Immunohistochemical distribution of calcium binding proteins in the cochlear nuclear complex of circling mice / Distribución inmunohistoquímica de proteínas de unión a calcio en el complejo nuclear coclear de ratones circulantes

SUMMARY: The term "circling mouse" refers to an animal model of deafness, in which the mouse exhibits circling, head tossing, and hyperactivity, with pathological features including degenerated spiral ganglion cells in the cochlea, and the loss of the organ of Corti. The cochlear nuclear (CN) complex, a part of the auditory brain circuit, is essential to process both ascending and descending auditory information. Considering calcium's (Ca2+) importance in homeostasis of numerous biological processes, hearing loss by cochlear damage, either by ablation or genetic defect, could cause changes in the Ca2+ concentration that might trigger functional and structural alterations in the auditory circuit. However, little is known about the correlation of the central nervous system (CNS) pathology in circling mice, especially of the auditory pathway circuit and Ca2+ changes. This present study investigates the distribution of Ca2+- binding proteins (CaBPs), calbindin D-28k (CB), parvalbumin (PV), and calretinin (CR) by using a free floating immunohistochemical method inthe CN of the wild-type mouse (+/+), the heterozygous mouse (+/cir), and the homozygous (cir/cir) mouse. CaBPs are well known to be an important factor that regulates Ca2+ concentrations. Compared with the dorsal and ventral cochlear nuclei of +/+ and +/ cirmice, prominent decreases of CaBPs' immunoreactivity (IR) in cir/cirmice were observed in the somas, as well as in the neuropil. The present study reportson the overall distribution and changes in the immunoreactivity of CaBPs in the CN of cir/cirmice because ofa hearing defect. This data might be helpful to morphologically elucidate CNS disorders and their relation to CaBPs immunoreactivity related to hearing defects.

RESUMEN: El término "ratón circulante" se refiere a un modelo animal con sordera, en el que el ratón exhibe hiperactividad, movimientos circulares y movimientos de la cabeza, con características patológicas que incluyen células ganglionares espirales degeneradas en la cóclea, un canal de Rosenthal vacío y la pérdida del órgano de Corti. El complejo nuclear coclear (CN), una parte del circuito cerebral auditivo, es esencial para procesar la información auditiva tanto ascendente como descendente. Considerando la importancia del calcio (Ca2+) en la homeostasis de numerosos procesos biológicos, la hipoacusia por daño coclear, por ablación o por defecto genético, podría provocar cambios en la concentración de Ca2+que pueden desencadenar alteraciones funcionales y estructurales en el circuitoauditivo. Sin embargo, existe poca información de la correlación de la patología del sistema nervioso central (SNC) en ratones circulantes, especialmente del circuito de la víaauditiva y los cambios de Ca2+. Este estudio nvestiga la distribución de proteínas de unión a Ca2+ (CaBP), calbindina D-28k (CB), parvalbúmina (PV) y calretinina (CR) mediante el uso de un método inmunohistoquímico de flotaciónlibre en el CN del ratón de tiposalvaje (+/+), el ratón heterocigoto (+/cir) y el ratón homocigoto (cir/cir). Se sabe que los CaBP son un factor importante que regula las concentraciones de Ca2+. En comparación con los núcleos cocleares dorsal y ventral de los ratones +/+ y +/ cir, se observaron disminuciones prominentes de la inmunorreactividad (IR) de CaBPs en los ratonescir/cir en los somas, asícomo en el neuropilo. El presente estudio informa sobre la distribución general y los cambios en la inmunorreactividad de CaBP en el CN de ratones cir/cir debido a un defecto auditivo. Estos datos podrían ser útiles para dilucidar morfológicamente los trastornos del SNC y su relación con la inmunorreactividad de CaBP relacionada con los defectosauditivos.

Effect of periapical inflammation on calcium binding proteins and ERK in the trigeminal nucleus / Efecto de la inflamación periapical sobre las proteínas fijadoras de calcio y ERK en el núcleo trigeminal

Peripheral inflammation induces plastic changes in neurons and glia which are regulated by free calcium and calcium binding proteins (CaBP). One of the mechanisms associated with the regulation of intracellular calcium is linked to ERK (Extracellular Signal-Regulated Kinase) and its phosphorylated condition (pERK). ERK phosphorylation is important for intracellular signal transduction and participates in regulating neuroplasticity and inflammatory responses. The aim of this study is to analyse the expression of two CaBPs and pERK in astrocytes and neurons in rat trigeminal subnucleus caudalis (Vc) after experimental periapical inflammation on the left mandibular first molar. At seven days post-treatment, the periapical inflammatory stimulus induces an increase in pERK expression both in S100b positive astrocytes and Calbindin D28k positive neurons, in the ipsilateral Vc with respect to the contralateral side and control group. pERK was observed coexpressing with S100b in astrocytes and in fusiform Calbindin D28k neurons in lamina I. These results could indicate that neural plasticity and pain sensitization could be maintained by ERK activation in projection neurons at 7 days after the periapical inflammation.

La inflamación periférica induce cambios plásticos en las neuronas y en la glía, los cuales están regulados por el calcio libre y las proteínas fijadoras calcio (CaBP). Uno de los mecanismos asociados con la regulación del calcio intrace-lular está vinculado con la fosforilación de la pro teína quinasa ERK. Asimismo, ERK fosforilado es importante para la trans-ducción de señales intracelulares y participa en la regulación de la neuroplasticidad y las respuestas inflamatorias. El objetivo de este estudio es analizar la expresión de dos CaBPs y pERK en astrocitos y neuronas del subnúcleo caudal del trigémino (Vc) después de una inflamación periapical experimental en el primer molar inferior izquierdo en ratas. A los siete días posteriores al tratamiento, el estímulo inflamatorio periapical induce un aumento en la expresión de pERK, en el número de astrocitos positivos para la proteína marcadora astroglial S100b y en neuronas positivas para Calbindina D28k, en el Vc ipsilateral respecto del lado contralateral y el grupo de control. Además, se observó coexpresión de pERK tanto en astrocitos S100b positivos, como en neuronas fusiformes Calbindin D28k positivas, de la lámina I. Estas observaciones podrían indicar que la neuroplasticidad y la sensibilización al dolor podrían mantenerse mediante la activación de ERK en las neuronas de proyección a los 7 días de la inflamación periapical.

The distribution of calbindin-D28k, parvalbumin, and calretinin immunoreactivity in the inferior colliculus of circling mouse / 대한해부학회지

The circling mice with tmie gene mutation are known as an animal deafness model, which showed hyperactive circling movement. Recently, the reinvestigation of circling mouse was performed to check the inner ear pathology as a main lesion of early hearing loss. In this trial, the inner ear organs were not so damaged to cause the hearing deficit of circling (cir/cir) mouse at 18 postnatal day (P18) though auditory brainstem response data indicated hearing loss of cir/cir mice at P18. Thus, another mechanism may be correlated with the early hearing loss of cir/cir mice at P18. Hearing loss in the early life can disrupt the ascending and descending information to inferior colliculus (IC) as integration site. There were many reports that hearing loss could result in the changes in Ca²⁺ concentration by either cochlear ablation or genetic defect. However, little was known to be reported about the correlation between the pathology of IC and Ca²⁺ changes in circling mice. Therefore, the present study investigated the distribution of calcium-binding proteins (CaBPs), calbindin-D28k, parvalbumin, and calretinin immunoreactivity (IR) in the IC to compare among wild-type (+/+), heterozygous (+/cir), and homozygous (cir/cir) mice by immunohistochemistry. The decreases of CaBPs IR in cir/cir were statistically significant in the neurons as well as neuropil of IC. Thus, this study proposed overall distributional alteration of CaBPs IR in the IC caused by early hearing defect and might be helpful to elucidate the pathology of central auditory disorder related with Ca²⁺ metabolism.

Calbindin-D28K Prevents Staurosporin-induced Bax Cleavage and Membrane Permeabilization

Calbindin-D28K has been implicated in the regulation of neuronal cell death. Previously, we demonstrated that calbindin-D28K prevents staurosporine (STS)-induced caspase activation and subsequent apoptosis in a neuronal cell line. However, the role of calbindin-D28K in STS-induced activation of calpain and necrotic cell death was not identified. Staurosporine induced the elevation of intracellular calcium after 1 hr of treatment. Overexpression of calbindin-D28K and presence of a calcium chelator, BAPTA, prevented the increase of calcium in STS-treated cells. Cleavage of Bax by calpain was prevented by the overexpressed calbindin-D28K. Permeabilization of the plasma membrane, a factor in necrosis, as well as apoptotic change of the nucleolus induced by STS, was prevented by calbindin-D28K. Thus, our study suggests that calbindin-D28K may exert its protective functions by preventing calpain activation in necrotic cell death, in addition to its effect on the caspase-apoptosis pathway.

Time-dependent changes of calbindin D-28K and parvalbumin immunoreactivity in the hippocampus of rats with streptozotocin-induced type 1 diabetes

The hippocampus is affected by various stimuli that include hyperglycemia, depression, and ischemia. Calcium-binding proteins (CaBPs) have protective roles in the response to such stimuli. However, little is known about the expression of CaBPs under diabetic conditions. This study was conducted to examine alterations in the physiological parameters with type 1 diabetes induced with streptozotocin (STZ) as well as time-dependent changes in the expression of two CaBPs changes of were being evaluated. Rats treated with STZ (70 mg/kg) had high blood glucose levels (>21.4 mmol/L) along with increased food intake and water consumption volumes compared to the sham controls. In contrast, body weight of the animals treated with STZ was significantly reduced compared to the sham group. CB-specific immunoreactivity was generally increased in the hippocampal CA1 region and granule cell layer of the dentate gyrus (DG) 2 weeks after STZ treatment, but decreased thereafter in these regions. In contrast, the number of PV-immunoreactive neurons and fibers was unchanged in the hippocampus and DG 2 weeks after STZ treatment. However, this number subsequently decreased over time. These results suggest that CB and PV expression is lowest 3 weeks after STZ administration, and these deficits lead to disturbances in calcium homeostasis.

Immunohistochemical study on the expression of calcium binding proteins (calbindin-D28k, calretinin, and parvalbumin) in the cerebellum of the nNOS knock-out(-/-) mice / 대한해부학회지

Nitric Oxide (NO) actively participates in the regulation of neuronal intracellular Ca2+ levels by modulating the activity of various channels and receptors. To test the possibility that modulation of Ca2+ buffer protein expression level by NO participates in this regulatory effect, we examined expression of calbindin-D28k, calretinin, and parvalbumin in the cerebellum of neuronal NO synthase knock-out (nNOS(-/-)) mice using immunohistochemistry. We observed that in the cerebellar cortex of the nNOS(-/-) mice, expression of calbindin-D28k and parvalbumin were significantly increased while expression of calretinin was significantly decreased. These results suggest another mechanism by which NO can participate in the regulation of Ca2+ homeostasis.

Effect of Electroacupuncture on the Expression of Hippocampal Calbindin-D28K in Hyperlipemia Rats with Concurrent Cerebral Ischemia / 针刺研究

Objective To observe the effect of electroacupuncture(EA) on Calbindin D28K(CB) expression and apoptosis in hippocampus in hyperlipemia rats with concomitant cerebral ischemia(CI).Methods Forty male SD rats were rando-mized into control,hyperlipemia+CI(model),acupuncture Ⅰ,and acupuncture Ⅱ groups,with 10 cases in each.Hyperlipemia model was established by feeding the animals with high fat forage for 6 weeks and CI model established by occlusion of the unila-teral middle cerebral artery.For rats of acupuncture Ⅰ group,EA(1-3 mA,15 Hz) was applied to bilateral "Sanyinjiao"(SP 6) and "Fenglong"(ST 40) for 20 min every time,once daily for 10 days before CI.For rats of both acupuncture Ⅰ and Ⅱ groups,after CI,EA was applied to SP 6,ST 40,"Baihui"(GV 20) and "Shuigou"(GV 26) were punctured with filiform needles and stimulated by twirled the needle with hand continuously for 1 min,once daily for 7 days.The apoptotic cells of hippocampal CA 3 area were displayed by TUNEL(terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling) method.CB antibody staining with immunohistochemistry was used to show CB-positive cells which were observed by optic microscope.Results In comparison with control group,the percentage of apoptotic cells of the hippocampal CA 3 area in model group increased considerably(P

The Effect of 835 MHz Radiofrequency Radiation Exposure on the Immunohistochemical Distribution of Calbindin D28k and Calretinin in the Mouse Cerebellar Cortex / 체질인류학회지

Widespread use of mobile phones and subsequent electromagnetic field (EMF) exposure have raised crucial question of their possible biological effects on the nervous system. The study on the effect of radiofrequency(RF) radiation on the nervous system, however, did not precede enough to determine the biological hazard to brain. Until now, several studies have reported decreases in neuron number and neuronal damage in the cortex, hippocampus, and basal ganglia in the brains of animals exposed to RF radiation. However, there were few reports about the cerebellum, the main voluntary motor control center. In this regard, by using immunohistochemisty, current study intended to investigate the changes in the calbindin D28k (CB) and calretinin (CR)-immunoreactivity (IR) in the mouse cerebellar cortex after EMF exposure at 835 MHz for different exposure times and absorption rates, 1 h/day for 5 days at 1.6 W/kg, 1 h/day for 5 days at 4.0 W/kg, 5 h/day for 1 day at 1.6 W/kg, 5 h/day for 1 day at 4.0 W/kg, daily exposure for one month at 1.6 W/kg. Among groups, most prominent CB IR was observed in the Purkinje cell layer followed by molecular and granular layer. The highest CB IR was noted in 5 h/day for 1 day at 1.6 W/kg in the entire three layers while the lowest was noted in one month at 1.6 W/kg. Similarly CR IR was maximum in one month at 1.6 W/kg whilst the lowest was observed in 1 h/day for 5 days at 4.0 W/kg. EMF exposure for 5 days at 1.6 W/kg reduced CB-IR. The CR-IR was mainly localized in small cells in the granular layer, with maximum IR observed after one month exposure. Therefore, the present study suggest the possibility of alterations of calcium ion concentration, which play a role in maintaining metabolic homeostasis, in the cerebellum after long-term exposure to 835 MHz of RF radiation, which might lead to thedisruption of normal trait.

Optimized Immunohistochemical Analysis of Cerebellar Purkinje Cells Using a Specific Biomarker, Calbindin D28k

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 micrometer 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.

Changes in Calcium-binding Proteins in the Rat Hippocampus after Involuntary Treadmill Exercise / 대한해부학회지

In this study, we investigated the effects of treadmill exercise on hippocampal levels of calcium-binding proteins - calbindin D-28k (CB), calretinin (CR) and parvalbumin (PV) - using immunohistochemistry and Western blot analysis. At 6 weeks of age, male Wistar rats were put on a treadmill with or without running for 1 h/day/5 consecutive days at a pace of 22 m/min for a period of 5 weeks. In sedentary and exercise groups, CB immunoreaction was detected in granule cells of the dentate gyrus, mossy fibers, and CA1 pyramidal cells. In addition, CB immunoreaction was observed in interneurons of the CA1-3 region. Exercise significantly increased CB immunoreactivity in dentate granule cells, CA1 pyramidal cells and CA1-3 interneurons. CR immunoreaction was mainly observed in interneurons of the dentate gyrus and CA1-3 regions. Similar number of CR-immunoreactive neurons was observed in the exercise and sedentary groups. PV immunoreaction was detected in interneurons of the dentate gyrus and CA1-3 regions. PVimmunoreactive fibers were significantly increased in all regions of the hippocampus in the exercise group, as compared to the sedentary group. Similar to the immunohistochemical findings, protein levels of CB and PV were also increased in the exercise group compared to the sedentary group. These increases in CB and PV in the hippocampus may induce neuronal plasticity after treadmill exercise and may be related to the enhancement of synaptic plasticity in the hippocampus by exercise.

Expression of Calbindin-D28K in Ethanol-Induced Brain Damage in Rats Cerebellum and Effect of Anisodamine on It / 中国康复理论与实践

@#Objective To investigate the effect of anisodamine on calbindin-D28K(CaBP) expression in the ethanol-induced brain damage in rat cerebellum.Methods2 months aged male Sprague-Dawley rats were injected intraperitoeally with ethanol,normal saline,saline+anisodamine and ethanol+anisodamine respectively for 8 d.They were evaluated with Morris water maze.The counts,average area and density of CaBP positive neurons in cerebellum were measured with immunohistochemical technique and image analytical system.Results The latency of Morris water maze was significantly longer in the ethanol group than in the others(P<0.05),while the distance was significantly longer in the ethanol group than in the saline group and saline+anisodamine group(P<0.05).There is not significant difference between ethanol group and ethanol+anisodamine group(P>0.05),but is seemed some longer.The counts,average area and density of CaBP positive Purkinje cell were all significantly less in ethanol group than in the others(P<0.05).There Pwas not significant difference among ethanol+anisodamine group,saline group and saline+anisodamine group(P>0.05) in the counts,but the average areas and density in ethanol+anisodamine group were less than those in saline group and saline+anisodomine group(P<0.05).Conclusion The ethanol can reduce the CaBP expression in the Purkinje cells of the rats cerebellum.Anisodamine can protect the rats cerebellum from it.

Immunocytochemical Study of Calbindin D(28k) Positive Cells in the Developing Rat Kidney / 대한해부학회지

Calbindin D(28k),a calcium binding protein,is found in various tissues,including some cells in the distal nephron.It plays an important role in the regulation of calcium reabsorption. We previously reported the expression of calbindin D(28k) in adult rat kidney.However,the exact time of expression during differentiation in the embryonic kidney is not known.During development,intercalated cells are deleted from the medullary collecting duct by two distinct mechanisms.However,the reason for the different modes of cell death is not known.As calbindin is reported to protect cells against apoptosis,we examined the expression of calbindin D(28k) in the developing rat kidney.Kidneys from 16-,17-,18-and 20-day-old fetuses and 1-,3-,5-,7-,14-and 21-day-old pups and adult Sprague awley rats were processed for immunohistochemistry using a monoclonal antibody against calbindin D(28k) .Intercalated cells were identified by immunostaining for H+ -ATPase and by electron microscopy.Calbindin D(28k) immunoreactivity first appeared in subpopulations of cells in the connecting tubule and medullary collecting duct in the 17-day-old fetus.In the connecting tubule,calbindin D(28k) was expressed only in H+ -ATPase negative connecting tubule cells,and there was no labeling of intercalated cells.In the medullary collecting duct,calbindin D(28k) immunostaining was observed in a few cells with apical H+ -ATPase,characteristic of type A intercalated cells.The numbers of calbindin D(28k) -positive type A intercalated cells increased from day 18 of gestation.In contrast,there was little or no calbindin D(28k) immunoreactivity in the type B intercalated cells or principal cells.During the first two weeks after birth,calbindin D(28k) -positive type A intercalated cells were lost from the terminal part of the medullary collecting duct by simple extrusion. After two weeks,calbindin D(28k) immunostaining decreased in the type A intercalated cells throughout the medullary collecting duct.However,the immunoreactivity of calbindin D(28k) in the cortical collecting duct was increased in some of the type A intercalated cells and the adult pattern was observed in 21-day-old pups.Thus,we propose that the different expression of calbindin D(28k) in type A and type B intercalated cells may be responsible-at least partly-for the different modes of cell death demonstrated in these cells during kidney development.

Immunohistochemical localization of calcium binding proteins and some neurotransmitters in myenteric plexus of goat stomach

To understand the neurochemical properties of the gastric myenteric plexus of ruminants, the expression patterns of calbindin D-28k (CB), calretinin (CR), substance P (SP) and calcitonin gene-related peptide (CGRP) were explored in the Korean native goat. In gastric myenteric plexus, CB and SP immunoreactivity were observed in round- or ovalshaped neurons. CR and CGRP immunoreactivity were detected only in the nerve fibers. This immunohistochemical localization of CB, CR, CGRP and SP in the myenteric plexus of the goat stomach exhibited species-specific patterns. These findings suggest that these substances may be directly or indirectly related to the gastric functions of the goat stomach.

Morphological Studies on the Calbindin D-28K and Parvalbumin Immunoreactive Neurons in the Medulla Oblongata and Ventral Horn of the Spinal Cord Gray Matter after Spinal Cord Injury in Rats / 체질인류학회지

This study was examined and compared the immunocytochemical distribution of the two calcium-binding proteins calbindin D-28K and parvalbumin immunreactive neurons in the medulla oblongata and spinal cord after transection of spinal cord in rats. In this experiment, calbindin D-28K immnunoreactive neurons were mainly found in many pyramidal cells distributed medulla oblongata and spina1 cord of rats. Parvalbumin immunoreactive cells were demonstrated in all lamina of the gray matter of the spinal cord. These immunoreactive cells had the most high density in the severa1 nuclei of the ventra1 horn of the all segments of the spina1 cord. Calbindin D-28K neuropil labeling was strongly noted in spina1 all segments of the spinal cord. In contrast parva1bumin immunoreactive, little differences were found in distribution, size and morphology of calbindin D-28K cell body or neuropil staining in the spinal cord. The number of parvalbumin immunoreactive cells were more than twice in the medulla oblongata and spinal cord compared to the calbindin D-28K immunoreactive cells. Calbindin D-28K and parvalbumin-immmoreactive somata were round, ova1, spind1e and polygona1 in shape, and the immunoreactive neurons were unipolar, bipolar, multipolar and horizontal in shape. The diameters of the somata of the two immunoreactive neurons were 40 ~50 micrometer, respectively. Also dendrites of two immunoreactive neurons were densely arrayed in network. These results suggest that CB-IR and PV-IR most high density in the of the VII~X layers in the ventra1 horn of the all segments of the spina1 cord.

Immunohistochemical Studies on the Calbindin D -28K and Parvalbumin Positive Neurons in the Brain Stem and Spinal Cord after Transection of Spinal Cord of Rats / 체질인류학회지

This studies were examined and compared the immunohistochemical distribution of the two calcium -binding proteins calbindin D -28K and parvalbumin positive neurons in the brain stem and spinal cord after transection of spinal cord in rats. In this experiment, calbindin D -28K immunoreactive neurons were mainly found in many pyramidal cells distributed in the brain stem and spinal cord of rats. Calbindin D -28K neuropil labeling was strongly noted in brain stem and in spinal all segments of the spinal cord. In contrast to parvalbumin, little differences were found in distribution, size and morphology of calbindin D -28K cell body or neuropil staining in the brain stem and spinal cord. Parvalbumin immunoreactive cells were demonstrated in all lamina of the gray matter of the spinal cord. These immunoreactive cells had the most high density in the layer I and II dorsal horn and several nuclei of the ventral horn of the all segments of the spinal cord. These immunoreactive cells between the brain stem and spinal cord were quite different markedly in number, cell size and morphology The number of parvalbumin positive cells were more than twice in the brain stem and spinal cord compared to the calbindin D -28K positive cells. Calbindin D -28K and parvalbumin -immunoreactive somata were round, oval, spindle and polygonal in shape, and the positive neurons were unipolar, bipolar, multipolar and horizontal in shape. The diameters of the somata of the two positive neurons were 30 ~40 micrometer, respectively. Also dendrites of two positive neurons were densely arrayed in arborization.

Changes of Calbindin -D28k Expression Levels After Transient Ischemic Damage in Rat Cerebellar Purkinje Cells / 체질인류학회지

Cerebellar Purkinje cells are selectively vulnerable to ischemia, although the reasons for this are not known. Moreover, an intracellular Ca 2 +/-overload induced by excitotoxicity (toxic glutamate receptor activation) is considered to be a key mediator of central neuronal loss consequent to ischemic damage. Calbindin -D28k is an intracellular calcium binding protein that is expressed in nearly all Purkinje cells of the rat cerebellum. Its major role is presumed to be associated with intracellular Ca 2 +/-buffering. In the present work, In -Situ Hybridization and Western blot methods were used to investigate the changes of calbindin -D28k and its gene expression levels in the rat cerebellum at various times after transient global ischemia. Both level of calbindin -D28k and its mRNA expression level in the cerebellum decreased after ischemic insult, whereas the number of cerebellar Purkinje cells was unaltered after ischemia. In the light of our finding of lower levels of calbindin -D28k and its mRNA in the cerebellum, altered intracellular calcium buffering capacity in the cerebellar Purkinje cell might be presumed. It is believed that this may lead to calcium - mediated cytotoxic events after ischemic insults in cerebellum.

PROJECTIONS OF CALBINDIN D-28K-LIKE,SUBSTANCE P RECEPTOR AND Fos-POSITIVE NEURONS FROM THE LUMBEAR SPINAL CORD TO THE LATERAL PARABRACHIAL NUCLEUS OF THE RAT / 解剖学报

Objective To observe the relationships among the direct projection neurons from the lumbar spinal cord to the lateral parabrachial nucleus(LPB),calbindin D-28K(CB)-like immunoreactive (-LI) neurons,peripheral noxious information transmission as well as substance P receptor(SPR)-LI neurons. Methods Triple-labeled techniques were used by tetramethyl rhodamine(TMR) retrograde tracing combined with immunofluorescence histochemistry for CB-,SPR- or Fos protein.The stained sections were observed under a confocal laser-scanning microscope. Results 1.After injecting TMR to the unilateral LPB,a number of TMR retrogradely labeled neurons were mainly distributed in the lamina Ⅰ,lateral spinal nucleus(LSN),and regions around the central canal of the spinal cord(lamina X) of the ipsilateral spinal cord;2.CB-LI neurons were mainly found in the laminae Ⅰ and Ⅱ of the lumbar spinal cord,especially in the lamina Ⅱ in the dense distribution; 3.SPR-LI neurons were also mainly seen in the lamina Ⅰ,LSN and lamina X of the spinal cord.A few of the SPR-LI neurons were also distributed in the lamina Ⅱ;4.Fos-positive neurons were detected in the laminae Ⅰ and Ⅱ,lateral aspect of the lamina Ⅴ to Ⅶ of the lumbar spinal cord by injecting 5% formalin into the ipsilateral hindpaw;5.Triple-labeled neurons for TMR/CB/SPR or TMR/CB/Fos were mainly found in lamina Ⅰ,while a few of the triple-labeled neurons were also found in lamina Ⅱ of the dorsal horn.TMR/CB/SPR triple-labeled neurons accounted for 103%,98% and 146% of total population of TMR-,CB- or SPR-LI neurons in laminae Ⅰ and Ⅱ,respectively.On the other hand,TMR/CB/Fos triple-labeled neurons formed 118%,106% and 158% of the total population of TMR-,CB-LI or Fos-positive neurons in the laminae Ⅰ and Ⅱ,respectively.Conclusion\ The results indicated that in the laminae Ⅰ and Ⅱ of the lumbar spinal cord some neurons with CB-Like immunoreactivity transmitting the peripheral noxious information and projecting directly to the LPB might receive SPergic primary afferents.

Postnatal Development of Parvalbumin and Calbindin D-28k Immunoreactivities in the Canine Anterior Cingulate Cortex: Transient Expression in Layer V Pyramidal Cells / 대한해부학회지

We have examined the ontogeny of parvalbumin and calbindin D-28k immunoreactivities in the canine anterior cingulate cortex from the day of birth (P0) through P180. At P7, parvalbumin immunoreactivity appears firstly in layer VI multipolar cells. The parvalbumin immunoreactivity in GABAergic interneurons appears to follow an 'inside-out' gradient of radial mergence and reaches an adult-like pattern by the end of the 6th postnatal month. Immunoreactivity is limited mainly to developing nonpyramidal cells, whereas pyramid-like parvalbumin immunoreactive cells are transiently observed in layer V from the P14 to the P90. The developmental pattern of calbindin D-28k immunoreactivity differs from that of parvalbumin immunoreactivity. Calbindin D-28k immunoreactivity develops firstly in layer V pyramidal cells from P0, which continues through the third postnatal month. Calbindin D-28k immunoreactive interneurons are located in the infragranular layers and white matter at P0 and increase in both the supragranular and infragranular layers by P14. This is followed by an adult-like pattern at the P180. These data suggested that parvalbumin and calbindin D-28k may play a role in protecting immature neurons from intracelluar calcium influx during postnatal development.

Chronological Alteration of Calbindin D-28k and Calretinin Immunoreactivity in the Gerbil Trigeminal Ganglion After Ischemia-Reperfusion / 대한해부학회지

Many researches have focused upon temporal changes of neurotransmitters and/or neuromodulators in the central nervous system after ischemic insult. In sensory neurons, the spatial and temporal alterations of neurotransmitters have been little studied. Calbindin D-28k (CB) and calretinin (CR) have been suggested to play a role in the transmission of neurotransmitters. Therefore, in the present study we investigated the chronological alteration of CB and CR immunoreactivity in the trigeminal ganglion cells of the Mongolian gerbil after ischemic insult. In the sham operated group, CB and CR immunoreactivities were found in small -, medium -and large -sized neurons. One and two days after ischemia-reperfusion, small and large-sized CB immunoreactive neurons increased significantly. Thereafter, number of the CB immunoreactive neurons decreased markedly. Furthermore, five days after ischemia -reperfusion, CB immunoreactivity was detected in a few neurons, and its immunoreactivity was also very weak in the cytoplasm. Number of the large -sized CR immunoreactive neurons increased significantly one day after ischemia -reperfusion. Thereafter, the number of the large -sized CR immunoreactive neurons decreased. Especially, the number of the medium-sized CR immunoreactive neurons increased dramatically 4 days after ischemia-reperfusion. These results suggest that an increase of CB and CR may play an important role in modulating the mechanoception 1 day after ischemia-reperfusion, because the immunoreactivities increased in large -sized neurons which have the myenlinated A fibers. These results also suggest that significant increase of CR expression in medium -sized neurons 4 and 5 days after ischemia-reperfusion may provoke CR in modulating the nociception or thermoception because the medium-sized neurons which have the myenlinated A sigma or C fibers.

Immunohistochemical Study on Parvalbumin and Calbindin D-28k Immunoreactive Neurons in the Dog Olfactory Bulb / 대한해부학회지

We have studied the distribution of calcium-binding proteins parvalbumin (PV -IR) and calbindin D-28k (CB -IR) immunoacitivity in the mongrel dog olfactory bulb using monoclonal antibodies and the avidin -biotin -immunoperoxidase method. The possible coexistence of both markers was determined by segmental histochemical and immunohistochemical double labelling of the same section. In the olfactory bulb of mongrel dog, PV-IR and CB-IR were mainly located in the external plexiform layer, and a few scattered in the glomerular layer, mitral cell layer, and glanule cell layer in the order of thier existence. In addition, three neuronal types were observed in the glomerular layer and external plexiform layer border. Horizontal cells and vertical cells of Cajal were also observed after both PV-IR and CB-IR labeling. Distict groups of PV-IR and CB-IR, differing in size, shape, dendritic branching pattern, and staining intensity, were distinguished in the all layers of the olfactory bulb. Specific neuronal populations were positive for both PV-IR and CB-IR markes. No cell colocalized both stains in the mongrel dog olfactory bulb. The number of PV-IR were more than abundant in olfactory bulb compared to the CB-IR cells. The PV-IR and CB-IR postitive cell somata were round, oval, spindle and polygonal in shape, and they were unipolar, bipolar and multipolar in type. The diameters of the somata of the PV-IR and CB-IR neurons were 20~40 micro meter, respectively. Also dendrites of the these neurons were densely arrayed in arborization.