• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.75-75
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• 2003

Since the establishment of embryonic stem cell, pluripotency of the cells was known to allow differentiation of the cells into various cell types consisting whole body. Several protocols have been developed to induce expression of specific genes.. However, no precise protocol that will generate a single type of the cells from stem cells has been reported. In order to produce cells suitable for transplantion into brain of PD animal model, which arouse due to a progressive degeneration of dopaminergic neurons in midbrain, human embryonic stem cell (hESC, MB03) was transfected with cDNAs cording for tyrosine hydroxylase (TH). Successful transfection was confirmed by western immunoblotting. Newly transfected cell line (TH#2/MB03) was induced to differentiate by the two neurogenic factors retinoic acid (RA) and b-FGF. Exp. I) Upon differentiation using RA/ascorbic acid (AA), embryoid bodies (EB, for 4days) derived from hES cells were exposed to RA (10$^{-6}$ M)/AA (50 mM) for 4 days, and were allowed to differentiate in N2 medium for 7, 14, 21, or 28 days. Exp. II) When bFGF was used, neuronal precursor cells were selected for 8 days in N2 medium after EB formation. After selection, cells were expanded at the presence of bFGF (20 ng/ml) for another 6 days followed by a final differentiation in N2 medium for 7, 14, 21 or 28 days. By indirect immunocytochemical studies, proportion of cells expressing NF200 increased rapidly from 20% at 7 days to 70 % at 28 days in RA/AA-treated group, while those cells expressing NF160 decreased from 80% at 7 days to 10% at 28 days upon differentiation in N2 medium. However, in differentiation by RA/AA treatment system, there was a significant increase in proportion of neuron maturity (73%) at day 14 after N2 medium. TH#2/MB03 cells expressing TH are >90% when matured at the absence of either bDNF or TGF-$\alpha$. These results suggested that TH#2/MB03 cells could be differentiated in vitro into mature neurons by RA/AA.

• 대한수의학회지
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• 제59권4호
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• pp.201-205
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• 2019

This study presents neurogenesis and neuronal migration patterns of gamma-aminobutyric acid-ergic (GABAergic) neurons during mesencephalic development of mouse. After neurons from embryonic day (E) 10-16 were labelled by a single injection of 5-bromo-2'-deoxyuridine (BrdU), immunohistochemistry was performed. Neurogenesis were mainly generated in the mesencephalic region at E10 to E13. After E14, BrdU positive cells were observed only in the dorsal mesencephalon. GABAergic neurons were mainly originated in the ventrolateral region of the mesencephalon at the early embryonic stage, especially at E11 to E13. E10-labeled cells showed positive for GABAergic neuron in the basal plate of the mesencephalon at E13. At E15, GABAergic neurons were observed in the entire basal plate and some regions of the ventral and dorsal mesencephalon. They were present in the whole basal plate, the ventral and dorsal mesencephalon of E17, spreading more outward of the mesencephalon at P0. Our study demonstrates that major neurogenesis of GABAergic neurons occurs at E11 to E13. However, neuronal migration continues until neonatal period during mesencephalic development.

• Molecules and Cells
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• 제45권2호
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• pp.65-75
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• 2022

Hypothalamus is a brain region that controls food intake and energy expenditure while sensing signals that convey information about energy status. Within the hypothalamus, molecularly and functionally distinct neurons work in concert under physiological conditions. However, under pathological conditions such as in diet-induced obesity (DIO) model, these neurons show dysfunctional firing patterns and distorted regulation by neurotransmitters and neurohormones. Concurrently, resident glial cells including astrocytes dramatically transform into reactive states. In particular, it has been reported that reactive astrogliosis is observed in the hypothalamus, along with various neuroinflammatory signals. However, how the reactive astrocytes control and modulate DIO by influencing neighboring neurons is not well understood. Recently, new lines of evidence have emerged indicating that these reactive astrocytes directly contribute to the pathology of obesity by synthesizing and tonically releasing the major inhibitory transmitter GABA. The released GABA strongly inhibits the neighboring neurons that control energy expenditure. These surprising findings shed light on the interplay between reactive astrocytes and neighboring neurons in the hypothalamus. This review summarizes recent discoveries related to the functions of hypothalamic reactive astrocytes in obesity and raises new potential therapeutic targets against obesity.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2007년도 Proceedings of The Convention of The Korean Society of Applied Pharmacology
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• pp.147-153
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• 2007

Central to developing new treatment strategies for late onset sporadic Parkinson's disease (PD) and early onset familial PD is resolving the enigma of the specific vulnerability exhibited by substantia nigra dopamine (DA) neurons despite multiple risk factors. Neuropathological evidence from both human and experimental models of PD firmly supports a significant role for oxidative stress (OS) and mitochondrial dysfunction in the death of nigral DA neurons. Largely unknown are the genes underlying selective susceptibility of nigral DA neuron to OS and mitochondrial dysfunction and how they effect nigral DA cell death. To overcome the paucity of nigral DA neurons as well as the dilution effect of non-DA cells in brain tissues, we have developed wild type DA cell line model, SN4741 and mutant DJ-1 (-/-) DA cells, appropriate for microarray analysis and differential mitochondrial proteomics. Mutations in the DJ-1 gene (PARK7), localized in cytoplasm and mitochondria, cause autosomal recessive early onset PD. Through microarray analysis using SN4741 cells followed by validation tests, we have identified a novel phylogenically conserved neuroprotective gene, Oxi-a, which is specifically expressed in DA neurons. The knockdown of the gene dramatically increased vulnerability to as. Importantly as down-regulated the expression level of the gene and recovery of its expression via transient transfection exerted significant neuroprotection against as insult. We also have identified altered expression of mitochondrial proteins and other familial PD genes in DJ-1 (-/-) mutant cells by differential mitochondrial proteomics. In DJ-1 (-/-) cells the knockdown of the other familial PD genes (Parkin and PINK1) dramatically increased susceptibility to as. Thus, further functional characterization of the Oxi-$\alpha$ gene family and the mitochondrial alteration in the DJ-1 (-/-) cell model will provide the rationale for the neuroprotective therapy against both sporadic and familial PD.

• The Korean Journal of Physiology and Pharmacology
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• 제11권6호
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• pp.247-251
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• 2007

Changes of single unit activity of CA1 hippocampus region were investigated in anesthetized Mongolian gerbils for six days following transient ischemia. Ischemia was produced immediately before the implantation of micro-wire recording electrodes. In control animals receiving pseudo-ischemic surgery, neither spontaneous neuronal activities ($5.70{\pm}0.4Hz$) nor the number of recorded neurons per animal changed significantly for six days. Correlative firings among simultaneously recorded neurons were weak (correlation coefficient > 0.6) in the control animals. Animals subjected to ischemia exhibited a significant elevation of neural firing at post-ischemic 12 hr ($9.95{\pm}0.9Hz$) and day 1 ($8.48{\pm}0.8Hz$), but a significant depression of activity at post-ischemic day 6 ($1.84{\pm}0.3Hz$) when compared to the activities of non-ischemic control animal. Ischemia significantly (correlation coefficient > 0.6) increased correlative firings among simultaneously recorded neurons, which were prominent especially during post-ischemic days 1, 2 and 6. Although the numbers of spontaneously active neurons recorded from control group varied within normal range during the experimental period, those from ischemic group changed in post-ischemic time-dependent manner. Temporal changes of the number of cells recorded per animal between control group and ischemic group were also significantly different (p = 0.0084, t = 3.271, df = 10). Cresyl violet staining indicated significant loss of CA1 cells at post-ischemic day 7. Overall, we showed post-ischemic time-dependent, differential changes of three characteristics, including spontaneous activity, network relationship and excitability of CA1 cells, suggesting sustained neural functions. Thus, histological observation of CA1 cell death till post-ischemic day 7 may not represent actual neuronal death.

• 대한의생명과학회지
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• 제16권4호
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• pp.259-264
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• 2010

This study was designed to investigate the effects of nitric oxide on the neuronal activity of rat cerebellar Purkinje cells. Sprague-Dawley rats aged 14 to 16 days were decapitated under ether anesthesia. After treatment with pronase and thermolysin, the dissociated Purkinje cells were transferred into a chamber on an inverted microscope. Spontaneous action potentials and potassium current were recorded by standard patch-clamp techniques under current and voltage-clamp modes respectively. 15 Purkinje cells revealed excitatory responses to $20\;{\mu}M$ of sodium nitroprusside (SNP) and 4 neurons (20%) did not respond to SNP. Whole potassium currents of Purkinje cells were decreased by SNP (n=10). Whole potassium currents of Purkinje cells were also decreased by L-arginine, substrate of nitric oxide (n=10). These experimental results suggest that nitric oxide increases the neuronal activity of Purkinje cells by altering the resting membrane potential and after hyperpolarization.

• The Korean Journal of Physiology and Pharmacology
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• 제2권1호
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• pp.9-19
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• 1998

The present study was primarily carried out to characterize the properties of the spinomesencephalic tract (SMT) neurons that project from the upper cervical spinal segments to the midbrain. It was also investigated whether these neurons received convergent afferent inputs from other sources in addition to cervical inputs. Extracellular single unit recordings were made from neurons antidromically activated by stimulation of midbrain. Recording sites were located in lamina $I{\sim}VIII\;of\;C1{\sim}C3$ segments of spinal cord. Receptive field (RF) and response properties to mechanical stimulation were studied in 71 SMT neurons. Response profiles were classified into six groups: complex (Comp, n=9), wide dynamic range (WDR, n=16), low threshold (LT, n=5), high threshold (HT, n=6), deep/tap (Deep, n=10), and non- responsive (NR, n=25). Distributions of stimulation and recording sites were not significantly different between SMT groups classified upon their locations and/or response profiles. Mean conduction velocity of SMT neurons was $16.7{\pm}1.28\;m/sec$. Conduction velocities of SMTs recorded in superficial dorsal horn (SDH, n=15) were significantly slower than those of SMTs recorded in deep dorsal horn (DDH, n=18), lateral reticulated area (LRA, n=21), and intermediate zone and ventral horn (IZ/VH, n=15). Somatic RFs for SMTs in LRA and IZ/VH were significantly larger than those in SDH and DDH. Five SMT units (4 Comps and 1 HT) had inhibitory somatic RFs. About half (25/46) of SMT units have their RFs over trigeminal dermatome. Excitabilities of 5/12 cells and 9/13 cells were modulated by stimulation of ipsilateral phrenic nerve and vagus nerve, respectively. These results suggest that upper cervical SMT neurons are heterogenous in their function by showing a wide range of variety in location within the spinal gray matter, in response profile, and in convergent afferent input.

• 대한한의학회지
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• 제24권2호
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• pp.204-212
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• 2003

Current therapy for acute ischemic stroke is highly focused on neuroprotective agents, and many herbal medicines have been challenged for experimental models. The aim of this study is to investigate whether Angelicae gigantis Radix can protect nerve cells against ischemic neural damage of middle cerebral artery occlusion (MCAO) in rats' brains. Rats were treated with Angelicae gigantis Radix immediately after 2 hours of MCAO for 7 days. On the 7th day, the brains of the rats were sliced through the hippocampus and dyedby c-Fos immunohistochemistry stain and cresyl violet stain for microscopic examination. The number of viable neurons and c-Fos immunoreactive cells in CA1 regions was counted. MCAO caused significant decrease in density of neurons and c-Fos immunoreactive cells compared to those of sham-operated rats. Administration of Angelicae gigantis Radix significantly elevated MCAO-induced decrease in density of neurons and c-Fos immunoreactive cells. These results suggest that the neuroprotective effect of Angelicae gigantis Radix against focal cerebral ischemia is related to c-Fos gene expression. Thus, these findings indicate that Angelicae gigantis Radix can be used for treatment and prevention of cerebral ischemia.

• 생명과학회지
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• 제23권6호
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• pp.804-811
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• 2013

Melanopsin은 옵신과 비슷한 광색소로 망막 광민감성 신경절 세포에서 적은 비율로 발견 된다. Melanopsin은 일주기 리듬조절에 관여 하고 동공반사를 조절한다. 이번 연구의 목적은 melanopsin이 망막 이외의 중추 시각계 에서도 발견 되는지를 알아 보는 것이다. 우리는 생쥐 시각 피질에 존재하는 melanopsin을 가지는 신경세포를 면역조직화학법을 통해 염색하여 melanopsin의 분포와 형태를 분석하였다. Melanopsin 면역반응은 핵을 제외한 신경세포의 세포체에서 일어났다. 우리는 melanopsin이 성체 생쥐의 시각 피질의 2,3층에서 높은 밀도로 모여서 존재하는 것을 확인했다. 시각 피질 1층에서 melanopsin 면역반응을 보인 신경세포는 드물게 발견 되었다. 생쥐 시각 피질에서 발견된 melanopsin의 세포종류는 주로 round/oval 세포였으나, vertical fusiform 그리고 pyramidal 세포 등도 드물게 발견 되었다. 염색된 세포들의 분포는 특이적이지 않았다. 우리의 실험 데이터로 melanopsin이 시각피질에도 분포한다는 것을 알 수 있었다. 또한, melanopsin을 가지는 신경세포는 nitric oxide synthase, calbindin and parvalbumin과도 같이 발현 되는 것을 관찰했다. 이러한 실험결과는 망막 이외의 부분에서 발견되는 melanopsin의 기능에 대한 연구가 필요함을 야기한다.

• 대한한방내과학회지
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• 제22권2호
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• pp.161-174
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• 2001

Objectives : The purpose of this investigation is to evaluate the effect of Chungpesagan-Tang Extracts on reversible forebrain ischemia in Sprague-Dawley rats. Methods : the volume of cerebral infarction and edema, the pathohistological change of neurons, the number of survived neurons, neurotransmitters through immunohistochemical methods, proteins connected with neurotransmitters through immunohistochemical methods and the pathohistological change of neurons through electro-microscopy were investigated. From these reseach data, the protection of neurons and the activity of brain cells were examined. Results : 1. The infaction volume of the control group was 23.9%, and that of the sample group was 16%. 2. The brain edema volume of the control group increased by 17% compared to the normal group and that of the sample group increased by 10%. 3. The light microscopy revealed that the neurons in the ischemia-induced area and CA1 area of hippocampus were most heavily damaged and that the sample group was less damaged compared with the control group. Most pyramidal neurons died in 7 days when brain ischemia was induced. 4. The number of survived pyramidal neurons in the CA1 area of the hippocampus were studied. The normal group had 93 neurons/mm, survived the control group(after 3 days) had 21/mm, the control group(after 7 days) had 3/mm and the sample group 33/mm. 5. The immunohistochemical methods revealed that: (1) In the control group, the sensitivity of GABA, NOS, DBH were increased, and those of Synapsin, eEF-$1{\alpha}$ decreased. NOS and DBH had positive reactions in the control group, but negative in the normal group. (2) In thd sample group, the sensitivity of GABA, NOS, DBH were attenuated, and those of NPY, Synapsin, CaMKII, eEF-$1{\alpha}$ increased when compared to the control group. 6. The electro-microscopy revealed that most neurons died by necrosis and some neurons died by apoptosis. Several imflammation cells appeared in the injured area of neurons. The number of neurons in the sample group that died by ischemia decreased. But, the number that died by apoptosis did not significantly change. Conclusions : The data shows that the effect of Chungpesagan-Tang Extracts on reversible forebrain ischemia in Sprague-Dawley rats is significant.