• 생명과학회지
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• 제16권2호
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• pp.315-322
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• 2006

신경세포 간 정보교환이 이루어지고 있는 신경전달물질의 방출과정은 극히 복잡하여, 이 방면의 독창적인 연구를 수행하기 위해서는 신규작용을 갖는 특이적인 저분자 probe의 탐색은 필수적이다. PC12세포에 tritium-label된 norepinephrine ($[^3H]-NE$)을 incorporation시킨 후에 60 mM의 고농도의 $K^+$의 자극에 의해서 탈분극 후에 방출되는 $[^3H]-NE$의 양을 scintillation countering하여 생리 활성 물질을 탐색하기 위한 in vitro의 실험계를 세웠다. 이 탐색계를 이용하여 곰팡이, 방선균와 박테리아의 대사산물 1만 1000여 샘플을 탐색한 결과, PC12세포에서 고농도의 $K^+$의 자극에 의해서 탈분극 후에 유도되는 $[^3H]-NE$의 방출을 효과적으로 저해하는 FS11052를 방선균 유래의 대사산물로부터 얻었다. FS11052는 또한 PC12세포와 rat cortical neurons에서 동일한 고농도의 $K^+$의 자극에 의한 탈분극 후에 유도되는 신경전달 물질로서 ATP의 방출에도 유의한 저해효과를 나타냈으며, 이 저해 효과는 ionopore로 알려진 ionomycin ($1{\mu}M$)을 포함하는 저농도의 $K^+$의 버퍼를 처리하였을 때에도 보여졌다. 이틀 결과로부터 FS11052의 신경전달 물질의 방출에 대한 저해작용은 세포내 $Ca^{2+}$ 유입 이 후의 반응으로 추정하며 이 작용기구에 대한 해석을 하기위하여, 신경세포의 돌기신장 형태에 대한 영향을 관찰한 결과, 분화를 유도하는 적정 농도인 $5{\mu}g/ml$의 NGF 존재 하에서의 PC12 세포의 돌기 신장에 대하여서는 억제작용을 나타냈다. 또 rat의 대뇌 해마 세포에 대하여 특정적인 형태의 돌기를 내고 있어, FS11052 물질의 첨가에 의해 통상의 긴 축색돌기는 억제되고 얇은 침상의 돌기가 세포체로부터 돌출되어 있었으며, growth cone 를 갖고 있지 않은 뉴우런이 많이 관찰되었다. FS11052 물질의 작용에 관해서는, 탈분극된 synaptic membrane이 $Ca^{2+}$ 이온을 유입 후 활성화되어 신경전달물질을 방출에 중요한 역할을 하고 있는 synaptotagmin, syntaxin, synapsin, SNAP25 등의 synaptosome을 구성하는 단백질에 직접 혹은 이와 밀접한 관련을 갖고 있는 인자와 간접적으로 작용하며, 신경전달물질의 방출을 억제하여 growth cone의 전향과 신경세포의 가소성을 조절하는 물질로 사료되어, 이 물질이 $Ca^{2+}$ 이온을 유입 후 일어나는 exocytosis와 신경계의 기능연구를 위해 사용되어질 수 있을 것으로 기대된다.

• Animal cells and systems
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• 제5권2호
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• pp.157-161
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• 2001

Mice homozygous for the lethal autosomal recessive anorexia mutation (anx) present with premature death around postnatal day 22. The anorexia mutant mice also present phenotypes such as reduced body weight, decreased food intake, and abnormal behavior characteristics such as body tremors, hyperactivity, uncoordinated gait, and head weaving. In order to investigate the expression of c-Fos in the hippocampus of anorexia mutant mice, the immunohistochemistry was performed in this study. The anorexia mutant mice exhibited lower expression of c-Fos in the hippocampus regions thBn the control group. In the CA3 and dentate gyrus, the number of c-Fos-positive cells in anorexia mutant mice was noticeably lower than that in control mice. However, no significant difference was found in the number of c-Fos-positive cells in CA1 of the two groups. The result suggests that the phenotypic characteristics of anorexia mutant mice may be associated with the hippocampal deficits of c-Fos expression.

• 대한소아치과학회지
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• 제25권4호
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• pp.671-682
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• 1998

Electrophysiological phenomena of pyramidal cells in the CA1 area of the dorsal hippocampus were recorded from and filled with neurobiotin in anesthetized rats. The electropharmacological properties of membrane as well as the cellular-synaptic generation of rhythmic slow activity (theta) were examined. The intracellular response characteristics of these pyramidal cells were distinctly different from responses of interneurons. Pyramidal cells had a high resting membrane potential, a low input resistance, and a large amplitude action potential. A afterhyperpolarization was followed a single action potential. Most of pyramidal cells did not display a spontaneous firing. Pyramidal cells displayed weak inward rectification and anodal break excitation. The slope of the frequency-current relation was 53.4 Hz/nA for the first interspike interval and 15.9 Hz/nA for the last intervals, suggesting the presence of spike frequency adaptation. Neurobiotin-filled neurons showed pyramidal morphology. Cells were generally bipolar dendritc processes ramifying in stratum lacunosum-moleculare, radiatum, and oriens. Commissural stimulation discharged pyramidal cells, followed by excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs). The frequency of theta-related membrane potential oscillation was voltage-independent in pyramidal neurons. At strong depolarization levels (less than 30 mV) pyramidal cells emitted sodium spike oscillation, phase-locked to theta. The observations provide direct evidence that theta-related rhythmic hyperpolarization of principal cells is brought by the rhythmically discharging interneurons. Furthermore, the findings in which interneurons were also paced by rhythmic inhibitory postsynaptic potentials during theta suggest that they were periodically hyperpolarized by their GABAergic septal afferents.

• Journal of Ginseng Research
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• 제35권2호
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• pp.138-148
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• 2011

Exponential rise in the use of mobile communication devices has generated health concerns due to radiofrequency (RF) exposure due to its close proximity to the head. Calcium binding proteins like calretinin regulate the levels of calcium ($Ca^{2+}$) which plays an important role in biological systems. Ginseng is known for maintaining equilibrium in the human body and may play a beneficial radioprotectant role against electromagnetic field (EMF) exposure. In the present study, we evaluated the radioprotective effects of red ginseng (RG) extract in a mouse model. Calretinin (CR) expression was measured using a free-floating immunohistochemical method in the hippocampus of mice after 835 MHz EMF exposure for 5 h/d for 5 d at specific absorption rate=1.6 W/kg for the different experimental groups. The control animals were treated with NaCl while the experimental animals received 10 mg/kg ginseng, or 30 mg/kg; EMF exposed mice were also treated with NaCl, 10 mg/kg ginseng (E10), or 30 mg/kg (E30). Decreases in CR immunoreactivity (IR) along with loss of CA1 and CA3 interneurons and infragranular cells were observed in the ENaCl group while such losses were not observed in the E10 and E30 groups. CR IR significantly increased in the RG-treated group compared to control and EMF-exposed groups treated with NaCl. The study demonstrates that RG extract can serve as a radioprotective agent that maintains $Ca^{2+}$ homeostasis and prevents neuronal loss in the brain hippocampal region caused by RF exposure.

• 생명과학회지
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• 제17권2호통권82호
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• pp.198-203
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• 2007

형질전환은 유전자의 기능을 이해하는데 매우 중요한 기법이다. $Ca^{2+}$-인산 침전법은 시간과 비용이 저렴하여 가장 흔히 사용된다. 그러나 성숙 신경세포는 어린 신경세포나 다른 세포종에 비하여 형질전환이 어렵고 쉽게 죽는다. 본 연구에서는 Clontech사의 $CalPhos^{TM}$ Mammalian Transfection 방법을 수정하여 성숙한 신경세포를 효율적으로 형질전환할 수 있는 방법을 고안하였다. 대뇌 신경교세포를 DMEM/10% 말혈청에서 70-80% confluence까지 키우고 배지를 혈청이 첨가되지 않은 Neurobasal/Ara-C로 바꾸어 주어 더 이상 신경교세포가 분열하지 않게 한 다음, 여기에 E19 해마신경세포를 접종하여 배양하였다. $DNA/Ca^{2+}$-인산 침전물은 Clontech사의 $CalPhos^{TM}$ Mammalian Transfection Kit을 이용하여 크기($0.5-1\;{\mu}m$ in diameter) 및 농도(약 10 particles/$100\;{\mu}m^2$)를 배지에서 배양시간을 변화시켜 적당히 조절하였다. 이렇게 하면 in vitro에서 2주 이상 배양한 신경세포도 24-well plate 한 well당 10-15개의 형질전환된 건강한 신경세포를 얻을 수 있었다. 이 방법의 효용성을 검증하기 위하여 연접단백질인 $EGFP-CaMKII{\alpha}$ 융합단백질과 RFP 단백질 유전자(각각 $pEGFP-CaMKII{\alpha}$ 및 pDsRed2)를 형질전환한 결과 전자는 점박이 모양, 후자는 세포전체에 퍼진 양상의 표현을 관찰할 수 있었다. 따라서 본 연구는 성숙한 신경세포를 효율적으로 형질전환할 수 있는 방법을 제공한다.

• 생명과학회지
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• 제17권12호
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• pp.1627-1633
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• 2007

c-Jun N-terminal kinase (JNK)-interacting protein 1(JIP1)은 비계단백질(scaffold protein)로서 신경 세포와 ??장${\beta}$세포에서 많이 발현된다. 본 연구에서는 배양한 흰쥐 해마신경세포에서 JIP1, JIP-2 및 JIP-3을 모두 인식하는 항체를 이용하여 이들의 세포내 표현을 조사하였다. 전반적으로 JIP은 세포체와 가지돌기에 반점 모양으로 표현되었다. 이 JIP 반점들을 통계적으로 분석한 결과 흥분성 연접후표지인 PSD95 및 ${\alpha}CaMKII$ 반점 과 각각 $54.8{\pm}4.0%$ 및 $94.1{\pm}4.5%$가 겹쳐졌다. 반면에 억제성 연접후표지인 그리신수용체 및 gephyrin 반점과는 각각 단지 $8.6{\pm}0.5%$ 및 $7.3{\pm}0.5%$만 겹쳐졌다. 한편 lipid raft의 표지인 flotillin 반점의 상당부분$(29.3{\pm}1.0%)$이 JIP 반점과 겹쳐졌다. 또한, JIP은 일부 가지돌기의 끝부분에 매우 강하게 발편되었으며 축삭에는 표현이 미미하였다. 이 결과들은 JIP 단백질이 흥분성 연접후구역, 일부 lipid raft, 그리고 일부 가지돌기 끝부분에 주로 위치함을 의미한다.

• The Korean Journal of Physiology and Pharmacology
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• 제27권1호
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• pp.113-125
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• 2023

It has been reported that stressful events in early life influence behavior in adulthood and are associated with different psychiatric disorders, such as major depression, post-traumatic stress disorder, bipolar disorder, and anxiety disorder. Maternal separation (MS) is a representative animal model for reproducing childhood stress. It is used as an animal model for depression, and has well-known effects, such as increasing anxiety behavior and causing abnormalities in the hypothalamic-pituitary-adrenal (HPA) axis. This study investigated the effect of MS on anxiety or aggression-like behavior and the number of GABAergic neurons in the hippocampus. Mice were separated from their dams for four hours per day for 19 d from postnatal day two. Elevated plus maze (EPM) test, resident-intruder (RI) test, and counted glutamic acid decarboxylase 67 (GAD67) or parvalbumin (PV) positive cells in the hippocampus were executed using immunohistochemistry. The maternal segregation group exhibited increased anxiety and aggression in the EPM test and the RI test. GAD67-positive neurons were increased in the hippocampal regions we observed: dentate gyrus (DG), CA3, CA1, subiculum, presubiculum, and parasubiculum. PV-positive neurons were increased in the DG, CA3, presubiculum, and parasubiculum. Consistent with behavioral changes, corticosterone was increased in the MS group, suggesting that the behavioral changes induced by MS were expressed through the effect on the HPA axis. Altogether, MS alters anxiety and aggression levels, possibly through alteration of cytoarchitecture and output of the ventral hippocampus that induces the dysfunction of the HPA axis.

• Molecules and Cells
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• 제38권6호
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• pp.540-547
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• 2015

Attention deficit/hyperactivity disorder (ADHD) is one of the most common neurodevelopmental disorders, affecting approximately 5% of children. However, the neural mechanisms underlying its development and treatment are yet to be elucidated. In this study, we report that an ADHD mouse model, which harbors a deletion in the Git1 locus, exhibits severe astrocytosis in the globus pallidus (GP) and thalamic reticular nucleus (TRN), which send modulatory GABAergic inputs to the thalamus. A moderate level of astrocytosis was displayed in other regions of the basal ganglia pathway, including the ventrobasal thalamus and cortex, but not in other brain regions, such as the caudate putamen, basolateral amygdala, and hippocampal CA1. This basal ganglia circuit-selective astrocytosis was detected in both in adult (2-3 months old) and juvenile (4 weeks old) $Git1^{\check{s}/\check{s}}$ mice, suggesting a developmental origin. Astrocytes play an active role in the developing synaptic circuit; therefore, we performed an immunohistochemical analysis of synaptic markers. We detected increased and decreased levels of GABA and parvalbumin (PV), respectively, in the GP. This suggests that astrocytosis may alter synaptic transmission in the basal ganglia. Intriguingly, increased GABA expression colocalized with the astrocyte marker, GFAP, indicative of an astrocytic origin. Collectively, these results suggest that defects in basal ganglia circuitry, leading to impaired inhibitory modulation of the thalamus, are neural correlates for the ADHD-associated behavioral manifestations in $Git1^{\check{s}/\check{s}}$ mice.

• The Korean Journal of Physiology and Pharmacology
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• 제24권1호
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• pp.81-88
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• 2020

Regulator of calcineurin 1 (RCAN1) can be induced by an intracellular calcium increase and oxidative stress, which are characteristic features of temporal lobe epilepsy. Thus, we investigated the spatiotemporal expression and cellular localization of RCAN1 protein and mRNA in the mouse hippocampus after pilocarpine-induced status epilepticus (SE). Male C57BL/6 mice were given pilocarpine hydrochloride (280 mg/kg, i.p.) and allowed to develop 2 h of SE. Then the animals were given diazepam (10 mg/kg, i.p.) to stop the seizures and sacrificed at 1, 3, 7, 14, or 28 day after SE. Cresyl violet staining showed that pilocarpine-induced SE resulted in cell death in the CA1 and CA3 subfields of the hippocampus from 3 day after SE. RCAN1 immunoreactivity showed that RCAN1 was mainly expressed in neurons in the shammanipulated hippocampi. At 1 day after SE, RCAN1 expression became detected in hippocampal neuropils. However, RCAN1 signals were markedly enhanced in cells with stellate morphology at 3 and 7 day after SE, which were confirmed to be reactive astrocytes, but not microglia by double immunofluorescence. In addition, realtime reverse transcriptase-polymerase chain reaction showed a significant upregulation of RCAN1 isoform 4 (RCAN1-4) mRNA in the SE-induced hippocampi. Finally, in situ hybridization with immunohistochemistry revealed astrocytic expression of RCAN1-4 after SE. These results demonstrate astrocytic upregulation of RCAN1 and RCAN1-4 in the mouse hippocampus in the acute and subacute phases of epileptogenesis, providing foundational information for the potential role of RCAN1 in reactive astrocytes during epileptogenesis.

• 생물정신의학
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• 제8권1호
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• pp.3-19
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• 2001

Recent basic and clinical studies demonstrate a major role for neural plasticity in the etiology and treatment of depression and stress-related illness. The neural plasticity is reflected both in the birth of new cell in the adult brain(neurogenesis) and the death of genetically healthy cells(apoptosis) in the response to the individual's interaction with the environment. The neural plasticity includes adaptations of intracellular signal transduction pathway and gene expression, as well as alterations in neuronal morphology and cell survival. At the cellular level, repeated stress causes shortening and debranching of dendrite in the CA3 region of hippocampus and suppress neurogenesis of dentate gyrus granule neurons. At the molecular level, both form of structural remodeling appear to be mediated by glucocorticoid hormone working in concert with glutamate and N-methyl-D-aspartate(NMDA) receptor, along with transmitters such as serotonin and GABA-benzodiazepine system. In addition, the decreased expression and reduced level of brain-derived neurotrophic factor(BDNF) could contribute the atrophy and decreased function of stress-vulnerable hippocampal neurons. It is also suggested that atrophy and death of neurons in the hippocampus, as well as prefrontal cortex and possibly other regions, could contribute to the pathophysiology of depression. Antidepressant treatment could oppose these adverse cellular effects, which may be regarded as a loss of neural plasticity, by blocking or reversing the atrophy of hippocampal neurons and by increasing cell survival and function via up-regulation of cyclic adenosine monophosphate response element-binding proteins(CREB) and BDNF. In this article, the molecular and cellular mechanisms that underlie stress, depression, and action of antidepressant are precisely discussed.