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.

Distribution of D1 and D2-dopamine receptors in calcium-binding-protein expressing interneurons in rat anterior cingulate cortex / 生理学报

Dopamine plays an important role in cognitive functions including decision making, attention, learning and memory in the anterior cingulate cortex (ACC). However, little is known about dopamine receptors (DAR) expression patterns in ACC neurons, especially GABAergic interneurons. The aim of the present study was to investigate the expression of the most abundant DAR subtypes, D1 receptors (D1Rs) and D2 receptors (D2Rs), in major types of GABAergic interneurons in rat ACC, including parvalbumin (PV)-, calretinin (CR)-, and calbindin D-28k (CB)-containing interneurons. Double immunofluorescence staining and confocal scanning were used to detect protein expression in rat brain sections. The results showed a high proportion of PV-containing interneurons express D1Rs and D2Rs, while a low proportion of CR-positive interneurons express D1Rs and D2Rs. D1R- and D2R-expressing PV interneurons are more prevalently distributed in deep layers than superficial layers of ACC. Moreover, we found the proportion of D2Rs expressed in CR cells is much greater than that of D1Rs. These regional and interneuron type-specific differences of D1Rs and D2Rs indicate functionally distinct roles for dopamine in modulating ACC activities via stimulating D1Rs and D2Rs.

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.

Expression of Calcium Transporters According to Dietary Sodium in the Distal Tubule of Rat Kidneys / 대한신장학회지

PURPOSE: Hypercalciuria is a risk factor of renal calcium stone and proper initial management is dietary salt restriction. But the molecular mechanism responsible for this sodium calcium relationship remains unclear. The present study investigates the relationship between different amount of sodium intake and the expression level of transporters involved in the active calcium transport. METHODS: Sprague-Dawley rats were randomized into three groups: a normal salt group, a low salt group, and a high salt group. Expression of mRNA and protein of transient receptor potential vanilloid (TRPV) 5 and calbindin-D28K was determined by real-time quantitative PCR and western blots, respectively. RESULTS: Hematocrit and body weight showed no difference among the three groups. High salt diet led to significant increase in the amount of urinary calcium excretion and decreased mRNA expression of calbindin-D28K and TRPV5. Protein abundance of calbindin-D28K and TRPV5 was decreased but the result was statistically insignificant. Low salt diet decreased the amount of urinary calcium excretion without significant difference. Messenger RNA expression and protein abundance of calbindin-D28K and TRPV5 showed no difference, compared to those of normal salt group. CONCLUSION: These data suggest that high sodium intake increases urinary calcium excretion, which is accompanied by a decreased expression of calbindin-D28K mRNA.

Expression of calbindin-D28k in human fallopian tubes / 生理学报

The present study was aimed at investigating the expression of calbindin-D28k (CaBP-D28k) in human fallopian tube, which were collected from 33 childbearing age women undergoing abdominal hysterectomy with adnexectomy for benign disease in the pelvic cavity. These women had normal menstrual cycle and history of normal pregnancy. Isthmus, ampullary and umbrella segments of fallopian tubes were respectively collected. These specimens were divided into 6 groups based on their menstrual cycles: early-proliferative stage (n=6), mid-proliferative stage (n=5), late-proliferative stage (n=5), early-secretory stage (n=7), mid-secretory stage (n=5) and late-secretory stage (n=5). The expressions of CaBP-D28k protein and mRNA in fallopian tubes were determined by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) methods. Positive expressions of CaBP-D28k protein and mRNA were observed in human fallopian tubes. There was no significant difference in the expression of CaBP-D28k protein among the isthmus, ampulla and umbrella segments in the same phase of menstrual cycle (P>0.05). However, in the menstrual cycle, the expression level of CaBP-D28k protein in the epithelium was the lowest during the early- and mid-proliferative stages and increased in both the late-proliferative and early-secretory stages (P<0.05), and then decreased in the mid- and late-secretory stages (P<0.05). The expressed CaBP-D28k protein was disposed to gobbets or dispersed sheets in cytoplasm in the early- and mid- proliferative stages, and showed concentrated granules on the top of cells in the late-proliferative and early-, mid-secretory stages. Then in the late-secretory stage redistribution renewed as in the early- and mid-proliferative stages. The CaBP-D28k mRNA obviously increased in the late-proliferative and early-secretory stages (P<0.05). These findings indicate that the expressions of CaBP-D28k protein and mRNA exist in human fallopian tubes and exhibit a cyclic change.

Temporal Change of Calbindin-D28k Immunoreactivity in the Dentate Gyrus of Voluntary Running Mouse / 대한해부학회지

Voluntary running is known to dramatically increase the cell proliferation and neurogenesis in the dentate gyrus of the adult mouse hippocampus. However, it is crucial to realize that adding excitatory neurons could result in serious maladaptive outcomes for hippocampal circuit function. To investigate the response of mature granule cells on the increase of cell proliferation during voluntary running, we investigated the temporal change of calbindin-D28k (a marker for mature granule cells) using immunohistochemistry during voluntary running with upregulated neurogenesis. By using immunohistochemsitry for Ki-67 and doublecortin (DCX), we observed that the cell proliferation and differentiation of granule cells increased at 1 week of voluntary running. We found that, at 6 weeks of voluntary running, the cell proliferation and differentiation of granule cells returned to sedentary control levels. On the other hand, calbindin-D28k immunoreactivity decreased in the granular cell layer of the dentate gyrus and CA3 region of hippocampus after 1 week of voluntary running. At 6 weeks of voluntary running, the density of the calbindin-D28k in the granular cell layer and CA3 region was returned to the sedentary control level. These results demonstrate that the cell proliferation and differentiation are increased at early point of voluntary running, and the granule cell activity in the dentate gyrus is temporally changed for response to the increase of cell proliferation and differentiation during voluntary running.

Co-existence of calcium-binding proteins and gamma-aminobutyric acid or glycine in neurons of the rat medullary dorsal horn / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>We investigated the co-expression of calbindin-D28k (CB), calretinin (CR) and parvalbumin (PV, a combination of the three is referred to as CaBPs) with gamma-aminobutyric acid (GABA) or glycine in neurons of the rat medullary dorsal horn (MDH).</p><p><b>METHODS</b>Immunofluorescence histochemical double-staining for CaBPs and GABA or glycine was performed on the sections from rat MDH.</p><p><b>RESULTS</b>CB-, CR-, PV-, GABA- and glycine-like immunoreactive (LI) neurons were differentially observed in all layers of the MDH, but particularly in lamina II. Neurons that exhibited immunoreactivity for both CaBPs and GABA or glycine were also observed mainly in lamina II. A few of them were found in laminae I and III. The percentages of neurons which co-expressed CB/GABA or CB/glycine out of the total numbers of CB- and GABA-LI neurons or CB- and glycine-LI neurons were 5.3% and 12.1% or 4.1% and 10.0%, respectively. The ratios of CR/GABA or CR/glycine co-existing neurons out of the total numbers of CR- and GABA-LI neurons or CR- and glycine-LI neurons were 5.8% and 7.6% or 4.4% and 7.1%, respectively. The rates of PV/GABA or PV/glycine co-localized neurons out of the total numbers of PV- and GABA-LI neurons or PV- and glycine-LI neurons were 11.1% and 5.1% or 9.9% and 5.1%, respectively.</p><p><b>CONCLUSION</b>The results indicate that some neurons in the MDH contain both CaBPs and GABA or glycine.</p>

Somatic and visceral nociceptive inputs from the orofacial area and the upper alimentary tract converge onto CB-containing neurons in interstitial nucleus of the spinal trigeminal tract in rats / 生理学报

The interstitial nucleus of the spinal trigeminal tract (INV) contains many calbindin-D28k-containing neurons (CB-neurons) receiving convergence information from the somatic and visceral structures. The purpose of the present study was to confirm whether the primary afferent terminals from the inferior alveolar nerve (IAN) make close contact and synaptic connections with the same CB-neurons receiving visceral nociceptive signals in INV. Biotinylated dextran amine (BDA) and horseradish peroxidase (HRP) tracing combined with CB and Fos proteins immunohistochemistry were used. After injections of BDA and formalin into unilateral IAN and upper alimentary tract, respectively, the transganglionic labeled afferent fibers and terminals from IAN were observed in the ipsilateral INV, especially in its enlarged part. A large number of CB- and Fos-like immunoreactive (LI) neurons were found in bilateral INV. These CB- and Fos-LI neurons mostly overlapped with BDA-labeled terminals in the enlarged part of INV. About one half of the CB-LI neurons were double labeled with Fos-LI nuclei (74/153). The terminals from IAN were to made close contacts with many CB/Fos-double labeled or CB-single labeled neurons. After injection of HRP into IAN, HRP-labeled fibers and terminals in INV were similar to that labeled with BDA. Under the electron microscope, a large number of CB-LI dendrites and a few soma in the enlarged part of INV were found to form asymmetrical axo-dendritic and axo-somal synapses with the HRP-labeled axon terminals. These results indicate that the orofacial somatic inputs from IAN and the visceral nociceptive inputs from the upper alimentary tract converge onto the same CB-containing neurons in INV. These CB-containing neurons in INV probably play an important role in information integration as well as visceral and cardiovascular activity.

Expression of an Anion Exchanger Pendrin in Human Kidney / 대한해부학회지

It has been reported that new apical anion exchanger perndrin, encoded by the pendred syndrome (PDS/pds, Slc26A4) gene, was expressed in the AE1-negative intercalated cells of rat and mouse kidneys. The purpose of this study was performed that expression of pendrin in the subtypes of intercalated cells in human kidney. The normal human renal tissues obtained from nephrotomized kidneys for renal cell carcinoma were fixed in periodate-lysine-paraformalde-hyde, and processed for immunohistochemistry. Subtypes of intercalated cells were identified by using antibodies for H(+)-ATPase and AE1, and connecting tubule cells and principal cells of collecting duct were identified using antibodies for calbindin D28K and AQP2, respectively. In human kidney, pendrin was expressed in the apical domain of AE1-negative intercalated cells including type B cells with diffuse and/or basolateal H(+)-ATPase, non A-non B (non -A/B) type intercalated cells with apical H(+)-ATPase and bipolar type of intercalated cells with apical and basolateral H(+)-ATPase. The AQP2-positive principal cells of cortical collecting duct were also had apical pendrin immunoreactivity. However, there was no pendrin immunoreactivity in AE1-positive type A intercalated cells, calbindin D28K-positive connecting tubule cells, and AQP2-positive medullary collecting duct. These results suggest that pendrin is an apical anion exchanger not only in the AE1-negative intercalated cells (type B, non-A/B and bipolar cells) but also in the principal cells of cortical collecting duct, and has an essential role in HCO3-secretion in human kidney.

Seizure -Related Change of NADPH -diaphorase and Calcium Binding Protein Positive Neurons in the Brain of Rats / 체질인류학회지

Nitric oxide (NO) is a gaseous messenger that plays a role in neurotransmission, long term potentiation, depression and cerebral blood flow. Increases in intracellular calcium levels activate the enzyme NOS, and the NO released then diffuse to adjacent cells and activate guanylate cyclase. NO mediates the increase in cerebral blood flow during seizure activity. Therefore, the present study was aimed to investigate the change of NOS and calcium binding proteins in the rat cerebral cortex following seizure. Rats were injected with kainate (KA) and killed at 6 hours, 1, 3, 5 and 10 days after seizure. Expressional change of nNOS, calbindin D28k and parvalbumin was assessed by histochemistry, immunohistochemistry and microdensitometry in the rat brain. The intensity of the NADPH -d staining in rat cortical neurons showed a marked susceptibility to KA administration. At 6 hours and 3 days after seizure, the optical density of the NADPH -d staining was increased relative to the signal in saline treated control rats. At 5 and 10 days after seizure, the optical density of NADPH -d staining was not significantly different in most cortical regions compared to controls. In the hippocampus, the optical density of NADPH -d staining was highest at 5 days after seizure. The optical densities of calbindin D28k and parvalbumin positive neurons were various in the cerebral cortex, hippocampus and caudatoputamen during postseizure period. These results indicate that the calcium binding proteins investigated here are not essential for determining the activation of nNOS/NADPH -d positive neurons in the cerebral cortex and striatum.

Seizure-Related Change of NADPH-diaphorase and Calbindin D28k Positive Neurons in the Cerebral Cortex of Rats / 대한해부학회지

Administration of kainate (KA) results in the induction of epileptiform activity and limbic motor seizures. Nitric oxide (NO) is a gaseous messenger that plays a role in neural transmission, long term potentiation, depression and cerebral blood flow. NO is formed by NO synthase (NOS) from arginine. NO mediates the increase in cerebral blood flow during seizure activity. However, the production site of NO has not been clearly defined. Recent report showed that constitutive NOS may be induced under certain conditions. Therefore, the present study was aimed to investigate the change of NOS and calbindin D28k in the rat cerebral cortex following seizure. Rats were injected with KA and killed at 6 hours, 1, 3, 6 and 12 days after seizure. Expressional change of nNOS and calbindin D28k was assessed by histochemistry, immunohistochemistry and RT-PCR in the rat brain. Induced NADPH-d positive neurons were observed in the cerebral cortex of 1, 3, 6 and 12 days after seizure and found in specific cortical areas, such as motor cortex, somatosensory cortex, auditory cortex, visual cortex, ectorhinal cortex and perirhinal cortex. The level of nNOS mRNA increased at 1, 3, 6 and 12 days after seizure compared with control group. Induced calbindin D28k positive neurons were observed in motor cortex and somatosensory cortex 1 and 3 days after seizure. The level of calbindin D28k mRNA in the cerebral cortex was slightly decreased at 1 day after seizure. Therefore, in this study, the induced NADPH-d, calbindin D28k positive neurons and upregulated NADPH-d positive neurons may influence the cerebral blood flow and neuronal activity in the cerebral cortex during post-seizure period.

Postnatal development of the Ca2+ -binding proteins parvalbumin and calbindin D-28k containing neurons in the rat amygdala-An Immunohistochemical Study / 대한해부학회지

The maturation pattern of the calcium binding proteins parvalbumin (PV) and calbindin-D28K (CB) from the day of birth, postnatal day 0 (P0) to 30 days (P5, P10, P15, P20, P30) and adult was studied in the rat amygdala using immunohistochemistry. PV and CB immunoreactivities in the amygdala of the rat showed very different patterns during postnatal development. The first PV-immunoreactive neurons appeared in the cortical amygdaloid nuclei and the basolateral amygdaloid nucleus at P5, and then in the lateral amygdaloid nucleus and the basomedial amygdaloid nucleus at P10. Adult patterns of PV-immunoreactive neurons were reached at P20. In contrast, CB-immunoreactive neurons were already found at birth in all amygdaloid nuclei except the intercalated nucleus. The intensity and number of immunoreactivity of CB-containing neurons increased during the first 10 days of postnatal life but dramatically decreased at P15. Mature patterns CB-immunoreactive neurons were achieved at P20. These two calcium binding proteins exhibited a non-homogeneous distribution in the adult amygdala, PV-immunoreactive neurons were mainly localized in the basolateral nuclear group but not in the medial amygdaloid nucleus, the cental amygdaloid nucleus and the intercalated nucleus. In contrast, CB-immunoreactive neurons were distributed in almost all amygdaloid nuclei except the intercalated nucleus. The present results showing different postnatal maturation patterns such as time of appearance, the number and distribution of immunoreactive cells suggest that PV and CB may play a different functional role during the postnatal development of the amygdala.

Synaptic Reorganization of Dentate Mossy Fibers and Expression of Calcium Binding Proteins in Hippocampal Sclerosis of Temporal Lobe Epilepsy

This study was designed to identify expression of calcium-binding proteins and synaptic reorganizations of dentate mossy fibers in hippocampal sclerosis of human temporal lobe epilepsy. Hippocampal neuronal density was quantitively analyzed in temporal lobe epilepsy group (n=50) to investigate the degree of hippocampal sclerosis and it was compared with that of autopsy control (n=3). To verify the distribution of calcium-binding proteins in neurons of epileptic hippocampi, the parvalbumin (PV)-immunoreactive and calbindin-D28K (CB)-immunoreactive neurons were quantitively analyzed in each area of Ammon's horn by immunohistochemical stain. Also, to clarify synaptic reorganizations of the dentate mossy fibers, a part of each hippocampus was examined under light microscopy and transmission electron microscopy using Timm sulphide silver method. In epileptic hippocampi, severity of hippocampal sclerosis (HS) was graded four, which consisted of 3 cases with no HS, 6 mild HS, 12 moderate HS, and 29 severe HS. The hippocampal neuronal loss was most prominent in CA1, followed by CA4 and CA2. Expression of calcium-binding proteins was more prevalent in CA2 of all groups. The proportion of PV-immunoreactive neurons in CA1 and CA4 significantly increased in the moderate and severe HS group, whereas the proportion of CB-immunoreactive neurons did not correlated with the severity of HS. Timm granules were noted in inner molecular supragranular layer of dentate gyrus of epileptic hippocampi and they tended to increase in proportion along with the severity of hippocampal sclerosis. Transmission electron microscopy showed that supragranular Timm granules corresponded to synaptic terminals of mossy fibers. These results suggest that parvalbumin appears to have more protective effect against neuronal loss and that mossy fiber synaptic reorganization seems to play a major role in pathogenesis of hippocampal sclerosis of human temporal lobe epilepsy.

Immunocytochemical Study of Calcium Binding Protein in the Distal Nephron of Rat Kidney / 대한해부학회지

Calbindin-D28k, a vitamin D-dependent calcium binding protein, plays a cardinal role in transport of calcium in kidney. Previous studies have demonstrated calbindin-D28k immunoreactivity in the distal nephron of mammalian kidney. However, it is well known that in most species including rat and human, there is a gradual transition from the distal convoluted tubule to the cortical collecting duct, and that the connecting segment do no tclearly demarcated, because of intermingling of distal convoluted tubule cells, connecting tubule cells, principal cells and at least two configurations of intercalated cells. In this study, to identify the cell types of calbindin-D28k-positive cells in distal nephron of rat kidney, we used double immunostaining with an antibody against calbindin-D28k and antibodies against thiazide sensitive Na+/Cl- cotransporter for distal convoluted tubule or H+-ATPase for intercalated cells. In the distal convoluted tubule, most of the distal convoluted tuble cells were calbindin-D28k-positive, whereas the intercalated cells were calbindin-D28k-negative. In the connecting tubule, 68% of the cells were calbindin-D28k-positive, and about 97% of the positive cells were connecting tubule cells and only 3% of them were intercalated cells. In the cortical collecting duct, and outer medullary collecting duct of outer stripe and inner stripe, only 8.6%, 11.8% and 4.4% of cells were weak positive for calbindin-D28k respectively. These weak positive cells in the collecting duct are mainly identified as intercalated cells. These findings indicate that calbindin-D28k is involved in not only transcellular transport of calcium but also processes regulating intracellular calcium in rat kidney.