u0026ntilde;iguez et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Taken together, we propose that sirtuin 1/EKR1/2 signaling converges on the mTORC1 signaling pathway. To confirm this, further studies using the DEX-induced 3D neural spheroid model with pharmacological (inhibitor or activator) or genetic (siRNA or vial-mediated gene transfer) tools to modulate the sirtuin 1 function are needed.\u003c/p\u003e \u003cp\u003eThis study provides preliminary insights into the potential of 3D neural spheroid models as a tool for investigating the cellular mechanisms underlying depression. Further research is needed to validate this model using various antidepressant treatments, including SSRIs and ketamine, to confirm its relevance for studying drug effects \u003cem\u003ein vitro\u003c/em\u003e. Additionally, examining the electrophysiological properties of the spheroids, such as synaptic activity and neuronal communication, would enhance our understanding of their functionality and potential as a model system.\u003c/p\u003e \u003cp\u003eOverall, while this 3D model offers advantages over conventional 2D systems, its application as a model for depression still requires further exploration. Future studies could use this system to investigate the molecular actions of rapid-acting antidepressants, such as ketamine, which modulates the mTORC1 pathway to improve synaptic plasticity. The search for alternative antidepressants with fewer side effects remains a critical area of investigation, and the DEX-induced 3D neural spheroid model may contribute to this effort.\u003c/p\u003e \u003cp\u003eIn conclusion, we developed a 3D \u003cem\u003ein vitro\u003c/em\u003e model of depression using primary cortical cell-based spheroid cultures. DEX treatment led to a reduction in neuroplasticity-related markers, including downregulation of the BDNF/ERK1/2/mTORC1 signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). While this 3D model reflects certain aspects of the brain microenvironment, further studies are needed to fully assess its utility in exploring the molecular mechanisms underlying neuroplasticity. The system offers a potential tool for investigating antidepressant mechanisms, although additional validation is required. Overall, the findings from this study may contribute to a better understanding of depression's pathophysiology and could inform future research into therapeutic strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eCompeting interest\u003c/strong\u003e \u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthical approval\u003c/strong\u003e \u003cp\u003e All experimental procedures were performed in accordance with the guidelines for the care and use of laboratory animals for scientific purposes with protocols approved by the Committee for Animal Experimentation and the Institutional Animal Laboratory Review Board of Inje Medical College (approval no. 2021-010).\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eStatement on ARRIVE Guidelines\u003c/h2\u003e \u003cp\u003e We declared that this study was carried out in compliance with the ARRIVE guidelines.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government Ministry of Science and ICT (MSIT) (NRF-2023R1A2C2005016 to S.W. Park and NRF-2021R1A2C1003591 to J.G. Lee). And, this research was supported by Basic Science Research Program through the NRF funded by the Ministry of Education (NRF-2021R1I1A3061265 to S. Jeong).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: Mi Kyoung Seo, Sehoon Jeong, Jung Goo Lee, and Sung Woo Park; Methodology: Mi Kyoung Seo, Woo Seok Cheon, Dong Yun Lee, Sumin Lee, Gyu-Hui Lee, Deok-Gyeong Kang, and Dae-Hyun Seog; Formal analysis and investigation: Mi Kyoung Seo, Woo Seok Cheon, and Dae-Hyun Seog; Writing \u0026ndash; original draft preparation: Sehoon Jeong, Jung Goo Lee, and Sung Woo Park; Writing \u0026ndash; review and editing: Mi Kyoung Seo, Woo Seok Cheon, Dae-Hyun Seog, and Seong-Ho Kim; Funding acquisition: Sehoon Jeong, Jung Goo Lee, and Sung Woo Park; Resources: Sehoon Jeong and Sumin Lee; Supervision: Jung Goo Lee and Sung Woo Park. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe data presented in this study are available on request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbe-Higuchi N, Uchida S, Yamagata H, Higuchi F, Hobara T, Hara K, Kobayashi A, Watanabe Y (2016) Hippocampal Sirtuin 1 Signaling Mediates Depression-like Behavior. 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Front Cell Neurosci 14:25. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi.org/10.3389/fncel\u003c/span\u003e\u003cspan address=\"10.3389/fncel\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 2020.00025\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Depression, Dexamethasone, Micro-spheroid device, mTORC1 signaling, Neuroplasticity, Spheroid","lastPublishedDoi":"10.21203/rs.3.rs-5413832/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5413832/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e models are useful for exploring the molecular mechanisms underlying impaired neuroplasticity in depression. In this study, we developed a three-dimensional spheroid model using the synthetic glucocorticoid dexamethasone to investigate its effects on key pathways involved in neuroplasticity, specifically focusing on BDNF, sirtuin 1, and mTORC1 signaling. A micro-spheroid device was fabricated using photolithography and soft lithography, and cortical spheroids were generated from primary rat cortical cells. These spheroids were treated with varying concentrations of dexamethasone, and their structure included neurons, astrocytes, microglia, and oligodendrocytes. Dexamethasone treatment (100, 200, and 300 \u0026micro;M) resulted in a dose-dependent reduction in cell viability, BDNF mRNA expression, and neurite outgrowth. At 100 \u0026micro;M, dexamethasone reduced the expression of BDNF and sirtuin 1 and decreased phosphorylation of ERK1/2. It also lowered the phosphorylation levels of mTORC1, 4E-BP1, and p70S6K, as well as synaptic proteins such as PSD-95 and GluA1. Dexamethasone treatment inhibited pathways related to neuroplasticity. While the dexamethasone-treated spheroids may serve as a basis for developing an \u003cem\u003ein vitro\u003c/em\u003e model of depression, further validation is required to confirm its broader applicability.\u003c/p\u003e","manuscriptTitle":"Establishment of a depression model using dexamethasone-treated three-dimensional cultured rat cortical cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-27 16:22:29","doi":"10.21203/rs.3.rs-5413832/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3bd7bd63-9913-47df-9d7b-fa1cd566e1ef","owner":[],"postedDate":"November 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-24T11:23:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-27 16:22:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5413832","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5413832","identity":"rs-5413832","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}