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Highlights • c-Fos expression in the MPNc increases after ejaculation in male rats. • c-Fos expression in non-calbindin MPNc neurons decreases with sexual experience. • The MPNc may change functionally with sexual experience. Abstract The medial preoptic area (MPN) plays an important role in the control of male sexual behavior. In rats, the central part of the MPN (MPNc) is sexually dimorphic and contains a sexually dimorphic nucleus composed of neurons expressing calbindin-D28 K (CALB-SDN). Although the functions of the MPNc are not well understood, surgical destruction of the MPNc adversely affects the performance of sexual behavior in sexually naive males, but not in sexually experienced males, supporting the notion that the MPNc changes functionally with sexual experience. In this study, we aimed to determine the effects of sexual experience on the neuronal activity of the MPNc and CALB-SDN. Sexual behavior in sexually inexperienced males that had no experience of ejaculation, and experienced males that had ejaculated once was observed. After they displayed sexual behavior, the brains were sampled, and immunohistochemical analysis of c-Fos, a neuronal activity marker, in the MPNc and CALB-SDN was performed. The numbers of c-Fos-immunopositive cells with or without calbindin-D28K-immunoreactivity increased significantly in the MPNc and CALB-SDN following ejaculation in both sexually inexperienced and experienced males, although the numbers did not change significantly with exposure to estrous female odors, the first mount, and the first intromission before and after the first ejaculation. We further found that the number of c-Fos-immunopositive and calbindin-D28K-immunonegative cells in the MPNc, but not in the CALB-SDN, was significantly smaller in sexually experienced males than in sexually inexperienced males. These results suggest that a population of MPNc neurons, which is located outside the CALB-SDN and do not express calbindin-D28 K, are activated during the first copulation and then silent after acquisition of sexual experience. [ABSTRACT FROM AUTHOR]

Calbindin‑D28k (CB), calretinin (CR) and parvalbumin (PV), which regulate cytosolic free Ca2+ concentrations in neurons, are chemically expressed in γ‑aminobutyric acid (GABA)ergic neurons that regulate the degree of glutamatergic excitation and output of projection neurons. The present study investigated age‑associated differences in CB, CR and PV immunoreactivities in the somatosensory cortex in three species (mice, rats and gerbils) of young (1 month), adult (6 months) and aged (24 months) rodents, using immunohistochemistry and western blotting. Abundant CB‑immunoreactive neurons were distributed in layers II and III, and age‑associated alterations in their number were different according to the species. CR‑immunoreactive neurons were not abundant in all layers; however, the number of CR‑immunoreactive neurons was the highest in all adult species. Many PV‑immunoreactive neurons were identified in all layers, particularly in layers II and III, and they increased in all layers with age in all species. The present study demonstrated that the distribution pattern of CB‑, CR‑ and PV‑containing neurons in the somatosensory cortex were apparently altered in number with normal aging, and that CB and CR exhibited a tendency to decrease in aged rodents, whereas PV tended to increase with age. These results indicate that CB, CR and PV are markedly altered in the somatosensory cortex, and this change may be associated with normal aging. These findings may aid the elucidation of the mechanisms of aging and geriatric disease. [ABSTRACT FROM AUTHOR]

Neuronal calcium-binding protein 1 and -2 (NECAB1/2) localize to multiple excitatory neuron populations in the mouse spinal cord. Here, we analyzed rat and human spinal cord, combining in situ hybridization and immunohistochemistry, complementing newly collated data on mouse spinal cord for direct comparisons. Necab1/2 mRNA transcripts showed complementary distribution in rodent's spinal cord. Multiple-labeling fluorescence histochemistry with neuronal phenotypic markers localized NECAB1 to a dense fiber plexus in the dorsal horn, to neurons mainly in superficial layers and to commissural interneurons in both rodent species. NECAB1-positive (+) motor neurons were only found in mice. NECAB1 distribution in the human spinal cord was similar with the addition of NECAB1-like immunoreactivity surrounding myelinated axons. NECAB2 was mainly present in excitatory synaptic boutons in the dorsal horn of all three species, and often in calbindin-D28k neuronal somata. Rodent ependymal cells expressed calbindin-D28k. In humans, they instead were NECAB2 and/or calretinin. Our results reveal that the association of NECAB2 to excitatory neuronal circuits in the spinal cord is evolutionarily conserved across the mammalian species investigated so far. In contrast, NECAB1 expression is more heterogeneous. Thus, our study suggests that the phenotypic segregation of NECAB1 and -2 to respective excitatory and inhibitory spinal systems can underpin functional modalities in determining the fidelity of synaptic neurotransmission and neuronal responsiveness, and might bear translational relevance to humans. [ABSTRACT FROM AUTHOR]

The localization of calbindin-D28k was examined in rat kidney at different stages of development. Calbindin was found specifically in the distal convoluted tubules (DCT). In the 10 day old rat kidney many cells apparently belonging to the collecting tubules were positive. In the neonatal nephrons there was also localization of calbindin-D28k to the limb of Henle, suggesting a difference in the regulation of intracellular calcium during maturation. Proximal tubule, renal corpuscle and macula densa gave no specific localization of calbindin-D28k.

Calcium binding proteins play important roles in all aspects of neural functioning in the central nervous system. In the present study, we examined age-related changes of three different calcium binding proteins calbindin-D28k (CB), calretinin (CR) and parvalbumin (PV) immunoreactivities in the striatum of young (1 month), adult (6 months) and aged (24 months) ages in three species of rodents (mouse, rat and gerbil) using immunohistochemistry and Western blotting. Our results show that the number of CB-immunoreactive neurons was highest in the adult mouse and rat; however, in the gerbil, the number of CB-immunoreactive neurons was not significantly different from each group although the CB immunoreactivity was significantly decreased in the aged group compared with the adult group. The number of CR-immunoreactive neurons in the striatum was significantly highest in all the adult groups, and, especially, the number of CR-immunoreactive neurons and CR immunoreactivity in the aged gerbil were significantly decreased in the aged group compared with the other groups. Finally, we did not found any significant difference in the number of PV-immunoreactive neurons in the striatum with age among the three rodents. On the other hand, we found that protein levels of three calcium binding proteins in all the mouse groups were similar to the immunohistochemical data. These results indicate that the distribution pattern of calcium binding proteins is different according to age; the adult might show an apparent tendency of high expression in the striatum. [ABSTRACT FROM AUTHOR]

Calcium levels in the lens rise with increasing age and increased intracellular calcium accumulation is known to be a risk factor for cataract formation. Calbindin-D28K (CALB1) is an intracellular calcium buffer. It is not clear whether CALB1 levels change in response to the Ca2+ accumulation in the lens that occurs with age. The present study investigated the distribution of CALB1 in the lenses of Sprague-Dawley (SD) rats and whether this changed with age. Lenses were isolated from SD rats at 1, 6, 12 and 18 months of age. CALB1 distribution was examined using immunohistochemistry. Lens epithelial cells were counted in median sagittal plane slices from the hematoxylin and eosin-stained lens and quantified using western blot analysis. Calb1 gene expression was examined using reverse transcription-quantitative polymerase chain reaction. CALB1 was distributed in the epithelial and fiber cells of the lens. CALB1 levels declined significantly with increasing age, whilst there was no significant accompanying decrease in the number of lens cells. A similar reduction was noted in CALB1 mRNA levels. To the best of our knowledge, this is the first study to demonstrate that CALB1 expression and CALB1 protein levels in SD rat lens decrease with age. This reduction does not reflect a reduction in lens cell numbers but a genuine reduction in gene expression within these cells. Thus, CALB1 may be important in changes occurring in the lens in older age, in particular in the development of cataracts. [ABSTRACT FROM AUTHOR]

The article discusses a study which examines the distribution of the calbindin-D28k, an intracellular protein with a high affinity for calcium, in the pineal gland of porcupine. The result reveals that two different kinds of cells were positive of calbindin-D28k. It also reveals that immunoreative cells were detected throughout the pineal gland without any preferential location.

Abstract: Expression of CB in the sympathetic ganglia was investigated by immunohistochemistry. The distribution of CB immunoreactivity was studied in the superior cervical ganglion (SCG), stellate ganglion (SG) and celiac ganglion (CG) from rats and cats of different ages (newborn, 10-day-old, 20-day-old, 30-day-old, two-month-old, six-month-old). We observed that the percentage of CB-immunoreactive (IR) neurons decreased during early postnatal development in rats and cats. In all studied ganglia of both species, the percentage of CB-IR neurons was high in newborn and 10-day-old animals and significantly decreased up to 30days of life. In rats of all ages, the largest percentage of CB-IR neurons was observed in the SG compared to the SCG and CG. In the cat sympathetic ganglia, the number of CB-IR neurons decreased more rapidly during the first two months of life, and only scattered CB-IR neurons were found in the sympathetic ganglia of two-month-old and six-month-old cats. In cats, the highest percentage of CB-IR neurons was observed in the SG, while the lowest percentage was found in the CG. The difference in size between CB+ and CB− neurons equally changed during development. Finally, the changes in the size and percentages of CB-IR neurons were complete in rats at the first month of life, and in cats at the end of the second month. [Copyright &y& Elsevier]

Kidney is a major target organ in preclinical studies. In recent years, intense research has been undertaken to characterize novel renal toxicity biomarkers. In this context, we studied nephron segment specific antibodies against aquaporin-1 (AQP-1), α-glutathione-S-transferase (alpha-GST), Tamm-Horsfall protein (TH), calbindin-D28K (CalD), and aquaporin-2 (AQP-2), using an immunoperoxidase method on formalin-fixed paraffin-embedded kidney tissues of dogs, rats, mice, and Cynomolgus monkeys. AQP-1 was specific for proximal tubules and thin descending limbs of Henle’s loops and AQP-2 for connecting and collecting ducts in dogs, rats, mice, and Cynomolgus monkeys. Alpha-GST stained the straight part of proximal tubules in dogs and proximal convoluted tubule and straight part of proximal tubules in rats. TH was specific for thick ascending limbs of Henle’s loops in mice, rats, dogs, and Cynomolgus monkeys and stained additionally scattered cells in cortical connecting/collecting ducts of dogs. CalD was found in distal convoluted tubules and cortical connecting and collecting ducts of dogs, rats, and mice and in distal convoluted tubules, connecting ducts, and cortical and medullary collecting ducts of Cynomolgus monkey. This panel of antibodies may be a helpful tool to identify renal tubules by light microscopy in preclinical studies and to validate new biomarkers of renal toxicity. [ABSTRACT FROM PUBLISHER]

During tooth development, the inner and outer enamel epithelia fuse by mitotic activity to produce a bilayered epithelial sheath termed Hertwig's epithelial root sheath (HERS). The epithelial rests of Malassez (ERM) are the developmental residues of HERS and remain in the adult periodontal ligament (PDL). Although the cellular regulation of the Ca-binding proteins parvalbumin, calbindin-D28k, and calretinin has been reported in the inner and outer enamel epithelia during tooth development, an involvement of Ca-binding proteins in the ERM has not so far been characterized. Among the three Ca-binding proteins tested (calbindin D28k, parvalbumin, calretinin), we have only been able to detect calretinin in a subpopulation of adult rat molar ERM, by using quantitative immunohistochemical and confocal immunofluorescence techniques. TrkA (a marker for ERM) is present in numerous epithelial cell clusters, whereas calretinin has been localized in the cytosol and perinuclear region of a subpopulation of TrkA-positive cells. We conclude that, in inner and outer enamel epithelial cells, Ca is regulated by calbindin, parvalbumin, and calretinin during tooth development, whereas in the ERM of adult PDL, Ca is regulated only by calretinin. The expression of Ca-binding proteins is restricted in a developmental manner in the ERM. [ABSTRACT FROM AUTHOR]

Objective: To investigate expression and localization of Calbindin-d28k (CaBP-d28k) in human ovary. Methods: The expression of the CaBP-d28k protein and gene in 23 human ovarian samples was measured by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) methods. Results: Positive expression of the CaBP-d28k protein and mRNA was observed in human ovary. The ovarian expression of the CaBP-d28k protein appeared in the cytoplasm of oocytes, granulose cells, theca cells, and two types of cells of corpus luteum. From primordial follicle, primary follicle to secondary follicle, increased CaBP-d28k levels were showed (P<0.05), and the CaBP-d28k expression in secondary follicle had the highest level. Conclusion: Human ovarian tissue locally expresses CaBP-d28k, and its expression level increases with follicular development. [ABSTRACT FROM AUTHOR]

The present study dealt with the localization of different calcium-binding proteins (CaBPs) in the pancreatic tissue of non-diabetic and diabetic rats and in rat insulinoma β-cells (INS-1). Transcripts of CaBPs displayed different expression levels in rat pancreatic tissue and INS-1 cells. Immunohistochemistry demonstrated that three of these proteins, calmodulin, calreticulin and calbindin-D28k, were located predominantly in the pancreatic islets (in both α- and β-cells) of rats, showing weaker labeling of exocrine tissue. Secretagogin was exclusively found within islets. All CaBPs were also immunohistochemically detected in INS-1 cells. Immunohistochemical analysis demonstrates differences in CaBP distributions when comparing the pancreatic tissues of diabetic Goto-Kakizaki rats and non-diabetic Wistar rats. Pancreatic tissue in type 2 diabetic Goto-Kakizaki rats showed significantly higher transcript levels of all CaBPs compared to those in Wistar rats. These results indicate that alterations of CaBPs in pancreatic islets are associated with metabolic disturbances related to type 2 diabetes. [ABSTRACT FROM AUTHOR]

Objective: To investigate the expression of Calbindin-d28k (CaBP-d28k) in human endometrium. Methods: Thirty-three samples of human normal endometrial tissues were divided into 6 groups: 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 expression and change of CaBP-d28k protein and gene were determined by immunohistochemistry and reverse transcription polymerase chain reaction methods. Results: In endometrial samples, the expression of CaBP-d28k protein was mainly observed in the cytoplasm of luminal and glandular epithelium. In the menstrual cycle, the level of CaBP-d28k protein in the epithelium was the lowest during the early and mid proliferative stages, and was the highest during the mid secretory stage, then decreased in the late secretory stage (P<0.05). In the stroma, the expressed type of CaBP-d28k protein was the same as in the epithelium, but was lower than that in the epithelium(P<0.05). The CaBP-d28k mRNA was at the lowest level in the early proliferative stage(P<0.05), and significantly increased in the late proliferative, and early, mid secretory stages (P<0.05). Conclusion: Both CaBP-d28k protein and gene were expressed in human endometrium, and their expression had cyclic changes. [Copyright &y& Elsevier]

Abstract: We used immunohistochemical methods to examine the distribution of the calcium channel α2 (CCα2) subunit in the chicken spinal cord and dorsal root ganglion (DRG) neurons and determine its relationship with calbindin-D28k (CB) in the DRG neurons. In the spinal cord, CCα2 subunit was detected in nerve terminals, which were observed as dot-like structures, and in laminae I, II, III and Lissauer''s tract in the dorsal horn. In the DRG neurons, approximately 65% of the total neurons were CCα2 subunit positive, and most (86%) of these neurons were small to medium sized, suggesting that the CCα2 subunit and/or a complex of the CCα2 and δ subunits is possibly localized in a number of nociceptive neurons. A majority (77%) of the positive neurons showed CB immunoreactivity and most (88%) of these neurons were small to medium sized. This may indicate a close correlation between the CCα2 subunit and CB in the nociceptive neurons. Thus, it is postulated that the mode of nociceptive transmission may involve a cellular Ca2+-regulating system that consists of both Ca2+ entry via calcium channels with the α2δ subunit and intracellular Ca2+-binding activity of CB in the nociceptive neurons of the DRG. [Copyright &y& Elsevier]

Abstract: The purpose of the present study was to elucidate regional differences in the vulnerability of cerebellar foliations of rats exposed to X-irradiation. Effects of X-irradiation on foliations were examined with respect to histological changes in Purkinje cells and Bergmann glial fibers by calbindin-D28k (CB) and glial fibrillary acidic protein (GFAP) immunohistochemistry, respectively. Wistar rats were exposed to X-irradiation (1.5Gy) on postnatal day (PND) 1. At 3 weeks of age, the cerebellum was examined. The cerebella of rats exposed to X-irradiation showed smaller and abnormal foliations compared with controls. Fewer cerebellar foliations due to fusion with neighboring folia were observed in folia I–III and VIa–VII. Moreover, the extent of such abnormalities was more severe in the latter folia. CB-immunoreactive (IR) Purkinje cells exhibited thin, short, disoriented dendrites that had invaded the granular layer or white matter. On the other hand, GFAP-IR Bergmann glial fibers had not extended their processes into the molecular layer perpendicular to the pial surface, and they appeared thin and disoriented. Accordingly, the above cerebellar abnormalities were more severe in folia I–III, VIa–VII and X than in other regions. In contrast to the histological alterations in these folia, there were no apparent qualitative differences in folia IV–V between X-irradiated and controls. These findings indicate regional difference in the vulnerability of cerebellar folia to X-irradiation. Such differences might be attributed to the cerebellar neurogenetic gradient. [Copyright &y& Elsevier]

Abstract: The rat medial prefrontal cortex, an area considered homologous to the human prefrontal cortex, is a region in which neuronal structural plasticity has been described during adulthood. Some plastic processes such as neurite outgrowth and synaptogenesis are known to be regulated by the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). Since PSA-NCAM is present in regions of the adult CNS which are undergoing structural remodeling, such as the hypothalamus or the hippocampus, we have analyzed the expression of this molecule in the medial prefrontal cortex of adult rats using immunohistochemistry. PSA-NCAM immunoreactivity was found both in cell bodies and in the neuropil of the three divisions of the medial prefrontal cortex. All cell somata expressing PSA-NCAM corresponded to neurons and 5′ bromodeoxyuridine labeling after long survival times demonstrated that these neurons were not recently generated. Many of these PSA-NCAM immunoreactive neurons in the medial prefrontal cortex could be classified as interneurons on the basis of their morphology and glutamate decarboxylase, isoform 67 expression. Some of the PSA-NCAM immunoreactive neurons also expressed somatostatin, neuropeptide Y and calbindin-D28K. By contrast, pyramidal neurons in this cortical region did not appear to express PSA-NCAM. However, some of these principal neurons appeared surrounded by PSA-NCAM immunoreactive puncta. Some of these puncta co-expressed synaptophysin, suggesting the presence of synapses. Since the etiology of some psychiatric disorders has been related to alterations in medial prefrontal cortex structural plasticity, the study of PSA-NCAM expression in this region may open a new approach to the pathophysiology of these mental disorders. [Copyright &y& Elsevier]

This study analyses by immunohistochemical methods the effects of the deletion of the Otx1 gene on 12 areas of the cerebral cortex and on neurons expressing Ca-binding proteins (CaBP), such as parvalbumin (Pv) and calbindin-D28K (Cb). We found that the deletion of the Otx1 gene modified differently the various cortical areas. The decrease in cortical thickness ranged from 29.35 to 9.85% and the reduction in cellular population from 35.90 to 3.65% in the different cortical areas. The influence of the Otx1 gene concerns all cortical layers with variable effects on different cortical areas. The cellular population of cerebral cortex considered as a whole was reduced by 20.67%, Pv-positive (Pv+) cells by 58.01% and Cb-positive (Cb+) cells by 51.54%. The quantitative distribution of Pv+ and Cb+ cells varied independently in the different cortical areas. Topographic analysis of CaBP cells in Otx1-null mice ( Otx1−/−) showed that Pv+ cells were principally distributed in layers IV and V and Cb+ cells in layers V and VI. Given that in the development of wild-type mice both cell types first appear in deep layers and later spread to superficial ones, the segregation of CaBP neurons in inner layers of Otx1−/− animals is an index of the immaturity of the cerebral cortex of these animals. This study showed that the Otx1 gene has a more complex role than previously reported, as it is involved in the maturation and differentiation of various cerebral cortices, and, specifically, in the development of CaBP cells. [ABSTRACT FROM AUTHOR]

• Calbindin neurons can influence the activity of the amygdala. • Four types of CB cells were described: multipolar, spherical, pyramidal and halo. • The halo type is specific to the marmoset monkey, located in the basal and central nuclei. • Many variations in fibers and neuropils are observed in the amygdala nuclei. • CB neurons and fibers were found in the myelin tracts that surrounded the amygdala. The location and distribution of the calcium-binding protein calbindin-D28k (CB) has been considered to be of great value as a neuronal marker for identifying distinct brain regions and discrete neuronal populations. In the amygdaloid complex (AC), the balance of excitatory and inhibitory inputs is controlled by CB immunoreactive interneurons. Alterations of inhibitory mechanisms in the AC may play a role in the emotional symptomatology of neurological diseases like Alzheimer's and psychiatric disorders like posttraumatic stress disorder. The present investigation examined the distribution and morphology of CB-containing neurons, neuropils and fibers in marmoset monkey ACs by using immunohistochemical and morphometrical methods. We recognized four types of CB cells in the AC: type 1 (multipolar), type 2 (spherical or bipolar), type 3 (pyramidal) and type 4 (halo cells), a cell type specific to the marmoset located in the basal and central nuclei. We detected CB cells in all nuclei and areas of the AC, where most of the cells were present in the deep nuclei (lateral, basal, accessory basal and paralaminar). In the superficial nuclei (the nucleus of the lateral olfactory tract, medial nucleus, periamygdaloid cortex and cortical nuclei), the CB cells were abundant in layers 2 and 3. The intercalated nuclei contained small densely packed cells. The CB neuropils were particularly dense in layer 1 of the superficial nuclei, in the deep nuclei and in the amygdalohippocampal area. Large CB immunoreactive neurons in the white matter and fibers with varicosities were found in the myelin tracts that surrounded the AC. These findings are the first step in determining whether some of these cells are specifically disrupted in pathological states. [ABSTRACT FROM AUTHOR]