• Molecules and Cells
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• v.41 no.5
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• pp.454-464
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• 2018

Crosstalk between G-protein signaling and glutamatergic transmission within the brain reward circuits is critical for long-term emotional effects (depression and anxiety), cravings, and negative withdrawal symptoms associated with opioid addiction. A previous study showed that Regulator of G-protein signaling 4 (RGS4) may be implicated in opiate action in the nucleus accumbens (NAc). However, the mechanism of the NAc-specific RGS4 actions that induce the behavioral responses to opiates remains largely unknown. The present study used a short hairpin RNA (shRNA)-mediated knock-down of RGS4 in the NAc of the mouse brain to investigate the relationship between the activation of ionotropic glutamate receptors and RGS4 in the NAc during morphine reward. Additionally, the shRNA-mediated RGS4 knock-down was implemented in NAc/striatal primary-cultured neurons to investigate the role that striatal neurons have in the morphine-induced activation of ionotropic glutamate receptors. The results of this study show that the NAc-specific knock-down of RGS4 significantly increased the behaviors associated with morphine and did so by phosphorylation of the GluR1 (Ser831) and NR2A (Tyr1325) glutamate receptors in the NAc. Furthermore, the knock-down of RGS4 enhanced the phosphorylation of the GluR1 and NR2A glutamate receptors in the primary NAc/striatal neurons during spontaneous morphine withdrawal. These findings show a novel molecular mechanism of RGS4 in glutamatergic transmission that underlies the negative symptoms associated with morphine administration.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.84-84
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• 2002

This study was to investigate generation of the specific neuronal cell in vitro from the neural progenitors derived from human embryonic stem (hES, MB03) cells. For the neural progenitor cell formation, we produced embryoid bodies (EB: for 5 days, without mitogen) from hES cells and then neurospheres (for 7-10 days, 20 ng/㎖ of bFGF added N2 medium) from EB. And then for the differentiation into neuronal cells, neural progenitor cells were cultured in N2 medium (without bFGF) supplemented with brain derived neurotrophic factor (BDNF, 5 ng/㎖) or platelet derived growth factor-bb (pDGF-bb, 20ng/㎖) for 2 weeks. (omitted)

• YAKHAK HOEJI
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• v.49 no.1
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• pp.30-37
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• 2005

Xuesaitong Ruanjiaonang (XR), a soft capsule containing Panax notoginseng saponins as main ingredients, is believed to remove extravasated blood and increase cerebral blood flow by improving blood circulation, and therefore, has been used in China to treat ischemic stroke or hemiplegia caused by cerebral thrombosis. To characterize pharmacological actions of XR, the present study evaluated its effects on neuronal cell damage induced by various oxidative insults or excitotoxic amino acids in primary cultured rat cortical cells. The neuronal cell viability was not affected by XR with the exposure for 2 h at the concentrations tested in this study ($10{\sim}1000\;{\mu}g/ml$). However, significant reduction of the cell viability was observed when the cultured cells were exposed to XR at $1000\;{\mu}g/ml$ for 24 h. XR was found to concentration-dependently inhibit the oxidative neuronal damage induced by $H_{2}O_2$, xanthine/xanthine oxidase or $Fe^{2+}$/ascorbic acid. In addition, it dramatically inhibited the excitotoxic damage induced by glutamate or N-methyl-D-aspartate (NMDA). We found that the NMDA-induced neurotoxicity was inhibited more effectively and potently than the glutamate-induced toxicity. Moreover, XR was found to exert mild inhibition of lipid peroxidation induced by $Fe^{2+}$/ascorbic acid in rat brain homogenates and some 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. Taken together, these results demonstrate neuroprotective and antioxidant effects of XR, showing inhibition of oxidative and excitotoxic damage in the cultured cortical neurons, as well as inhibition of lipid peroxidation and its radical scavenging activity. Considering that excitotoxicity and oxidative stress pl ay crucial roles in neuronal cell damage during ischemia and reperfusion, these results may provide pharmacological basis for its clinical usage to treat ischemic stroke.

• Journal of Veterinary Science
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• v.22 no.6
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• pp.86.1-86.13
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• 2021

Background: Conditioned medium is the medium obtained from certain cultured cells and contained secretome from the cells. The secretome, which can be in the form of growth factors, cytokines, exosomes, or other proteins secreted by the cells, can induce the differentiation of cells that still have pluripotent or multipotent properties. Objectives: This study examined the effects of conditioned medium derived from E17 rat brain cells on cells with pluripotent properties. Methods: The conditioned medium used in this study originated from E17 rat brain cells. The CM was used to induce the differentiation of primary colonies of mice blastocysts. Primary colonies were stained with alkaline phosphatase to analyze the pluripotency. The morphological changes in the colonies were examined, and the colonies were stained with GFAP and Neu-N markers on days two and seven after adding the conditioned medium. Results: The conditioned medium could differentiate the primary colony, beginning with the formation of embryoid-body-like structure; round GFAP positive cells were identified. Finally, neuron-like cells testing positive for Neu-N were observed on the seventh day after adding the conditioned medium. Conclusions: Conditioned medium from different species, in this case, E17 rat brain cells, induced and promoted the differentiation of the primary colony from mice blastocysts into neuron-like cells. The addition of CM mediated neurite growth in the differentiation process.

• Korean Journal of Biological Psychiatry
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• v.8 no.1
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• pp.85-95
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• 2001

This study was designed to examine the effects of two antidepressant drugs on the expression of c-fos mRNA in cultured embryonic rat hippocampal neurons. The drugs used were imipramine and amitriptyline. On the fourth day of culture, hippocampal neurons were treated with variable concentrations of each drug. Competitive RT-PCR(Reverse Transcriptase-PCR) analysis was used to quantify the c-fos mRNA expression induced by each drug. Experimental results showed that acute and direct treatment with imipramine and amitriptyline with relatively low concentrations(imipramine ${\leq}10{\mu}M$, amitriptylne ${\leq}10{\mu}M$) had no inductive effect on the expression of c-fos mRNA in the rat hippocampal neurons. However, after treatment with relatively high concentrations(imipramine ${\geq}100{\mu}M$, amitriptyline ${\geq}100{\mu}M$) c-fos mRNA was not detected. These findings suggest the followings. Firstly, the action mechanisms of these drugs on the hippocampal neurons might not be mediated by c-fos but by other immediate-early genes(IEGs). Secondly, their actions may be mediated indirectly via other areas of the brain. Thirdly, the expression of c-fos might be inhibited by high concentrations of these drugs, or the high concentrations could induce cell death. Finally, though cell death remains to be confirmed, the inhibition of c-fos induction or cell death could play a role in the cognitive impairments known to be adverse effects of some antidepressants. This study is believed to be a first step toward understanding the mechanisms of learning and memory. Further studies are needed to investigate the expression of various IEGs and changes in the hippocampal neurons of rat resulting from chronic treatment with antidepressant drugs.

• Journal of Life Science
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• v.17 no.1 s.81
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• pp.12-17
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• 2007

Curcumin is a natural phenolic yellow curry spice, derived from the tumeric, which has been used for the treatment of diseases associated with oxidative stress and inflammation. Curcumin is known to have both anti-oxidative and anti-inflammatory properties. These properties can be beneficial to protect the brain from the neurodegenerative diseases. We now report the neuroprotective effects of curcumin pretreatment in primary hippocampal neurons to glutamate-induced excitotoxicity. Pretreatment of embryonic mouse hippocampal cell cultures with low does of curcumin protected neurons against glutamate-induced death, however, this neuroprotection was not correlated with the modulation of oxidative stress. Interestingly, high dose of curcumin showed the cytotoxicity in primary cultured hippocampal neurons. Immunoblot analyses showed that levels of stress response. protein HSP70 were significantly elevated in neurons exposed to low dose of curcumin, whereas levels of cleaved PARP were increased in neurons exposed to high dose of curcumin. These findings show that curcumin can modulate neuronal responses to glutamate, and suggest possible use of curcumin and related compounds in the prevention and/or treatment of neurodegenerative disorders.

• Journal of Life Science
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• v.17 no.12
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• pp.1627-1633
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• 2007

c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP1), also known as Islet-brain 1 (IB1), is a scaffold protein that is highly expressed in neurons and pancreatic ${\beta}-cells$. In this study subcellular localization of JIP was investigated in cultured rat hippocampal neurons using an antibody that recognize all variants of JIP1, JIP-2 and JIP-3. The overall expression profile of JIP is punctate throughout soma and dendrites. Statistic analysis showed that $54.8{\pm}4.0%\;and\;94.1{\pm}4.5%$ of total JIP immunopuncta overlapped with those of excitatory postsynaptic markers SD-95 and ${\alpha}Camik$, respectively. In contrast, only $8.6{\pm}0.5%\;and\;7.3{\pm}0.5%$ of JIP clusters overlapped with those of inhibitory postsynaptic markers glycine receptor (GlyR) and gephyrin, respectively. JIP clusters overlapped or juxtaposed with SV2 but not GAD, markers for general and inhibitory nerve terminals, respectively. A substantial fraction $(29.3{\pm}1.0%)$ of flotillin immunopuncta, a marker for lipid rafts, clusters overlapped with those of JIP. In addition, JIP was highly expressed in some select ends of dendrites but minimal in axons. These data suggest important roles of JIP in excitatory postsynaptic sites, lipid rafts and dendritic ends.

• International Journal of Stem Cells
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• v.15 no.1
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• pp.95-103
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• 2022

Background and Objectives: In recent years, brain organoid technologies have been the most innovative advance in neural differentiation research. In line with this, we optimized a method to establish cerebral organoids from feeder-free cultured human pluripotent stem cells. In this study, we focused on the consistent and robust production of cerebral organoids comprising neural progenitor cells and neurons. We propose an optimal protocol for cerebral organoid generation that is applicable to both human embryonic stem cells and human induced pluripotent stem cells. Methods and Results: We investigated formation of neuroepithelium, neural tube, and neural folding by observing the morphology of embryoid bodies at each stage during the cerebral organoid differentiation process. Furthermore, we characterized the cerebral organoids via immunocytochemical staining of sectioned organoid samples, which were prepared using a Cryostat and Vibratome. Finally, we established a routine method to generate early cerebral organoids comprising a cortical layer and a neural progenitor zone. Conclusions: We developed an optimized methodology for the generation of cerebral organoids using hESCs and hiPSCs. Using this protocol, consistent and efficient cerebral organoids could be obtained from hiPSCs as well as hESCs. Further, the morphology of brain organoids could be analyzed through 2D monitoring via immunostaining and tissue sectioning, or through 3D monitoring by whole tissue staining after clarification.

• Journal of Embryo Transfer
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• v.32 no.2
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• pp.39-45
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• 2017

Mesenchymal stem cells (MSCs) have been considered an alternative source of neuronal lineage cells, which are difficult to isolate from brain and expand in vitro. Previous studies have reported that MSCs expressing Nestin ($Nestin^+$ MSCs), a neuronal stem/progenitor cell marker, exhibit increased transcriptional levels of neural development-related genes, indicating that $Nestin^+$ MSCs may exert potential with neurogenic differentiation. Accordingly, we investigated the effects of the presence of $Nestin^+$ MSCs in bone-marrow-derived primary cells (BMPCs) on enhanced neurogenic differentiation of BMPCs by identifying the presence of $Nestin^+$ MSCs in uncultured and cultured BMPCs. The percentage of $Nestin^+$ MSCs in BMPCs was measured per passage by double staining with Nestin and CD90, an MSC marker. The efficiency of neurogenic differentiation was compared among passages, revealing the highest and lowest yields of $Nestin^+$ MSCs. The presence of $Nestin^+$ MSCs was identified in BMPCs before in vitro culture, and the highest and lowest percentages of $Nestin^+$ MSCs in BMPCs was observed at the third (P3) and fifth passages (P5). Moreover, significantly the higher efficiency of differentiation into neurons, oligodendrocyte precursor cells and astrocytes was detected in BMPCs at P3, compared with P5. In conclusion, these results demonstrate that neurogenic differentiation can be enhanced by increasing the proportion of $Nestin^+$ MSCs in cultured BMPCs.

• Biomolecules & Therapeutics
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• v.25 no.6
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• pp.593-598
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• 2017

The $Na^+/H^+$ exchanger-1 (NHE-1) is a ubiquitously expressed pH-regulatory membrane protein that functions in the brain, heart, and other organs. It is increased by intracellular acidosis through the interaction of intracellular $H^+$ with an allosteric modifier site in the transport domain. In the previous study, we reported that glutamate-induced NHE-1 phosphorylation mediated by activation of protein kinase C-${\beta}$ (PKC-${\beta}$) in cultured neuron cells via extracellular signal-regulated kinases (ERK)/p90 ribosomal s6 kinases (p90RSK) pathway results in NHE-1 activation. However, whether glutamate stimulates NHE-1 activity solely by the allosteric mechanism remains elusive. Cultured primary cortical neuronal cells were subjected to intracellular acidosis by exposure to $100{\mu}M$ glutamate or 20 mM $NH_4Cl$. After the desired duration of intracellular acidosis, the phosphorylation and activation of PKC-${\beta}$, ERK1/2 and p90RSK were determined by Western blotting. We investigated whether the duration of intracellular acidosis is controlled by glutamate exposure time. The NHE-1 activation increased while intracellular acidosis sustained for >3 min. To determine if sustained intracellular acidosis induced NHE-1 phosphorylation, we examined phosphorylation of NHE-1 induced by intracellular acidosis by transient exposure to $NH_4Cl$. Sustained intracellular acidosis led to activation and phosphorylation of NHE-1. In addition, sustained intracellular acidosis also activated the PKC-${\beta}$, ERK1/2, and p90RSK in neuronal cells. We conclude that glutamate stimulates NHE-1 activity through sustained intracellular acidosis, which mediates NHE-1 phosphorylation regulated by PKC-${\beta}$/ERK1/2/p90RSK pathway in neuronal cells.