• Development and Reproduction
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• v.12 no.1
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• pp.1-8
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• 2008

Specific protocols to increase the differentiation of neuronal cells from embryonic stem (ES) cells have been well established, such as retinoic acid induction and lineage selection of neuronal cells. For the neuropathological studies, ES-derived neurons (ES neurons) must show normal physiological characteristics related to cell death and survival and should be maintained in vitro for a sufficient time to show insults-specific cell death without spontaneous death. When mouse ES cells were plated onto astrocytes monolayer after retinoic acid induction, most ES cells differentiated into neuronal cells, which were confirmed by the presence of specific neuronal markers, and the cultures were viable for at least four weeks. When these cultures were examined for vulnerability to glutamate excitotoxicity, ES neurons were vulnerable to excitotoxic insults mediated by agonist-specific receptors. The vulnerability to excitotoxic death increased with developmental age of ES neurons in vitro. Specific receptors for Neurotrophin and GDNF family ligands were present in ES neurons. GDNF and NT-3 could modulate the survival and excitotoxic vulnerability of ES neurons. The vulnerability and resistance to toxic insults, which are essential requirements of model culture systems for neuropathological studies, make ES neurons to a useful model culture system. Especially ES cell are highly amenable to genetic modification unlikely to primary neuronal cells, which will give us a chance to answer more complicated neurophysiological questions. Recently there was an outstanding attempt to explore the cellular toxicity using human ES cells (Schrattenholz & Klemm, 2007) and it suggested that ES cells could be a new model system for neurophysiological studies soon and go further a large-scale screening system for pharmacological compounds in the future.

• Progress in Medical Physics
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• v.15 no.4
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• pp.228-236
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• 2004

Retinal ganglion cells transmit visual scene as an action potential to visual cortex through optic nerve. Conventional recording method using single intra- or extra-cellular electrode enables us to understand the response of specific neuron on specific time. Therefore, it is not possible to determine how the nerve impulses in the population of retinal ganglion cells collectively encode the visual stimulus with conventional recording. This requires recording the simultaneous electrical signals of many neurons. Recent advances in multi-electrode recording have brought us closer to understanding how visual information is encoded by population of retinal ganglion cells. We examined how ganglion cells act together to encode a visual scene with multi-electrode array (MEA). With light stimulation (on duration: 2 sec, off duration: 5 sec) generated on a color monitor driven by custom-made software, we isolated three functional types of ganglion cell activities; ON (35.0$\pm$4.4%), OFF (31.4$\pm$1.9%), and ON/OFF cells (34.6$\pm$5.3%) (Total number of retinal pieces = 8). We observed that nearby neurons often fire action potential near synchrony (< 1 ms). And this narrow correlation is seen among cells within a cluster which is made of 6～8 cells. As there are many more synchronized firing patterns than ganglion cells, such a distributed code might allow the retina to compress a large number of distinct visual messages into a small number of ganglion cells.

• Development and Reproduction
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• v.13 no.4
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• pp.227-237
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• 2009

Recently, adipose mesenchymal stem cells (AdMSC) that are similar to bone marrow MSC and blood derived MSC are thought to be another source for stem cell therapy. However, the diseases that can be applied for stem cells therapy are age-dependent degenerative diseases. Accordingly, the present study investigated the growth and differentiation potential to neural cells of human AdMSC (hAdMSC) obtained from aged thirty, forty and fifty. The growth of cells and cell viability were measured by passage and neural differentiation of hAdMSC was induced in neural differentiation condition for 10 days. Our results demonstrated that cell number, viability and morphology were not different from hAdMSC by age and passage. Immunofluorescence analysis of neural cell marker (TuJ1, NSE, Sox2, GFAP or MAP2) demonstrated no significant differences in neural cell differentiation by age and passage. As the number of passage was increased, the mRNA level of MAP2 and Sox2 was decreased in hAdMSC from age of 50 compared to hAdMSC from age of 30. In conclusion, the present study demonstrated that ability of neural cell differentiation of hAdMSC was maintained with ages, suggesting that autologous stem cells from aged people can be applied for stem cell therapy with age-dependent neural disease with the same stem cell quality and ability as stem cell derived from young age.

• KSBB Journal
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• v.20 no.4
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• pp.323-327
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• 2005

RT-PCR is an useful method to investigate the expression of target gene as detection tools. Although RT-PCR is the powerful detection method for tissues, it was difficult to amplify the target gene product using the single cell. To clarify the expression level of the genes related to Parkinson's disease (PD), I performed the laser dissection of single cell from Substantia nigra. I examined the mRNA expression level in the dopaminergic neuron isolated from the PD patients by the single cell RT-PCR method. It is known that tyrosine hydroxylase (TH), DOPA decarboxylase (DDC) are involved in biosynthesis of the catecholamine such as dopamine. Little has been known about the gene expression features of these enzymes in single dopaminergic neuron. I could detect the specific gene products in single cell level. The different expression was observed in PD-related gene products from the single neuron of PD patients. Interestingly, TH gene expression was significantly decreased with comparing the ratio of decrease in other PD-related genes. Hence, I represented data that indicate the RT-PCR method described in this report is an effective method in detecting a specific single-cell mRNA level related with diseases.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2001.10a
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• pp.626-629
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• 2001

인간의 뇌에는 천 억개 이상의 신경세포들이 있다. 이들은 신경작용의 매우 복잡한 네트워크를 통해 서로서로 연결되어져 있다. 하나의 신경세포로부터 다른 신경세포로 신호가 전달되는 과정은 다른 화학 전달물질들에 의해 이루어지며 신호 전이는 시냅스라고 불리는 신경세포간의 특정 접촉부 위에서 일어난다. 뉴런의 신호전달 체계에 대한 연구는 20세기 초반에 본격적으로 이루어져 왔으며, 현재는 각 뉴런에 대한 정확한 신호전달 원리를 밝히는데 많은 연구가 이루어지고 있다. 최근에 연구가 활발하게 이루어지고 있는 신경전달 물질중 하나인 일산화 질소는 인간의 세포에 노출되었을 경우 세포막을 기준으로 농도 차가 발생하여 근육이 이완되는 현상을 유발한다. 이런 세포막을 기준으로 한 운동신경 변화, 심장박동의 변화, 근육의 이완철상 및 치명적인 이상을 초래하는 현상을 GENESIS를 이용하여 시뮬레이션 해 보았다.

• Journal of Life Science
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• v.28 no.12
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• pp.1397-1405
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• 2018

Mitochondria have multiple functions in cells: providing chemical energy, storing cellular $Ca^{2+}$, generating reactive oxygen species, and regulating apoptosis. Through these functions, mitochondria are also involved in the maintenance, proliferation, and differentiation of stem/progenitor cells. In the brain, the subventricular zone (SVZ) is one of the neurogenic regions that contains neural stem cells (NSCs) throughout a lifetime. However, reports on the role of mitochondria in SVZ NSCs are scarce. Here, we show that rotenone, a complex I inhibitor of mitochondria, inhibits the proliferation and differentiation of SVZ NSCs in different ways. In proliferating NSCs, rotenone decreases mitosis as measured through phosphorylated histone H3 detection; moreover, apoptosis is not induced by rotenone at 50 nM. In differentiating NSCs, rotenone blocks neurogenesis and oligodendrogenesis while glial fibrillary acidic protein-positive astrocytes are not affected. Interestingly, in this study there were more cells in the differentiating NSCs treated with rotenone for 4-6 days than in the vehicle control group which was a different effect from the reduced number of cells in the proliferating NSCs. We examined both apoptosis and mitosis and found that rotenone decreased apoptosis as detected by staining cleaved caspase-3 but did not affect mitosis. Our results suggest that functional mitochondria are necessary in both the proliferation and differentiation of SVZ NSCs. Furthermore, mitochondria might be involved in the mitosis and apoptosis that occur during those processes.

• Journal of Life Science
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• v.26 no.1
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• pp.8-16
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• 2016

Reactive oxygen species (ROS), at appropriate concentrations, mediate various normal cellular functions, including defense against pathogens, signal transduction, cellular growth, and gene expression. A recent study demonstrated that ROS and ROS-generating NADPH oxidase (Nox) are important in self-renewal and neuronal differentiation of subventricular zone (SVZ) neural stem cells in adult mouse brains. In this study, we found that endogenous ROS were detected in SVZ neural stem cells cultured from postnatal mouse brains. Nox4 was predominantly expressed in cultured cells, while the levels of the Nox1 and Nox2 transcripts were very low. In addition, the Nox4 gene was highly upregulated (by up to 10-fold) during neuronal differentiation. Immunocytochemical analysis detected the Nox4 protein mainly in neurons positive for the neuronal specific tubulin Tuj1. After differentiation, endogenous ROS were detected exclusively in neuron-like cells with processes. In addition, perturbation of the cellular redox state with N-acetyl cysteine, a ROS scavenger, during neuronal differentiation greatly inhibited neurogenesis. Lastly, knockdown of Nox4 using short hairpin RNA decreased neurogenesis. These findings suggest that Nox4 may be a major ROS-generating enzyme in postnatal SVZ neural stem cells, and Nox4-mediated ROS generation may be important in their neuronal differentiation.

• Journal of Life Science
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• v.19 no.7
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• pp.963-967
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• 2009

This study evaluated the neuroprotective effect of extracts from the root bark of Morus alba (MA) against glutamate-induced cytotoxicity in neuronal cells. Glutamate-induced cytotoxicity was shown by MTT reduction assay. The neuroprotective effects of methanol, ethanol, and acetone extracts from MA against glutamate-induced cytotoxicity were measured. Among the three extracts, the methanolic extracts showed the highest protective effect, as determined by the results of an morphological assay, a lactate dehydrogenase release assay. Furthermore, the methanol extracts were fractionated sequentially with hexane, diethyl ether, ethyl acetate, and water layer according to degree of polarity. The hexane fractions exhibited a neuroprotective effect against glutamate-stressed N18-RE-105 cells. Therefore, these results suggest that extracts of MA could be a new potential candidate as a protective substance against glutamate-induced cytotoxicity.

• Applied Microscopy
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• v.29 no.4
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• pp.479-491
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• 1999

This study was performed to investigate the distribution and differentiation of choline acetyltransferase (ChAT)-immunoreactive cells in the basal nucleus of Meynert of the postnatal and adult rat forebrains, utilizing techniques of immunocytochemistry. According to the cell shape and the ratio of long axis vs short axis of cell soma, the ChAT-immunoreactive nerve cells in the basal nucleus of Meynert of the adult rat were classified into six types. In the adult rat, the frequency distributions (FD) of round, oval, elongated, fusiform, triangular and polygonal cells were 9.4%, 35.5%, 32.1%, 5.9%, 9.1% and 8.0%, respectively. The FD of oval and round nerve cells on the postnatal day (PND) 14 were observed to be 18.7% and 51.5%, respectively. Those were shown to be progressively decreased during developmental process to the adult. Also, those of elongated and triangular nerve cells on the PND 21 were observed to be 30.4% and 10.1%, respectively. Those were shown to be same phenomenon a,1 those in the round and oval cells. Meanwhile, those of the triangular and polygonal nerve cells were progressively increased from the early postnatal stage to the adult. The total mean volumes of ChAT-immunoreactive cell somata in the PND 7 rat were the lowest $(1,083{\mu}m^3)$ and those in the PND 21 rat were shown to be the highest $(5,045{\mu}m^3)$. But in the adult, those were decreased to $(2,731{\mu}m^3)$. Those in the PND 21 rat were shown to be about 84.7% larger than those in the adult. On the electron micrography, the cell organelles such as ribosomes, polysomes, rough endoplasmic reticula (RER) and mitochondria were well developed in the PND 21 rat forebrains, but Golgi complexes were shown to be proliferating phase. Especially, ribosomes, polysomes and RER were immunoreactive in the tissues treated with 0.05% triton X-100. According to the observations in the present study, it is considered that the ChAT-immunoreactive nerve cells in the basal nucleus of Meynert of the rat forebrains are differentiated throughout the following processes of changes during the postnatal development: 1) increase of cell soma volumes with the differentiation of tell organelles and neurites, 2) increase in the FD of differentiated tell types and 3) cell schrinkage without cell loss. The ribosomes, polysomes and RER are considered to be closely related to the intracellular localization and biosynthesis of the ChAT but not Colgi complex.

• Journal of Life Science
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• v.16 no.5
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• pp.750-758
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• 2006

Calcium-binding protein calretinin is thought to play important roles in calcium buffering. Recently, we reported on the distribution, morphology of calretinin-immunoreactive (IR) neurons and the effects of eye enucleation on the immunoreactivity of calretinin in the superficial layers of the hamster superior colliculus (SC). In the present study, we describe the distributions and types of labeled cells and effects of enucleation in the deeper layers by immunocytochemistry. We also compare this labeling to that of GABA, the major inhibitory neurotransmitter in the central nervous system. In contrast to the superficial layers, the deeper layers contained many calretinin-IR neurons which formed two tiers. The first tier, which was very distinctive, was found within the intermediate gray layer. The second tier was found in the deep gray layer. Labeled neurons varied dramatically in morphology and included vertical fusiform, stellate, round/oval, and horizontal neurons. In contrast to the superficial layers, enucleation appeared to have no effect on the distribution of calretinin immunoreactivity in the deeper layers. Two-color immunofluorescence revealed that none of calretinin-IR neurons were labeled with an antibody to GABA. The present results demonstrate that calretinin identifies unique neuronal sublaminar organizations in the hamster SC. The present results also demonstrate that none of the calretinin-IR neurons in the hamster SC is GABAergic interneurons. As many calretinin-IR cells are GABAergic interneurons in most other brain areas, this phenomenon in hamster SC is exceptional.