Expression and roles of NUPR1 in cholangiocarcinoma cells / 대한해부학회지

Nuclear protein-1 (NUPR1) is a small nuclear protein that is responsive to various stress stimuli. Although NUPR1 has been associated with cancer development, its expression and roles in cholangiocarcinoma have not yet been described. In the present study, we found that NUPR1 was over-expressed in human cholangiocarcinoma tissues, using immunohistochemistry. The role of NUPR1 in cholangiocarcinoma was examined by its specific siRNA. NUPR1 siRNA decreased proliferation, migration and invasion of human cholangiocarcinoma cell lines (HuCCT1 and SNU1196 cells). From these results, we conclude that NUPR1 is over-expressed in cholangiocarcinoma and regulates the proliferation and motility of cancer cells.

Characterization of the expression of cytokeratins 5, 8, and 14 in mouse thymic epithelial cells during thymus regeneration following acute thymic involution / 대한해부학회지

The thymus is a central lymphoid organ for T cell development. Thymic epithelial cells (TECs) constitute a major component of the thymic stroma, which provides a specialized microenvironment for survival, proliferation, and differentiation of immature T cells. In this study, subsets of TECs were examined immunohistochemically to investigate their cytokeratin (CK) expression patterns during thymus regeneration following thymic involution induced by cyclophosphamide treatment. The results demonstrated that both normal and regenerating mouse thymuses showed a similar CK expression pattern. The major medullary TECs (mTEC) subset, which is stellate in appearance, exhibited CK5 and CK14 staining, and the minor mTEC subset, which is globular in appearance, exhibited CK8 staining, whereas the vast majority of cortical TECs (cTECs) expressed CK8 during thymus regeneration. Remarkably, the levels of CK5 and CK14 expression were enhanced in mTECs, and CK8 expression was upregulated in cTECs during mouse thymus regeneration after cyclophosphamide-induced acute thymic involution. Of special interest, a relatively high number of CK5+CK8+ TEC progenitors occurred in the thymic cortex during thymus regeneration. Taken together, these findings shed more light on the role of CK5, CK8, and CK14 in the physiology of TECs during mouse thymus regeneration, and on the characterization of TEC progenitors for restoration of the epithelial network and for concomitant regeneration of the adult thymus.

Expression of IL-17A during Thymus Regeneration in the Rat / 체질인류학회지

IL-17A is a pro-inflammatroy cytokine secreted by activated T cells. The IL-17 family consist of IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F. IL-17A and IL-17F are produced primarily in activated T cells. In contrast, IL-17B, IL-17C, IL-17D and IL-17E are expressed in a wide assortment of tissues. Their functions partially overlap those of IL-17A, although they have not been as thoroughly investigated. The receptor for IL-17A (IL-17R) is widely expressed in a variety of tissues. IL-17A and IL-17E mRNAs were expressed in only EL4 cells. IL-17C mRNA expression was observed in the thymic subcapsular/cortex epithelial cells (SNEC), cortex or cortical reticular cells (CREC), medullary epithelial cells (MEC), medullary interdigitating-like cells (MDC), thymocytes and EL4 cells. However, IL-17C mRNA was not expressed in RAW 264.7 cells. Immunohistochemical study also demonstrated not only the presence of IL-17A mainly in the thymic epithelial cells, but also the upregulated expression of IL-17A in the thymic epithelial cells of the regenerating thymus. Thus, the results of the present study suggest that IL-17A expressed in the thymocytes and thymic epithelial cells could play an important role in the development of new T cells to replace T cells damaged by cyclophosphamide treatment during thymus regeneration.

Vitamin D3 regulates cell viability in gastric cancer and cholangiocarcinoma / 대한해부학회지

A low serum level of vitamin D has been associated with an increased incidence of gastrointestinal tract cancers. However, the effects of vitamin D3 have not been investigated in gastric cancer and cholangiocarcinoma. In the present study, we found that vitamin D3 treatment significantly suppressed the viability of gastric cancer and cholangiocarcinoma cells. Moreover, vitamin D3 had a synergistic effect with other anti-cancer drugs, such as paclitaxel, adriamycin, and vinblastine, for suppressing cell viability. To determine the underlying mechanism involved in the regulation of viability by vitamin D3, we examined the effects of vitamin D3 on expression of hedgehog signaling target genes, which has been associated with gastric cancer and cholangiocarcinoma. Vitamin D3 treatment decreased the level of mRNA expression of patched1, Gli1, cyclin D1, and Bcl2, suggesting the possibility that vitamin D3 may act through regulation of hedgehog signaling. From the above results, we conclude that vitamin D3 regulates cell viability in gastric cancer and cholangiocarcinoma.

CDH3/P-Cadherin regulates migration of HuCCT1 cholangiocarcinoma cells / 대한해부학회지

Intrahepatic cholangiocarcinoma is the second most common subtype of primary hepatobilliary cancer. Despite advances in surgical and medical therapy, its survival rate remains poor. Compared to hepatocellular carcinoma (HCC), the most common liver malignancy, the underlying mechanisms of cholangiocarcinoma carcinogenesis are poorly characterized. P-cadherin (CDH3) is a cadherin super family member. Although CDH3 is frequently over-expressed in cholangiocarcinoma tissues, its roles have never been characterized. To determine the roles of CDH3 in cholangiocarcinoma, we investigated CDH3 function in HuCCT1 cells using specific siRNA. Transfection with CDH3 siRNA did not affect proliferation of HuCCT1 cells. However, cell migration and invasion were significantly reduced when CDH3 was down-regulated. In addition, expressions of several biomarkers for epithelial-mesenchymal transition (EMT) were not changed by CDH3 down-regulation. These results suggest that CDH3 regulates cell migration independent of EMT in cholangiocarcinoma cells.

Expression of 4-1BB and 4-1BBL in thymocytes during thymus regeneration

4-1BB, a member of the tumor necrosis factor receptor (TNFR) superfamily, is a major costimulatory receptor that is rapidly expressed on the surface of CD4+ and CD8+ T cells after antigen- or mitogen-induced activation. The interaction of 4-1BB with 4-1BBL regulates immunity and promotes the survival and expansion of activated T cells. In this study, the expression of 4-1BB and 4-1BBL was examined during regeneration of the murine thymus following acute cyclophosphamide-induced involution. Four-color flow cytometry showed that 4-1BB and 4-1BBL were present in the normal thymus and were preferentially expressed in the regenerating thymus, mainly in CD4+CD8+ double-positive (DP) thymocytes. Furthermore, the CD4loCD8lo, CD4+CD8lo and CD4loCD8+ thymocyte subsets, representing stages of thymocyte differentiation intermediate between DP and single-positive (SP) thymocytes, also expressed 4-1BB and 4-1BBL during thymus regeneration but to a lesser degree. Interestingly, the 4-1BB and 4-1BBL positive cells among the CD4+CD8+ DP thymocytes present during thymus regeneration were TCR(hi) and CD69+ unlike the corresponding controls. Moreover, the 4-1BB and 4-1BBL positive cells among the intermediate subsets present during thymus regeneration also exhibited TCRhi/int and CD69+/int phenotypes, indicating that 4-1BB and 4-1BBL are predominantly expressed by the positively selected population of the CD4+CD8+ DP and the intermediate thymocytes during thymus regeneration. RT-PCR and Western blot analyses confirmed the presence and elevated levels of 4-1BB and 4-1BBL mRNA and protein in thymocytes during thymus regeneration. We also found that the interaction of 4-1BB with 4-1BBL promoted thymocyte adhesion to thymic epithelial cells. Our results suggest that 4-1BB and 4-1BBL participate in T lymphopoiesis associated with positive selection during recovery from acute thymic involution.

Expression of the p75NTR Neurotrophin Receptor in the Thymic Medullary Epithelial Cells and Interdigitating Dendritic Cells during Rat Thymus Regeneration Following Acute Thymic Involution / 대한해부학회지

The existence of a functional link between the nervous and immune systems has been well established. The present study was to characterize the expression of p75NTR during thymus regeneration from acute involution induced by cyclophosphamide in the rat. Immunohistochemical and double immunofluorescence analyses demonstrated that expression of the p75NTR was decreased in the thymic medullary epithelial cells and interdigitating dendritic cells during thymus regeneration. The presence of p75NTR protein in extracts from the control and regenerating rat thymus was confirmed by western blot. Furthermore, RT-PCR analysis supported these results by demonstrating that thymic extracts contain p75NTR mRNA at lower levels during thymus regeneration. Thus, our results suggest that the p75NTR located on the thymic medullary epithelial cells and interdigitating dendritic cells could play a role in the development of new T cells to replace the thymocytes damaged during thymus regeneration

RANKL stimulates proliferation, adhesion and IL-7 expression of thymic epithelial cells

Abstract In many clinical situations which cause thymic involution and thereby result in immune deficiency, T cells are the most often affected, leading to a prolonged deficiency of T cells. Since only the thymic-dependent T cell production pathway secures stable regeneration of fully mature T cells, seeking strategies to enhance thymic regeneration should be a key step in developing therapeutic methods for the treatment of these significant clinical problems. This study clearly shows that receptor activator of NF-kappaB ligand (RANKL) stimulates mouse thymic epithelial cell activities including cell proliferation, thymocyte adhesion to thymic epithelial cells, and the expression of cell death regulatory genes favoring cell survival, cell adhesion molecules such as ICAM-1 and VCAM-1, and thymopoietic factors including IL-7. Importantly, RANKL exhibited a significant capability to facilitate thymic regeneration in mice. In addition, this study demonstrates that RANKL acts directly on the thymus to activate thymus regeneration regardless of its potential influences on thymic regeneration through an indirect or systemic effect. In light of this, the present study provides a greater insight into the development of novel therapeutic strategies for effective thymus repopulation using RANKL in the design of therapies for many clinical conditions in which immune reconstitution is required.

Effects of Indomethacin on Ischemia-induced Brain Injury / 대한해부학회지

Cerebral ischemia can have severe results and disrupt quality of life. Current medicine is not effective at overcoming these problems. To find out more effective therapies, it is necessary to understand the microenvironment of cerebral injury after the ischemia. In the present study, to investigate the effects of inflammatory reaction, indomethacin, an anti-inflammatory drug, was used in a photothrombotic focal infarction rat model. It was revealed that cerebral ischemia increased neurogenesis in the subventricular (SVZ) and subgranular zones (SGZ), and in the penumbral region. Indomethacin treatment reduced the cerebral ischemia-induced neurogenesis by 86.2%, 53.8%, and 52.8% respectively. Cerebral ischemia increased gliosis and angiogenesis in the penumbral region and indomethacin reduced gliosis and angiogenesis by 48.2% and 58.1%, respectively. These results suggest that indomethacin treatment after the cerebral ischemia can reduce neurogenesis, angiogenesis, and gliosis in the penumbral region.

Effects of retinoic acid on ischemic brain injury-induced eurogenesis

Neurogenesis can be induced by pathological conditions such as cerebral ischemia. However the molecular mechanisms or modulating reagents of the reactive neurogenesis after the cerebral ischemia are poorly characterized. Retinoic acid (RA) has been shown to increase neurogenesis by enhancing the proliferation and neuronal differentiation of forebrain neuroblasts. Here, we examined whether RA can modulate the reactive neurogenesis after the cerebral ischemia. In contrast to our expectation, RA treatment decreased the reactive neurogenesis in subventricular zone (SVZ), subgranular zone (SGZ) and penumbral region. Furthermore, RA treatment also decreased the angiogenesis and gliosis in penumbral region.

Expression of CD70 during Thymus Regeneration in the Rat / 대한해부학회지

The thymus is the central lymphoid organ for development of bone marrow-derived precursor cells into mature T-cells. The thymic stroma provides the specialized microenvironment for the proliferation, differentiation, and maturation of the immature T-cells, thymocytes. The CD27 is a tumor necrosis factor (TNF) receptor family member whose expression is limited to cells of the lymphoid lineage. CD70, the ligand of CD27, is a TNF related trans-membrane protein induced upon activation on T and B cells, dendritic cells and macrophages. CD70/CD27 interaction plays a key role in T dependent B cell responses and is responsible for plasma cell differentiation. This study was performed to investigate the expression of CD70 during regeneration following acute involution induced by cyclophosphamide (CY) in the rat thymus using RT-PCR analysis and single and double immunohistochemistry. The results from RT-PCR analysis showed that CD70 is expressed in mouse thymic medullary interdigitating (IDC)-like cells (MDC), DC2.4 and Raw264.7 but not expressed in the mouse thymic epithelial cells (subcapsular/cortex epithelial cells, cortical epithelial cells and medullary epithelial cells). Interestingly, upregulation of CD70 expression was observed in the thymic stromal cells and thymocytes isolated from rat thymus during thymus regeneration. Furthermore, immunohistochemistry demonstrated that CD70 was mainly expressed in the ED1 positive macrophages predominantly in the thymic cortex both in the normal thymus and during thymus regeneration. In line with the data obtained by biochemical analysis, CD70 immunoreactive macrophages is increased both in number and in size during thymus regeneration. Thus, the results of the present study suggest that CD70 expressed on the thymic macrophages could play a role in the development of new T cells to replace T cells damaged by cyclophosphamide treatment during thymus regeneration.

Morphological Changes of Anterior Horn Cells of Lumbar Spinal Cord after Anterior Root Avulsion in Adult Rat / 대한해부학회지

The morphological changes in the anterior horn of the L4 and L5 spinal segments were observed following anterior root avulsion in the adult male Sprague-Dawley rat (300~350 gm) at 5 days, 1 week, 2 weeks and 3 weeks postlesion. The animals were perfused with 4% paraformaldehyde, 0.15% picric acid in 0.1 M phosphate buffer solution and cryostat sections were prepared. Immunohistochemistry was used to identify changes of the phenotype in the anterior horn cells. Primary antibodies, goat anti-choline acetyltransferase (ChaT, 1 : 500, Chemicon), mouse antirat ED-1 (1 : 200, Serotec), rabbit anti-glial fibrillary acidic protein (GFAP, 1 : 200, DAKO) and rabbit anti-vascular endothelial growth factor (VEGF, 1 : 500, Santa Cruz Biotechnology) were used. Avidin-Biotin complex method was performed for immunohistochemical reaction and color reaction was developed with DAB-H2O2. Following results were observed in the anterior horn of lumbar spinal cord; 1. The number of ChaT-immunoreactive (ir) cells were reduced 20% level of control animals at 3 weeks after avulsion. 2. ED-1-ir microglia were significantly increased at 1 week and processes of ED-1-ir microglia surrounded around the axotomized neuronal cell bodies. 3. Gliosis defined by extensive GFAP immunoreactivity was observed both ipsilateral and contralateral side of lesion but the VEGF-ir cells were significantly increased in the ipsilateral side of lesion. Therefore, this study suggested that the majority of axotomized motor neurons were degenerated and the cellular proliferation and phenotype changes including glial cell activation were observed in the lumbar spinal cord after anterior root avulsion of adult rats.

Patterning of Ventral Neural Tube in Tg737(oprk) Mutant / 대한해부학회지

Polaris, which is encoded by Tg737 gene, has been associated with cilia formation. Recently pheno-types of ventral neural tube in mice who have abnormal cilia formation have been reported to be similar with those of sonic hedgehog (Shh)signaling mutants. These interesting findings lead us to further examine the patterning of ventral neural tube in Tg737(oprk) mice. In this study, we found that motor neuron and V2 interneuron were preserved whereas P3 progenitor domain and floor plate were missing in Tg737(oprk) mutant. V2 and motor neurons in Tg737(oprk) were ventralized and ixed with each other. Nkx6.1 and Olig2 expressions were preserved and the Olig2 expression was ventralized in Tg737(oprk). These penotypes are quite similar with those in Shh(-/-); Gli3(-/-) or Gli2(-/-) ; Gli3(-/-) mutants, suggesting that the function of Polaris might be involved in Shh signaling.

Expressions of BMP Antagonists in Shh -/- ; Gli3 -/- Mutant / 체질인류학회지

Sonic hedgehog (Shh) has been known as an essential morphogen for the generation of motor neuron in developing spinal cord. However, motor neuron can be generated in Shh -/- ; Gli3 -/- or Gli2 -/- ; Gli3 -/- mutants although these mutants don't have Shh signaling. To find out the compensatory mechanism for the generation of motor neuron in Shh -/- ; Gli3 -/- mutant, we studied bone morphogenetic protein (BMP) antagonists including follistatin, flik and noggin, and retinoic acid signaling in this mutant. To study expressions of BMP antagonists, we performed in situ hybridization. To examine an activity of retinoic acid, we measured beta -galactosidase activity in retinoic acid response element (RARE) transgenic mouse. The expression of follistatin was reduced at both levels of forelimb and hindlimb in Shh -/- mutant compared to wild type embryo. It was restored at the level of forelimb but reduced at the level of hindlimb in Shh -/- ; Gli3 -/- mutant compared to wild type. The expression of flik was similar with wild type embryo at both levels of forelimb and hindlimb in Shh -/- mutant. The expression of flik was similar with wild type embryo at the level of forelimb however reduced in hindlimb level in Shh -/- ; Gli3 -/- mutant. The expression of noggin, a BMP antagonist, was increased in Shh -/- mutant. Activity of retinoic acid signaling was not affected in Shh -/- or Shh -/- ; Gli3 -/- mutants. From these results, we conclude that retinoic acid but not follistatin and flik, may be involved in the generation of motor neuron in Shh -/- ; Gli3 -/- mutant.

Roles of Gli3 in Dorsal Neural Tube / 대한해부학회지

Essential roles of Gli3 in ventral neural tube have been stressed from studies of Shh(-/-) and Shh(-/-); Gli3(-/-) mutants. However, roles of Gli3 in dorsal neural tube have not been fully appreciated despite of its high expression. To find out roles of Gli3 in dorsal neural tube, we studied cell proliferation and neuronal differentiation in dorsal neural tube of Gli3(-/-) mutant. In Gli3(-/-) mutant, proliferation of progenitor cells in dorsal neural tube is increased compared to wild type embryos based on phosphohistone 3 immunohistochemistry and BrdU experiment. The appearances of HuC/D positive and Isl1 postive cells which represent postmitotic neurons and dI3 interneurons were delayed in Gli3(-/-) mutant compared to wild type embryo. The appearance of a proneural gene, Ngn2 was also delayed in Gli3(-/-) mutant compared to wild type embryo. Neuronal differentiation of progenitor cells in dorsal neural tube was delayed in Gli3(-/-) mutant compared to wild type embryos based on HuC/D, Isl1 and Ngn2 expressions. These results suggest that Gli3 plays important roles in cell proliferation and neuronal differentiation in dorsal neural tube. Thus our data shed a new light on the role of Gli3 in the development of neural tube.

Roles of Gli3 in the Establishment of Progenitor Domains of Developing Spinal Cord / 대한해부학회지

University Sonic hedgehog (Shh) signaling has been shown to play instructive roles in developing spinal cord. Depending on the Shh concentration gradient, different progenitor domains and ventral neurons are induced. However, the way how the Shh gradient is translated into different progenitor domains, is not clear. To investigate the translation of the Shh gradient, we studied expressions of homeoproteins which are critical for establishment of progenitor domains, in the ventral neural tube of Shh(-/-)and Shh(-/-);Gli3(-/-) mutants, using in situ hybridization. In Shh(-/-) mutant, the expressions of class II homeoproteins (Nkx6.1, Nkx6.2, Olig2, Nkx2.2 were totally repressed. The expressions of class I homeoproteins (Dbx1, Dbx2, Irx3, Pax6 were ventralized. In Shh(-/-);Gli3(-/-) mutant, the expressions of class II homeoproteins except Nkx2.2 were restored. The expressions of class I home-oproteins were restored to its original position although their restoration is not complete. From above results, we conclude that Gli3 can regulate the expressions of class II homeoproteins, which suggests that the Shh gradient will be translated into Gli activity in the developing spinal cord.

Expression of the ED3 Antigen and its Upregulation after Cyclophosphamide Treatment on Basal Epithelial Cells of Rat Stratified Squamous Epithelium / 대한해부학회지

Sialoadhesin (Sn) expression has been demonstrated on murine and rat macrophages in lymphatic organs and is recognized by the monoclonal antibody (mAb) ED3 in the rat. Sialoadhesin (Siglec-1), the ED3 antigen in the rat, is a subtype of sialic acid -binding Ig-like lectins (Siglecs) that bind specifically to sialic acid-containing structures such as selectins and was originally identified as the sheep erythrocyte receptor (SER) responsible for sialic acid-dependent binding of native sheep erythrocytes (SE) to resident murine bone marrow macrophages in rosetting assays. The aim of the present study was to investigate the expression and potential function of sialoadhesin in the stratified squamous epithelium of the rat tongue, esophagus and skin. The expression of sialoadhesin was demonstrated by immunohistochemical analysis with the mAb ED3. This study demonstrated not only the presence of sialoadhesin on the basal epithelial cells of the stratified epithelium in normal rat tongue, esophagus and skin but also its upregulated expression on these cells in CY-treated rats. The results of the present study shed some light on the potential function of sialoadhesin in the basal epithelial cells of the stratified epithelium. Further studies may provide more insight into the role of sialoadhesin in the epithelial stem cells.

Ultrastructure of Different Types of Thymic Epithelial Cells in the Rat Thymus / 대한해부학회지

Thymic epithelial cells constitute a major component of the thymic microenvironment. The thymus is involved in the regulation of the proliferation, maturation and differentiation of thymocytes. There is some controversy about the classification of thymic epithelial cell types. Traditionally, thymic epithelial cells have been divided into cortical and medullary epithelial cell types. In general, the thymic epithelium can be broadly subdivided into subcapsular, cortical and medullary epithelial cells, and Hassall's corpuscles by immunocytochemical methods. Although a few studies were performed on the ultrastructural characteristics of the different types of thymic epithelial cells, there is still some controversy about the classification of thymic epithelial cell subsets. Thus, the present study was performed to investigate the ultrastructural features of thymic epithelial cell subsets in adult male Sprague-Dawley rats, which are the most commonly used species of rat for biological researches, using transmission electron microscopy to shed more light on the heterogeneity of thymic epithelial cells. On the basis of ultrastructural features, we could identify and classify eight subsets of epithelial cells in normal rat thymus. In particular, this study provided a clear and easy way to identify the type 3 epithelial cells by their characteristic 'perinuclear arrangement pattern of relatively short bundles of tonofilaments'. This is an important finding since the type 3 epithelial cells has been considered to be the most difficult type to identify among various thymic epithelial cell types. The results of the present ultrastructural study of thymic epithelial cells provided more insight into the heterogeneity of thymic epithelial cells, and can contribute to the understanding of roles played by different types of thymic epithelial cells.

Three-Dimensional Arrangement of Muscle Fibers, Collagen and Elastin Fibers of the Proximal Prostatic Urethra in Benign Prostatic Hyperplasia / 대한비뇨기과학회지

PURPOSE: Anatomical change of the prostatic stroma, that is benign prostatic hyperplasia, results in a functional change which manifests as lower urinary tract symptoms (LUTS). We observed/investigated whether there are structural changes in the three-dimensional arrangement of muscle, collagen and elastin fibers of the prostatic urethra which can be another possible mechanism of LUTS. MATERIALS AND METHODS: Hyperplastic nodules, which were surgically en bloc resected, and normal prostate from radical cystectomy specimens were examined. Surgical specimens were fixed in formalin and sectioned serially in a transverse plane along the lumen of the upper segment of the prostatic urethra. Additional sagittal sections were also made serially. All tissue blocks were 2mm thick. All slides were prepared with hematoxylin-eosin, Masson-trichrome and Verhoeff's elastin stain. RESULTS: Muscle and collagen fibers surrounding prostatic acini seemed poorly oriented and blended with the outermost fibers of the urethra. Muscle and stromal fibers surrounding the prostatic urethra seemed to be stretched and oriented mainly in the longitudinal direction with small fibroblastic nodules in the periurethral stroma. A periurethral gland structure was observed between the urethral wall in which the fiber was arranged longitudinally. Circular muscle fibers were not observed in the periurethral area, indicating that the surgical capsule resulted from urethral muscle fibers. CONCLUSIONS: Apparent changes to the 3-dimensional arrangement of collagen, elastin and muscle fibers surrounding the acini and prostatic urethra were not observed in the proximal prostatic urethra or the bladder neck, except that the fibers surrounding the urethra were stretched.

Synaptic Localization of NMDA Receptor and Shank Protein in Hippocampal Neuron in Vitro / 대한해부학회지

The existence of NMDA receptor and a new organizer protein, Shank, in the postsynaptic density was studied with the cultured hippocampal neurons using by double immunofluorescence method. The hippocampi from embryonic 18 days were dissected and hippocampal neurons were obtained from dissociated hippocampi with 0.25% trypsin and 0.1% DNase in PBS. The hippocampal neurons were plated with density 3,600/cm2 on the poly-L-lysine coated coverglass and cultured 37degrees C, 5% CO2 incubator for 5 weeks. The N2 supplemented MEM was used as a culture medium. Following results are obtained from experiments: 1. The 3~5 minor processes from the cell body of hippocampal neurons were observed at 20 hr in vitro. One of the minor processes was elongated and looked like an axon, and another minor processes showed dendritic branching pattern with slender in thickness. 2. The excitatory NMDA receptor colocalized with PSD-95 which is the postsynaptic density protein. The presynaptic protein, synapsin 1, was closely apposed with PSD-95. 3. Shank which is an organizer protein colocalize with NMDA receptor/PSD-95 complex in the postsynaptic density. Shank proteins may be concerned with the cluster formation of NMDA receptor/PSD-95 in the postsynaptic membrane.