Expression of Nicotinic Acetylcholine Receptor α4 and β2 Subunits on Direction-Selective Retinal Ganglion Cells in the Rabbit

Lee, Jun-Seok,Kim, Hyun-Jin,Ahn, Chang-Hyun,Jeon, Chang-Jin JAPAN SOCIETY OF HISTOCHEMISTRY AND CYTOCHEMISTRY 2017 Acta histochemica et cytochemica Vol.50 No.1

<P>The direction selectivity of the retina is a distinct mechanism that is critical function of eyes for survival. The direction-selective retinal ganglion cells (DS RGCs) strongly respond to a preferred direction, but rarely respond to opposite direction or null directional visual stimuli. The DS RGCs are sensitive to acetylcholine, which is secreted from starburst amacrine cells (SACs) to the DS RGCs. Here, we investigated the existence and distribution of the nicotinic acetylcholine receptor (nAChR) α4 and β2 subunits on the dendritic arbors of the DS RGCs in adult rabbit retina using immunocytochemistry. The DS RGCs were injected with Lucifer yellow to identify their dendritic morphology. The double-labeled images of dendrites and nAChR subunits were visualized for reconstruction using high-resolution confocal microscopy. Although our results revealed that the distributional pattern of the nAChR subunits on the dendritic arbors of the DS RGCs was not asymmetric in the adult rabbit retina, the distribution of nAChR α4 and β2 subunits and molecular profiles of cholinergic inputs to DS RGCs in adult rabbit retina provide anatomical evidence for direction selectivity.</P>

Transplantation of Bone Marrow-Derived Mesenchymal Stem Cells into the Developing Mouse Eye

Lee, Eun-Shil,Yu, Song-Hee,Jang, Yu-Jin,Hwang, Dong-Youn,Jeon, Chang-Jin Japan Society of Histochemistry and Cytochemistry 2011 Acta histochemica et cytochemica Vol.44 No.5

<P>Mesenchymal stem cells (MSCs) have been studied widely for their potential to differentiate into various lineage cells including neural cells <I>in vitro</I> and <I>in vivo</I>. To investigate the influence of the developing host environment on the integration and morphological and molecular differentiation of MSCs, human bone marrow-derived mesenchymal stem cells (BM-MSCs) were transplanted into the developing mouse retina. Enhanced green fluorescent protein (GFP)-expressing BM-MSCs were transplanted by intraocular injections into mice, ranging in ages from 1 day postnatal (PN) to 10 days PN. The survival dates ranged from 7 days post-transplantation (DPT) to 28DPT, at which time an immunohistochemical analysis was performed on the eyes. The transplanted BM-MSCs survived and showed morphological differentiation into neural cells and some processes within the host retina. Some transplanted cells expressed microtubule associated protein 2 (MAP2ab, marker for mature neural cells) or glial fibrillary acid protein (GFAP, marker for glial cells) at 5PN 7DPT. In addition, some transplanted cells integrated into the developing retina. The morphological and molecular differentiation and integration within the 5PN 7DPT eye was greater than those of other-aged host eye. The present findings suggest that the age of the host environment can strongly influence the differentiation and integration of BM-MSCs.</P>

Immunocytochemical Localization of Calbindin D28K, Calretinin, and Parvalbumin in Bat Superior Colliculus

Jeong, Se-Jin,Kim, Hyun-Ho,Lee, Won-Sig,Jeon, Chang-Jin JAPAN SOCIETY OF HISTOCHEMISTRY AND CYTOCHEMISTRY 2014 Acta histochemica et cytochemica Vol.47 No.3

<P>The purpose of this study was to investigate the localization of cells containing the calcium-binding proteins (CBPs) calbindin D28K (CB), calretinin (CR), and parvalbumin (PV) in the superior colliculus (SC) of the bat using immunocytochemistry. CB-immunoreactive (IR) cells formed a laminar tier within the upper superficial gray layer (SGL), while CR-IR cells were widely distributed within the optic layer (OL). Scattered CR-IR cells were also found within the intermediate gray, white, and deep gray layers. By contrast, PV-IR cells formed a laminar tier within the lower SGL and upper OL. Scattered PV-IR cells were also found throughout the intermediate layers, but without a specific laminar pattern. The CBP-IR cells varied in size and morphology: While most of the CB-IR cells in the superficial layers were small round or oval cells, most CR-IR cells in the intermediate and deep layers were large stellate cells. By contrast, PV-IR cells were small to large in size and included round or oval, stellate, vertical fusiform, and horizontal cells. The average diameters of the CB-, CR-, and PV-IR cells were 11.59, 17.17, and 12.60 μm, respectively. Double-immunofluorescence revealed that the percentage of co-localization with GABA-IR cells was 0.0, 0.0, and 10.27% of CB-, CR-, and PV-IR cells, respectively. These results indicate that CBP distribution patterns in the bat SC are unique compared with other mammalian SCs, which suggest functional diversity of these proteins in visually guided behaviors.</P>

Expression of p75 ^NGFR , a Proliferative and Basal Cell Marker, in the Buccal Mucosa Epithelium during Re-epithelialization

Ishii, Akihiro,Muramatsu, Takashi,Lee, Jong-Min,Higa, Kazunari,Shinozaki, Naoshi,Jung, Han-Sung,Shibahara, Takahiko JAPAN SOCIETY OF HISTOCHEMISTRY AND CYTOCHEMISTRY 2014 Acta histochemica et cytochemica Vol.47 No.4

<P>We investigated the expression of p75<SUP>NGFR</SUP>, a proliferative and basal cell marker, in the mouse buccal mucosa epithelium during wound healing in order to elucidate the role of epithelial stem cells. Epithelial defects were generated in the epithelium of the buccal mucosa of 6-week-old mice using CO<SUB>2</SUB> laser irradiation. BrdU was immediately administered to mice following laser irradiation. They were then sacrificed after 1, 3, 7, and 14 days. Paraffin sections were prepared and the irradiated areas were analyzed using immunohistochemistry with anti-p75<SUP>NGFR</SUP>, BrdU, PCNA, and CK14 antibodies. During re-epithelialization, PCNA (–)/p75<SUP>NGFR</SUP> (+) cells extended to the wound, which then closed, whereas PCNA (+)/p75<SUP>NGFR</SUP> (+) cells were not observed at the edge of the wound. In addition, p75<SUP>NGFR</SUP> (–)/CK14 (+), which reflected the presence of post-mitotic differentiating cells, was observed in the supra-basal layers of the extended epithelium. BrdU (+)/p75<SUP>NGFR</SUP> (+), which reflected the presence of epithelial stem cells, was detected sparsely in buccal basal epithelial cells after healing, and disappeared after 7 days. These results suggest that p75<SUP>NGFR</SUP> (+) keratinocytes are localized in the basal layer, which contains oral epithelial stem cells, and retain the ability to proliferate in order to regenerate the buccal mucosal epithelium.</P>

Localization of Nitric Oxide Synthase-containing Neurons in the Bat Visual Cortex and Co-localization with Calcium-binding Proteins

Gu, Ya-Nan,Kim, Hang-Gu,Jeon, Chang-Jin JAPAN SOCIETY OF HISTOCHEMISTRY AND CYTOCHEMISTRY 2015 Acta histochemica et cytochemica Vol.48 No.4

<P><I>Microchiroptera</I> (microbats) is a suborder of bats thought to have degenerated vision. However, many recent studies have shown that they have visual ability. In this study, we labeled neuronal nitric oxide synthase (nNOS)—the synthesizing enzyme of the gaseous non-synaptic neurotransmitter nitric oxide—and co-localized it with calbindin D28K (CB), calretinin (CR), and parvalbumin (PV) in the visual cortex of the greater horseshoe bat (<I>Rhinolophus ferrumequinum</I>, a species of microbats). nNOS-immunoreactive (IR) neurons were found in all layers of the visual cortex. Intensely labeled neurons were most common in layer IV, and weakly labeled neurons were most common in layer VI. Majority of the nNOS-IR neurons were round- or oval-type neurons; no pyramidal-type neurons were found. None of these neurons co-localized with CB, CR, or PV. However, the synthesis of nitric oxide in the bat visual cortex by nNOS does not depend on CB, CR, or PV.</P>

Synaptic Pattern of KA1 and KA2 upon the Direction-Selective Ganglion Cells in Developing and Adult Mouse Retina

Lee, Jee-Geon,Lee, Kyoung-Pil,Jeon, Chang-Jin Japan Society of Histochemistry and Cytochemistry 2012 Acta histochemica et cytochemica Vol.45 No.1

<P>The detection of image motion is important to vision. Direction-selective retinal ganglion cells (DS-RGCs) respond strongly to stimuli moving in one direction of motion and are strongly inhibited by stimuli moving in the opposite direction. In this article, we investigated the distributions of kainate glutamate receptor subtypes KA1 and KA2 on the dendritic arbors of DS-RGCs in developing (5, 10) days postnatal (PN) and adult mouse retina to search for anisotropies. The distribution of kainate receptor subtypes on the DS-RGCs was determined using antibody immunocytochemistry. To identify their characteristic morphology, DS-RGCs were injected with Lucifer yellow. The triple-labeled images of dendrites, kinesin II, and receptors were visualized by confocal microscopy and were reconstructed from high-resolution confocal images. We found no evidence of asymmetry in any of the kainate receptor subunits examined on the dendritic arbors of both the On and Off layers of DS-RGCs in all periods of developing and adult stage that would predict direction selectivity.</P>

Re-epithelialization of the Buccal Mucosa after Alkaline Chemical Injury

Takaichi, Saneyuki,Muramatsu, Takashi,Lee, Jong-Min,Jung, Han-Sung,Shinozaki, Naoshi,Katakura, Akira,Yamane, Gen-yuki JAPAN SOCIETY OF HISTOCHEMISTRY AND CYTOCHEMISTRY 2014 Acta histochemica et cytochemica Vol.47 No.5

<P>Alkaline conditions in the oral cavity may be caused by a variety of stimuli, including tobacco products, antacids, alkaline drinking water and bicarbonate toothpaste. However, the effects of an alkaline pH on the oral mucosa had not been elucidated. The purpose of this study was to investigate how basal keratinocytes are actively involved in re-epithelialization after alkaline chemical injury. We generated epithelial defects in the oral mucosa of mice by applying an alkaline chemical, and the localization of cytokeratin 13, cytokeratin 14, PCNA and p63 was investigated during the re-epithelialization process. PCNA- and p63-positive staining was seen in basal cells covering the wound surface at 1 day after the chemical injury. Cytokeratin 14-positive and PCNA-negative basal keratinocytes were localized in a few layers of the wound epithelium during epithelial outgrowth. Cytokeratin 14-positive and PCNA-positive basal keratinocytes, indicating proliferation, were localized over the entire layer of the epithelium at the wound margin. These results imply that basal keratinocytes at the wound margin migrate to the wound surface, provoke differentiation and keratinization during epithelial outgrowth and that epithelial cells are supplied from the wound margin to the epithelial outgrowth after alkaline chemical injury.</P>

Localization of Rod Bipolar Cells in the Mammalian Retina Using an Antibody Against the α ₁ c L-type Ca ²⁺ Channel

Huh, Yu-Jin,Choi, Jae-Sik,Jeon, Chang-Jin JAPAN SOCIETY OF HISTOCHEMISTRY AND CYTOCHEMISTRY 2015 Acta histochemica et cytochemica Vol.48 No.2

<P>Bipolar cells transmit stimuli via graded changes in membrane potential and neurotransmitter release is modulated by Ca<SUP>2+</SUP> influx through L-type Ca<SUP>2+</SUP> channels. The purpose of this study was to determine whether the α<SUB>1</SUB>c subunit of L-type voltage-gated Ca<SUP>2+</SUP> channel (α<SUB>1</SUB>c L-type Ca<SUP>2+</SUP> channel) colocalizes with protein kinase C alpha (PKC-α), which labels rod bipolar cells. Retinal whole mounts and vertical sections from mouse, hamster, rabbit, and dog were immunolabeled with antibodies against PKC-α and α<SUB>1</SUB>c L-type Ca<SUP>2+</SUP> channel, using fluorescein isothiocyanate (FITC) and Cy5 as visualizing agents. PKC-α-immunoreactive cells were morphologically identical to rod bipolar cells as previously reported. Their cell bodies were located within the inner nuclear layer, dendritic processes branched into the outer plexiform layer, and axons extended into the inner plexiform layer. Immunostaining showed that α<SUB>1</SUB>c L-type Ca<SUP>2+</SUP> channel colocalized with PKC-α in rod bipolar cells. The identical expression of PKC-α and α<SUB>1</SUB>c L-type Ca<SUP>2+</SUP> channel indicates that the α<SUB>1</SUB>c L-type Ca<SUP>2+</SUP> channel has a specific role in rod bipolar cells, and the antibody against the α<SUB>1</SUB>c L-type Ca<SUP>2+</SUP> channel may be a useful marker for studying the distribution of rod bipolar cells in mouse, hamster, rabbit, and dog retinas.</P>

Transplantation of Adipose Derived Stromal Cells into the Developing Mouse Eye

Yu, Song-Hee,Jang, Yu-Jin,Lee, Eun-Shil,Hwang, Dong-Youn,Jeon, Chang-Jin Japan Society of Histochemistry and Cytochemistry 2010 Acta histochemica et cytochemica Vol.43 No.6

<P>Adipose derived stromal cells (ADSCs) were transplanted into a developing mouse eye to investigate the influence of a developing host micro environment on integration and differentiation. Green fluorescent protein-expressing ADSCs were transplanted by intraocular injections. The age of the mouse was in the range of 1 to 10 days postnatal (PN). Survival dates ranged from 7 to 28 post transplantation (DPT), at which time immunohistochemistry was performed. The transplanted ADSCs displayed some morphological differentiations in the host eye. Some cells expressed microtubule associated protein 2 (marker for mature neuron), or glial fibrillary acid protein (marker for glial cell). In addition, some cells integrated into the ganglion cell layer. The integration and differentiation of the transplanted ADSCs in the 5 and 10 PN 7 DPT were better than in the host eye the other age ranges. This study was aimed at demonstrating how the age of host micro environment would influence the differentiation and integration of the transplanted ADSCs. However, it was found that the integration and differentiation into the developing retina were very limited when compared with other stem cells, such as murine brain progenitor cell.</P>

Glutamate Receptors GluR1 and GluR4 in the Hamster Superior Colliculus: Distribution and Co-localization with Calcium-Binding Proteins and GABA

Choi, Jae-Sik,Lee, Jea-Young,Jeon, Chang-Jin Japan Society of Histochemistry and Cytochemistry 2009 Acta histochemica et cytochemica Vol.42 No.2

<P>We investigated the distributions of AMPA glutamate receptor subtypes GluR1 and GluR4 in the hamster superior colliculus (SC) with antibody immunocytochemistry and the effect of enucleation on these distributions. We compared these labelings to those of GluR2/3 in our previous report (Park <I>et al.</I>, 2004, Neurosci Res., 49:139–155) and calcium-binding proteins calbindin D28K, calretinin, parvalbumin, and GABA. Anti-GluR1-immunoreactive (IR) cells were scattered throughout the SC. By contrast, anti-GluR4-IR cells formed distinct clusters within the lower lateral stratum griseum intermediale (SGI) and lateral stratum album intermediale (SAI). The GluR1- and GluR4-IR neurons varied in size and morphology. The average diameter of the GluR1-IR cells was 13.00 µm, while the GluR4-IR cells was 20.00 µm. The large majority of IR neurons were round or oval cells, but they also included stellate, vertical fusiform and horizontal cells. Monocular enucleation appeared to have no effect on the GluR1 and GluR4 immunoreactivity. Some GluR1-IR cells expressed calbindin D28K (9.50%), calretinin (6.59%), parvalbumin (2.53%), and GABA (20.54%). By contrast, no GluR4-IR cells expressed calcium-binding proteins or GABA. Although the function of the AMPA receptor subunits in SC is not yet clear, the distinct segregation of the GluR subunits, its differential colocalization with calcium-binding proteins and GABA, and differential responses to enucleation suggest the functional diversity of the receptor subunits in visuo-motor integration in the SC.</P>