• Molecules and Cells
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• v.19 no.3
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• pp.382-390
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• 2005

Calcium-binding proteins are thought to play important roles in calcium buffering. The present study investigated the effects of ischemia and reperfusion on calbindin D28K, calretinin, and parvalbumin immunoreactivity in the ganglion cell layer of the rabbit. Rabbits were administered ischemic damage by increasing the intraocular pressure. After 60 and 90 min of ischemia, reperfusion (7 d) was allowed to occur. The b-wave of the electroretinogram (ERG) was reduced by more than 50% and almost 80% in retina given ischemia for 60 and 90 min, respectively. The oscillatory potential (OPs) wave was reduced approximately 50% at 60 min ischemia and 70% at 90 min ischemia. In both normal and ischemic-treated retina, calcium-binding protein immunoreactivity was seen in many cells in the ganglion cell layer. In eyes subjected to 60 min ischemia, there was a decrease of the density of calbindin D28K- (8.29%), calretinin- (14.44%), and parvalbumin- (26.83%) immunoreactive (IR) cells compared to the control retina. In eyes subjected to 90 min ischemia, there was a higher decrease of the density of calbindin D28K- (18.48%), calretinin- (33.59%), and parvalbumin- (54.26%) IR cells than at 60 min. Some calcium-binding protein-IR neurons, especially calretinin-IR neurons, showed aggregations that were abnormally packed together in retina subjected to ischemia for 90 min. The results show that calbindin D28K-, calretinin-, and parvalbumin-IR cells in the ganglion cell layer are susceptible to ischemic damage and reperfusion. The degree of reduction varied among different calcium-binding proteins and ischemic damage times. These results suggest that calbindin D28K-containing neurons are less susceptible to ischemic damage than calretinin- and parvalbumin-containing neurons in the ganglion cell layer of rabbit retina.

• Applied Microscopy
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• v.32 no.2
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• pp.175-183
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• 2002

In this study, we carried out immunostaining and immunogold labeling with rabbit anti-dopamine (TH) and rabbit anti-calbindin-$D_{28K}$ to examine the characteristics and functions of the neurons that secrete neurotransmitters in optic lobes of Todarodes pacificus and Octopus minor inhabiting the Korean waters. The obtained results are as follow. In the immunostaining with anti-dopamine, only a few of the large amacrine cells in an the upper part of an outer granule cell layer and the cells forming the islands of medulla showed positive reaction in Todarodes pacificus, while $2{\sim}3$ cells in the upper and middle parts of an outer granule cell layer and more than 5 cells in the islands of medulla reacted positively in Octopus minor. For the case of anti-calbindin case, $2{\sim}3$ small amacrine cells in the upper portion of the outer granule cell layer and $1{\sim}2$ cells which are located in the lower part of an inner granule cell layer showed positive reaction in Todarodes pacificus, while, in Octopus minor, 4 cells in the outer granule cell layer reacted positively, no immunoreactive cell being found in the inner granule cell layer. As a result of performing the immunogold labeling, relative large number ($17{\sim}26$) of gold particles were labeled per $0.5{\mu}m^2$ of the cytoplasm of the cells which showed the immunoreactivity to the anti-dopamine and anti-calbindin in Todarodes pacificus, however, small number (10) of gold particles were labeled in Octopus minor, which reach only half of the number in the Todarodes pacificus.

• Molecules and Cells
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• v.19 no.3
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• pp.408-417
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• 2005

Nitric oxide (NO) occurs in various types of cells in the central nervous system. We studied the distribution and morphology of neuronal nitric oxide synthase (NOS)-containing neurons in the visual cortex of mouse and rabbit with antibody immunocytochemistry. We also compared this labeling to that of calbindin D28K, calretinin, and parvalbumin. Staining for NOS was seen both in the specific layers and in selective cell types. The densest concentration of intense anti-NOS immunoreactive (IR) neurons was found in layer VI, while the weak anti-NOS-IR neurons were found in layer II/III in both animals. The NOS-IR neurons varied in morphology. The large majority of NOS-IR neurons were round or oval cells with many dendrites coursing in all directions. Two-color immunofluorescence revealed that only 16.7% of the NOS-IR cells were double-labeled with calbindin D28K in the mouse visual cortex, while more than half (51.7%) of the NOS-IR cells were double-labeled with calretinin and 25.0% of the NOS-IR cells were double-labeled with parvalbumin in mouse. By contrast, 92.4% of the NOS-IR neurons expressed calbindin D28K while only 2.5% of the NOS-IR neurons expressed calretinin in the rabbit visual cortex. In contrast with the mouse, none of the NOS-IR cells in the rabbit visual cortex were double-labeled with parvalbumin. The results indicate that neurons in the visual cortex of both animals express NOS in specific layers and cell types, which do not correlate with the expression of calbindin D28K, calretinin or parvalbumin between the two animals.

• Applied Microscopy
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• v.24 no.4
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• pp.61-77
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• 1994

The calcium-binding proteins (CaBP), parvalbumin (PV) and calbindin-D 28K (calbindin) are particularly abundant and specific in their distribution, and present in different subsets of neurons in many brain regions. Although their physiological roles in the neurons have not been elucidated, they are valuable markers of neuronal subpopulations for anatomical and developmental studies. This study is designed to characterize dorsal lateral geniculate nucleus (dLGN) neurons and axon terminals in terms of differential expression of immunoreactivity (IR) for two well-known CaBPs, PV and calbindin. The experiments were carried out on 6 adult monkeys. Monkeys were perfused under deep Nembutal anesthesia with 2% paraformaldehyde and 0.2% glutaraldehyde in 0.1M phosphate buffer. After removal, the brains were postfixed for 6-8 hr in 2% paraformaldehyde at $4^{\circ}C$ and infiltrated with 30% sucrose at $4^{\circ}C$. Thereafter, they were frozen in dry ice. Serial sections of the thalamus, at $20{\mu}m$, were made in the frontal plane with a sliding microtome. The sections were stained for PV and calbindin with indirect immunocytochemical methods. For electron microscopy, after infiltration with 30% sucrose the blocks of thalamus were serially sectioned at $50{\mu}m$ with a Vibratome in the coronal plane and stained immediately by indirect ABC methods without Triton X-100 in incubation medium. Stained sections were postfixed in 0.2% osmium tetroxide, dehydrated and flat-embedded in Spurr resin. The block was then trimmed to contain only a selected lamina or interlaminar space. The dLGN proper showed strong PV IR in fibers in all laminae and interlaminar zones. Particularly dense staining was noted in layers 1 and 2 that contain many stained fibers from optic tract. Neuronal cell body stained with PV was concentrated only in the laminae. In these laminae staining was moderate in cell bodies of all large and medium-sized neurons, and was strong in cell bodies of some small neurons together with their processes. Calbindin IR was marked in the neuronal cell body and neuropil in the S layers and interlaminar zones whereas moderate in the neuropil throughout the nucleus. Regional difference in distribution of PV and calbindin IR cell is distinct; the former is only in the laminae and the latter in both the S layer and interlaminar space. The CaBP-IR elements were confined to about $10{\mu}m$ in depth of Vibratome section. The IR product for CaBP was mainly associated with synaptic vesicle, pre- and post-synaptic membrane, and outer mitochondrial membrane and along microtubule. PV-IR was noted in various neuronal elements such as neuronal soma, dendrite, RLP, F, PSD and some myelinated or unmyelinated axons, and was not seen in the RSD and glial cells. Only a few neuronal components in dLGN was IR for calbindin and its reaction product was less dense than that of PV, and scattered throughout cytoplasm of soma of some relay neurons, and was also persent in some dendrite, myelinated axons and RLP. The RSD, F, PSD and glial elements were always non-IR for calbindin. Calbindin labelled RLP were presynaptic to unlabeled dendrite or dendritic spine and PSD. Calbindin-labeled dendrite of various sizes were always postsynaptic to unlabeled RSD, RLP or F. From this study it is suggested that dLGN cells of different functional systems and their differential projection to the visual cortex can be distinguished by differential expression of PV and calbindin.

• Molecules and Cells
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• v.21 no.1
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• pp.34-41
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• 2006

The neuronal localization of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor (GluR) subunits is vital as they play key roles in the regulation of calcium permeability. We have examined the distribution of the calcium permeable AMPA glutamate receptor subunit GluR1 in the mouse visual cortex immunocytochemically. We compared this distribution to that of the calcium-binding proteins calbindin D28K, calretinin, and parvalbumin, and of GABA. The highest density of GluR1-immunoreactive (IR) neurons was found in layers II/III. Enucleation appeared to have no effect on the distribution of GluR1-IR neurons. The labeled neurons varied in morphology; the majority were round or oval and no pyramidal cells were labeled by the antibody. Two-color immunofluorescence revealed that 26.27%, 10.65%, and 40.31% of the GluR1-IR cells also contained, respectively, calbindin D28K, calretinin, and parvalbumin. 20.74% of the GluR1-IR neurons also expressed GABA. These results indicate that many neurons that express calcium-permeable GluR1 also express calcium binding proteins. They also demonstrate that one fifth of the GluR1-IR neurons in the mouse visual cortex are GABAergic interneurons.

• Asian-Australasian Journal of Animal Sciences
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• v.9 no.1
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• pp.37-44
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• 1996

The present study was conducted to investigate the role of thyroid hormones in the regulation of gene expression of calbindin-$D_{28k}$ (CaBP-D28K) in the chicken. By employing slot blot and RIA analyses, levels of CABP-D28K mRNA and CaBP-D28K protein in the intestine, kidney, cerebellum and liver were measured 6 and 12 h after i.m. injection of 1, 25-dihydroxyvitamin $D_3$ [1, 25 $(OH)_2D_3$; 250 ng/chick] and 3, 5, 3'-triiodothyronine ($T_3$; 500 ng/chick) in one-day-old chicks. The abundant messages of CaBP-D28K mRNA were detected in the intestine, kidney and cerebellum while there was little message in the liver. After 1, 25 $(OH)_2D_3$ treatment (6 + 12 hours), levels of CaBP-D28K mRNA increased in the intestine, but there was no change in the mRNA levels in the kidney and cerebellum. Although $T_3$ alone had no effect on CaBP-D28K mRNA levels, simultaneous administration of $T_3$ enhanced the 1, 25 $(OH)_2D_3$ effect of levels of CaBP-D28K mRNA in the intestine both 6 and 12 h post-treatment, and in the kidney 12 h post-treatment. At a protein level, co-treatment with 1, 25 $(OH)_2D_3$ and $T_3$ elicited a significant increase in CaBP-D28K expression in the intestine 12 h post-treatment, as compared to treatment with only 1, 25 $(OH)_2D_3$, whereas no differences were observed in the CaBP-D28K protein levels in the kidney and cerebellum. These results suggest that thyroid hormones may play a synergistic role with 1, 25 $(OH)_2D_3$ for CaBP-D28K gene expression in the intestine and kidney in chicks.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.5
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• pp.373-378
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• 2009

Cerebellar Purkinje cells (PCs) play a crucial role in motor functions and their progressive degeneration is closely associated with spinocerebellar ataxias. Although immunohistochemical (IHC) analysis can provide a valuable tool for understanding the pathophysiology of PC disorders, the method validation of IHC analysis with cerebellar tissue specimens is unclear. Here we present an optimized and validated IHC method using antibodies to calbindin D28k, a specific PC marker in the cerebellum. To achieve the desired sensitivity, specificity, and reproducibility, we modified IHC analysis procedures for cerebellar tissues. We found that the sensitivity of staining varies depending on the commercial source of primary antibody. In addition, we showed that a biotin-free signal amplification method using a horseradish peroxidase polymer-conjugated secondary antibody increases both the sensitivity and specificity of ICH analysis. Furthermore, we demonstrated that dye filtration using a $0.22\;{\mu}m$ filter eliminates or minimizes nonspecific staining while preserving the analytical sensitivity. These results suggest that our protocol can be adapted for future investigations aiming to understand the pathophysiology of cerebellar PC disorders and to evaluate the efficacy of therapeutic strategies for treating' these diseases.

• 한국전자현미경학회:학술대회논문집
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• 1997.05a
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• pp.44-44
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• 1997

• Proceedings of the Korean Society of Life Science Conference
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• 2007.10a
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• pp.57.1-57.1
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• 2007

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• Applied Microscopy
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• v.41 no.4
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• pp.235-248
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• 2011

Traumatic brain injury (TBI) is one of the leading causes of death and disability in children and adults and is a major risk factor for the development of posttraumatic epilepsy (PTE). Recent studies have provided significant insight into the pathophysiological mechanisms underlying the development of epilepsy. Although the link between brain trauma and epilepsy is well recognized, the complex biological mechanisms that result in PTE following TBI have not been fully elucidated. Therefore, this study investigated in order to identify whether or not the abnormal expression of calcium-binding proteins in the lesioned hippocampus plays a role in neuronal damage by brain trauma and whether or not the expressions may change in the contralateral hippocampus during the adaptive stage as early time point following TBI. During early time point following TBI, both parvalbumin (PV) and calbindin D-28k (CB) immunoreactivities were decreased with in the lesioned hippocampus. However, these expressions were recovered to control levels as depend on time courses. On the other hand, PV immunoreactivity in contralateral hippocampus was transiently reduced as compared to the control levels, whereas CB expression was unchanged. These findings indicate that the alterations of the calcium-binding proteins, especially PV and CB, may contribute to the neuronal death and/or damage induced by abnormal inhibitory neurotransmission at early time period following brain trauma and the development of epileptogenesis in patients with traumatic brain injury.