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Gap junctions (GJs) are intercellular channels associated with cell-cell communication. Connexin 26 (Cx26) encoded by the GJB2 gene forms GJs of the inner ear, and mutations of GJB2 cause congenital hearing loss that can be syndromic or non-syndromic. It is difficult to predict pathogenic effects using only genetic analysis. Using ionic and biochemical coupling tests, we evaluated the pathogenic effects of Cx26 variants using computational analyses to predict structural abnormalities. For seven out of ten variants, we predicted the variation would result in a loss of GJ function, whereas the others would completely fail to form GJs. Functional studies demonstrated that, although all variants were able to function normally as hetero-oligomeric GJ channels, six variants (p.E47K, p.E47Q, p.H100L, p.H100Y, p.R127L, and p.M195L) did not function normally as homo-oligomeric GJ channels. Interestingly, GJs composed of the Cx26 variant p.R127H were able to function normally, even as homo-oligomeric GJ channels. This study demonstrates the particular location and property of an amino acid are more important mainly than the domain where they belong in the formation and function of GJ, and will provide information that is useful for the accurate diagnosis of hearing loss.

The present study aimed to investigate the influence of the host retinal microenvironment on cell migration and differentiation using Neuro2a (N2a) cells transduced with green fluorescent protein. N2a cells were transplanted into the vitreous cavities of developing mouse eyes (C57BL/6) on postnatal days 1, 5, and 10 (P1, 5, and 10). To analyze the effects of the host microenvironment on neural differentiation of N2a cells in vitro, cells were treated with a conditioned medium (CM) collected from retinal cells cultured at each developmental stage. We observed that numerous cells transplanted into P5 mice eyes migrated into all layers of the host retina, and the presence of processes indicated morphological differentiation. Some transplanted N2a cells expressed several neural markers. However, cells transplanted into the P1 and 10 mice eyes only proliferated within the vitreous cavity. Neurite length increased in N2a cells treated with CM collected from the cultured retinal cells from P5 and 10 mice, while western blotting revealed that the levels of proteins related to neural differentiation were not significantly altered in N2a cells treated with CM. We show that the migration and differentiation capacities of transplanted cells were differentially influenced by the microenvironment of the retinal postnatal ontogeny.

The purpose of this study was to identify tyrosine hydroxylase-immunopositive (TH+) cells in the sparrow retina using immunocytochemistry and quantitative analysis. All TH+ cells were conventional amacrine cells. Based on dendritic morphology, at least two types were observed. The first type had a single thick primary process that descended from the cell body and many densely beaded processes in substrata (s) 1, less beaded processes in s3, and spiny processes in s4/5 of the inner plexiform layer. The dendrites of the second type appeared similar in each layer, but it displayed several primary processes that spread laterally away from the soma before descending to the inner plexiform layer. The average density of TH+ cells was 37.48 ± 1.97 cells/mm2 (mean ± standard deviation; n = 4), and the estimated total number of TH+ cells was 3,061.25 ± 192.79. The highest and lowest densities of TH+ cells were located in the central dorsotemporal retina and periphery of the ventronasal retina, respectively. TH+ cells did not express calbindin-D28 K, calretinin, or parvalbumin. These results suggest that all TH+ cells in specific amacrine cell subpopulations are involved in retinal information processing in both the ON and OFF sublaminae in sparrow retina.

Calcium-binding proteins (CBPs) are important components in calcium-mediated cellular signal transduction. Among the many CBPs, at least three EF-hand CBPs, calbindin-D28K (CB), calretinin (CR), and parvalbumin (PV), have been extensively studied in the retina. In the present study, we investigated the expression patterns of these three CBPs in cholinergic starburst amacrine cells (SACs), which are the most important element for direction selectivity in the rabbit retina. Double-label immunocytochemical analysis of vibratome sections and single-cell injection after immunocytochemical analysis on whole mounts were carried out in rabbit retinas. We found that all SACs in the inner nuclear layer and the ganglion cell layer contained PV. However, none of the SACs in the inner nuclear layer or ganglion cell layer contained either CB or CR. These results suggest that PV, but not CR or CB, may act as a calcium-buffering protein in the SACs of the rabbit retina.

Melanopsin is an opsin-like photopigment found in the small proportion of photosensitive ganglion cells of the retina. It is involved in the regulation of the synchronization of the circadian cycle as well as in the control of pupillary light reflex. The purpose of the present study is to investigate whether melanopsin is also expressed in the other areas of the central visual system outside the retina. We have studied the distribution and morphology of neurons containing melanopsin in the mouse visual cortex with antibody immunocytochemistry. Melanopsin immunoreactivity was mostly present in neuronal soma, but not in nuclei. We found that melanopsin was present in a large subset of neurons within the adult mouse visual cortex with the highest density in layer II/III. In layer I of the visual cortex, melanopsin-immunoreactive (IR) neurons were rarely encountered. In the mouse visual cortex, the majority of the melanopsin-IR neurons consisted of round/oval cells, but was varied in morphology. Vertical fusiform and pyramidal cells were also rarely labeled with the anti-melanopsin antibody. The labeled cells did not show any distinctive distributional pattern. Some melanopsin-IR neurons in mouse visual cortex co-localized with nitricoxide synthase, calbindin and parvalbumin. Our data indicate that melanopsin is located in specific neurons and surprisingly widespread in visual cortex. This finding raises the need of the functional study of melanopsin in central visual areas outside the retina.

A calcium-binding protein, parvalbumin (PV), is widely distributed in the central nervous system and is expressed in the retinal neurons of various vertebrate species. The present study was aimed at describing the types and density of PV-containing retinal ganglion cells (RGCs) in rabbits by using single-cell injection after immunocytochemistry. PV-containing RGCs were first identified by immunocytochemistry and were then iontophoretically injected with a lipophilic dye, DiI. Subsequently, confocal microscopy was used to characterize the morphological classification of the PV-immunoreactive (IR) ganglion cells on the basis of their dendritic field size, branching pattern, and stratification within the inner plexiform layer. The results indicated that at least 8 morphologically different types of rabbit RGCs express PV. They were heterogeneous in terms of their morphology. The present study showed that the proportion of RGCs that contained PV was between 17% and 19% of the total number of ganglion cells. The density of PV-IR RGCs in the rabbit retina was 144 cells/mm(2). Also, it was found that PV was present in all cholinergic amacrine cells in the ganglion cell layer (GCL) and the inner nuclear layer (INL). This integrated approach of characterizing the cell morphology and the selective expression of a particular protein will lead to a better understanding of the properties of RGCs.

The calcium-binding protein parvalbumin (PV) occurs in the retinal ganglion cells (RGCs) of various vertebrate species. In the present study, we aimed to identify the types of PV-containing RGCs that project to the superior colliculus (SC) in the mouse. We injected retrograde tracer dextran into the mouse SC to label RGCs. PV-containing RGCs were first identified by immunocytochemistry and then neurons double-labeled with dextran and PV were iontophoretically injected with a lipophilic dye, DiI. Subsequently, confocal microscopy was used to characterize the morphologic classification of the PV-immunoreactive (IR) retinotectal ganglion cells on the basis of dendritic field size, branching pattern, and stratification within the inner plexiform layer. Among the 8 different types of PV-containing RGCs in the mouse retina, we found all 8 types of RGCs projecting to the SC. The RGCs were heterogeneous in morphology. The combined approach of using tracer injection and a single cell injection after immunocytochemistry on a particular protein will provide valuable data to further understand the functional features of the RGCs which constitute the retinotectal pathway.

Mesenchymal stem cells (MSCs) have been studied widely for their potential to differentiate into various lineage cells including neural cells in vitro and in vivo. 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.

We recently reported the distributions of AMPA (α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate) receptor subtypes glutamate receptors (GluR) 1 and GluR4 in the superior colliculi (SC) of hamsters with antibody immunocytochemistry and the effect of enucleation on these distributions. We also compared these labelings to those of calcium-binding proteins calbindin D28K, calretinin, parvalbumin, and GABA. In the present study, we investigated whether the GluR1- and GluR4-immunoreactive (IR) neurons are interneurons or projection neurons by injection of the retrograde tracer horseradish peroxidase (HRP) into one of each major ascending and descending pathways of the SC. HRP injections were made into a tecto-reticulospinal pathway (TRS) and dorsal lateral geniculate nucleus (dLGN). Animals were then allowed to recover and to survive for 48 hr before perfusion. Sections containing retrograde-labeled neurons were then treated for GluR-immunoreactivity. HRP injections proved that only a small population of the GluR1-IR cells project into TRS (1.4%) and dLGN (2.6%). However, a large subpopulation of GluR4-IR cells project into TRS (32.7%). The differential compositions of inter/projection neurons, along with our previous studies on the separate distribution of the GluR subunits, its differential co-localization with calcium-binding proteins and GABA, and differential reactions to enucleations, strongly imply the functional variety of the receptor subunits in visual behavior responses.

The purpose of this investigation is to characterize parvalbumin-immunoreactive (IR) neurons in the inner nuclear layer (INL) of zebrafish retina through immunocytochemistry, quantitative analysis, and confocal microscopy. In the INL, parvalbumin-IR neurons were located in the inner marginal portion of the INL. On the basis of dendritic stratification in the inner plexiform layer (IPL), at least two types of amacrine cells were IR for parvalbumin. The first one formed distinctive laminar tiers within s4 (PVs4) of the IPL, and the second within s5 (PVs5). The average number of PVs4 cells was 8263 cells per retina (n = 3), and the mean density was 1671 cells/mm2. The average number of PVs5 cells was 1037 cells per retina (n = 3), and the mean density was 210 cells/mm2. Quantitatively, 88.9% of anti-parvalbumin labeled neurons were PVs4 cells and 11.1% were PVs5 cells. Their density was highest in the midcentral region of the ventrotemporal retina and lowest in the periphery of the dorsonasal retina. The average regularity index of the PVs4 cell mosaic was 4.09, while the average regularity index of the PVs5 cell mosaic was 3.46. No parvalbumin-IR cells expressed calretinin or disabled-1, markers for AII amacrine cells, in several animals. These results indicate that parvalbumin-IR neurons in zebrafish are limited to specific subpopulations of amacrine cells and the expressional pattern of parvalbumin may not correspond to AII amacrine cells in several other animals. Their distribution suggests that parvalbumin-IR neurons are mainly involved in ON pathway information flow.

Parvalbumin occurs in various types of cells in the retina. We previously reported parvalbumin distribution in the inner nuclear layer of bat retina. In the present study, we identified the parvalbumin- immunoreactive neurons in the ganglion cell layer of the retina of a bat, Rhinolophus ferrumequinum, and investigated the distribution pattern of the labeled neurons. Parvalbumin immunoreactivity was found in numerous cell bodies in the ganglion cell layer. Quantitative analysis showed that these cells had medium to large-sized somas. The soma diameter of the parvalbumin-immunoreactive cells in the ganglion cell layer ranged from 12.35 to 19.12 ㎛ (n=166). As the fibers in the nerve fiber layer were also stained, the majority of parvalbumin-immunoreactive cells in the ganglion cell layer should be medium to large-sized retinal ganglion cells. The mean nearest neighbor distance of the parvalbumin-immunoreactive cells in the ganglion cell layer of the bat retina ranged from 59.57 to 62.45 ㎛ and the average regularity index was 2.95 ± 0.3 (n=4). The present results demonstrate that parvalbumin is expressed in medium to large-sized retinal ganglion cells in bat retina, and they have a well-organized distributional pattern with regular mosaics. These results should be important as they are applicable to a better understanding of the unsolved issue of a bat vision. This data will help to provide fundamental knowledge for the better understanding of the unique behavioral aspects of bat flight maneuverability.

Single-cell injection after immunocytochemistry is a reliable technique for classifying neurons by their morphological structure and their expression of a particular protein. The aim of the present study was to classify the morphological types of calbindin D28k-immunoreactive retinal ganglion cells in the mouse using single-cell injection after immunocytochemistry, to estimate the density of calbindin D28k-immunoreactive retinal ganglion cells in the mouse retina. Calbindin D28k is an important calcium-binding protein that is widely expressed in the central nervous system. Calbindin D28k-immunoreactive retinal ganglion cells were identified by immunocytochemistry and then iontophoretically injected with the lipophilic dye, DiI. Subsequently, the injected cells were imaged by confocal microscopy to classify calbindin D28k-immunoreactive retinal ganglion cells based on their dendritic ramification depth within the inner plexiform layer, field size, and morphology. The cells were heterogeneous in morphology: monostratified or bistratified, with small to large dendritic field size and sparse to dense dendritic arbors. At least 10 different morphological types (CB1–CB10) of calbindin D28k-immunoreactive retinal ganglion cells were found in the mouse retina. The density of each cell type was quite variable (1.98–23.76%). The density of calbindin D28k-immunoreactive cells in the ganglion cell layer of the mouse retina was 562 cells/mm2, 8.18% of calbindin D28k-immunoreactive cells were axon-less displaced amacrine cells, 91.82% were retinal ganglion cells, and approximately 18.17% of mouse retinal ganglion cells expressed calbindin D28k. The selective expression of calbindin D28k in cells with different morphologies may provide important data for further physiological studies of the mouse retina.

The purpose of the present study is to identify the dopaminergic amacrine (DA) cells in the inner nuclear layer (INL) of zebrafish retina through immunocytochemistry and quantitative analysis. Two types of tyrosine hydroxylase-immunoreactive (TH-IR) cells appeared on the basis of dendritic morphology and stratification patterns in the inner plexiform layer (IPL). The first (DA1) was bistratified, with branching planes in both s1 and s5 of the IPL. The second (DA2) was diffuse, with dendritic processes branched throughout the IPL. DA1 and DA2 cells corresponded morphologically to A(on)(-s1/s5) and A(diffuse)(-1) (Connaughton et al., 2004). The average number of total TH-IR cells was 1088±79cells per retina (n=5), and the mean density was 250±27cells/mm(2). Their density was highest in the mid central region of ventrotemporal retina and lowest in the periphery of dorsonasal retina. Quantitatively, 45.71% of the TH-IR cells were DA1 cells, while 54.29% were DA2 cells. No TH-IR cells expressed calbindin D28K, calretinin or parvalbumin, markers for the various INL cells present in several animals. Therefore the TH-IR cells in zebrafish are limited to very specific subpopulations of the amacrine cells.

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.

Calcium-binding proteins are present in a number of retinal cell types. Types and density of parvalbumin-immunoreactive (IR) retinal ganglion cells (RGCs) in the mouse retina were previously reported using a newly developed single-cell injection technique following immunocytochemistry [Kim, T.J., Jeon, C.J., 2006. Morphological classification of parvalbumin-containing retinal ganglion cells in mouse: single-cell injection after immunocytochemistry. Invest. Ophthalmol. Vis. Sci. 47, 2757-2764]. The present study was aimed at describing the types and density of calretinin-containing RGCs in the mouse. Calretinin-containing RGCs were first identified by immunocytochemistry and were then iontophoretically injected with a lipophilic dye, DiI. Subsequently, confocal microscopy was used to characterize the morphologic classification of the calretinin-IR ganglion cells on the basis of the dendritic field size, branching pattern, and stratification within the inner plexiform layer (IPL). The results indicated that at least 10 morphologically different types of RGCs express calretinin in the mouse retina. They were heterogeneous in morphology: monostratified to bistratfied, small-to-large dendritic field size, and sparse-to-dense dendritic arbors. The present study showed that 86.59% (38,842/44,857) of RGCs contained calretinin. The density of calretinin-IR ganglion cell in the mouse retina was 2795cells/mm(2). The combined approach of cell morphology and the selective expression of a particular protein would provide valuable data for further knowledge on functional features of the RGCs.

Recently, we reported the existence of AII "rod" amacrine cells in the retina of the greater horseshoe bat Rhinolophus ferrumequinum (Jeon et al., 2007). In order to enhance our understanding of bat vision, in the present study, we report on a quantitative analysis of cone and rod photoreceptors. The average cone density was 9,535 cells/mm2, giving a total number of cones of 33,538 cells/retina. The average rod density was 368,891 cells/mm2, giving a total number of rods of 1,303,517 cells. On average, the total populations of rods were 97.49%, and cones were 2.51% of all the photoreceptors. Rod: cone ratios ranged from 33.85:1 centrally to 42.26:1 peripherally, with a mean ratio of 38.96:1. The average regularity index of the cone mosaic in bat retina was 3.04. The present results confirm the greater horseshoe bat retina to be strongly rod-dominated. The rod-dominated retina, with the existence of AII cells discovered in our previous study, strongly suggests that the greater horseshoe bat retina has a functional scotopic property of vision. However, the existence of cone cells also suggests that the bat retina has a functional photopic property of vision.

We previously reported that Tat-Cu,Zn-superoxide dismutase (Tat-SOD), a major antioxidant enzyme, can be directly transduced into mammalian cells and skin [Kwon et al. (2000); Park et al. (2002)]. To enhance the therapeutic potential of Tat-SOD in the treatment of various disorders, we screened a number of natural products for their ability to increase transduction efficiency. Ginsenosides were effective with cultured HeLa cells and enhanced the penetration of Tat-SOD into both the epidermis and the dermis of the subcutaneous layer when sprayed on mice skin. Although their mechanism of action is not fully understood we believe that ginsenosides may be useful cofactors with this antioxidant enzyme in anti-aging cosmetics or as a therapeutic protein in disorders related to reactive-oxygen species.