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The diversity of the mammalian cerebral cortex demands technical approaches to map the spatial distribution of neurons with different biochemical identities. This issue is magnified in the case of the primate cortex, characterized by a large number of areas with distinctive cytoarchitectures. To date, no full map of the distribution of cells expressing a specific protein has been reported for the cortex of any primate. Here we have charted the 3-dimensional distribution of neurons expressing the calcium-binding protein calbindin (CB+ neurons) across the entire marmoset cortex, using a combination of immunohistochemistry, automated cell identification, computerized reconstruction, and cytoarchitecture-aware registration. CB+ neurons formed a heterogeneous population, which together corresponded to 10-20% of the cortical neurons. They occurred in higher proportions in areas corresponding to low hierarchical levels of processing, such as sensory cortices. Although CB+ neurons were concentrated in the supragranular and granular layers, there were clear global trends in their laminar distribution. For example, their relative density in infragranular layers increased with hierarchical level along sensorimotor processing streams, and their density in layer 4 was lower in areas involved in sensorimotor integration, action planning and motor control. These results reveal new quantitative aspects of the cytoarchitectural organization of the primate cortex, and demonstrate an approach to mapping the full distribution of neurochemically distinct cells throughout the brain which is readily applicable to most other mammalian species., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Atapour et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The cerebellum is involved in higher order cognitive function and is susceptible to age-related atrophy. However, limited evidence has directly examined the cerebellum's role in cognitive aging. To interrogate potential substrates of the relationship between cerebellar structure and memory in aging, here we target the Purkinje cells (PCs). The sole output neurons of the cerebellum, PC loss and/or degeneration underlie a variety of behavioral abnormalities. Using a rat model of normal cognitive aging, we immunostained sections through the cerebellum for the PC-specific protein, calbindin-D28k. Although morphometric quantification revealed no significant difference in total PC number as a function of age or cognitive status, regional cell number was a more robust correlate of memory performance in the young cerebellum than in aged animals. Parallel biochemical analysis of PC-specific protein levels in whole cerebellum additionally revealed that calbindin-D28k and Purkinje cell protein-2 (pcp-2) levels were lower selectively in aged rats with spatial memory impairment compared to both young animals and aged rats with intact memory. These results suggest that cognitive aging is associated with cerebellum vulnerability, potentially reflecting disruption of the cerebellum-medial temporal lobe network., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA. The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

Fundamental differences in excitatory pyramidal cells across cortical areas and species highlight the implausibility of extrapolation from mouse to primate neurons and cortical networks. Far less is known about comparative regional and species-specific features of neurochemically distinct cortical inhibitory interneurons. Here, we quantified the density, laminar distribution, and somatodendritic morphology of inhibitory interneurons expressing one or more of the calcium-binding proteins (CaBPs) (calretinin [CR], calbindin [CB], and/or parvalbumin [PV]) in mouse (Mus musculus) versus rhesus monkey (Macaca mulatta) in two functionally and cytoarchitectonically distinct regions-the primary visual and frontal cortical areas-using immunofluorescent multilabeling, stereological counting, and 3D reconstructions. There were significantly higher densities of CB+ and PV+ neurons in visual compared to frontal areas in both species. The main species difference was the significantly greater density and proportion of CR+ interneurons and lower extent of CaBP coexpression in monkey compared to mouse cortices. Cluster analyses revealed that the somatodendritic morphology of layer 2-3 inhibitory interneurons is more dependent on CaBP expression than on species and area. Only modest effects of species were observed for CB+ and PV+ interneuron morphologies, while CR+ neurons showed no difference. By contrast to pyramidal cells that show highly distinctive area- and species-specific features, here we found more subtle differences in the distribution and features of interneurons across areas and species. These data yield insight into how nuanced differences in the population organization and properties of neurons may underlie specializations in cortical regions to confer species- and area-specific functional capacities., (© 2023 Wiley Periodicals LLC.)

Anatomists have long expressed interest in neurons of the white matter, which is by definition supposed to be free of neurons. Hypotheses regarding their biochemical signature and physiological function are mainly derived from animal models. Here, we investigated 15 whole-brain human postmortem specimens, including cognitively normal cases and those with pathologic Alzheimer's disease (AD). Quantitative and qualitative methods were used to investigate differences in neuronal size and density, and the relationship between neuronal processes and vasculature. Double staining was used to evaluate colocalization of neurochemicals. Two topographically distinct populations of neurons emerged: one appearing to arise from developmental subplate neurons and the other embedded within deep, subcortical white matter. Both populations appeared to be neurochemically heterogeneous, showing positive reactivity to acetylcholinesterase (AChE) [but not choline acetyltransferase (ChAT)], neuronal nuclei (NeuN), nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), microtubule-associated protein 2 (MAP-2), somatostatin (SOM), nonphosphorylated neurofilament protein (SMI-32), and calcium-binding proteins calbindin-D 28K (CB), calretinin (CRT), and parvalbumin (PV). PV was more richly expressed in superficial as opposed to deep white matter neurons (WMNs); subplate neurons were also significantly larger than their deeper counterparts. NADPH-d, a surrogate for nitric oxide synthase, allowed for the striking morphological visualization of subcortical WMNs. NADPH-d-positive subcortical neurons tended to embrace the outer walls of microvessels, suggesting a functional role in vasodilation. The presence of AChE positivity in these neurons, but not ChAT, suggests that they are cholinoceptive but noncholinergic. WMNs were also significantly smaller in AD compared to control cases. These observations provide a landscape for future systematic investigations., (© 2023 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

The mammalian striatum has long been considered a homogeneous entity. However, neuroanatomical and histochemical studies reveal that the striatum is much more heterogeneous than previously suspected. The caudate (Cd) and putamen (Pu) are composed of two chemical compartments: the matrix and the striosomes. Striatal interneurons have been classified into a variety of morphological and neurochemical subtypes. In this study, we compared the distribution of multiple neurochemical markers in the striatum of marmosets and described the morphology of different types of striatum interneurons. The immunoreactivities of choline-acetyl transferase (ChAT), neuropeptide Y (NPY), nitric oxide synthase (NOS), calretinin (CR), parvalbumin (PV) were analyzed along the entire rostrocaudal extent of the marmoset striatum. Calbindin immunohistochemistry is useful in identifying medium spiny neurons (MSNs), with efficient soma staining. Based on the size of the CB-positive cells, considered medium-sized, as expected, cholinergic cells are larger in area and diameter than the other subpopulations investigated, followed by NOS, NPY, PV and CR. In adjacent CB and PV-stained sections, the matrix and striosomes were clearly distinguished. The matrix is strongly reactive to CB and PV neuropils, while the striosomes exhibit low reactivity, especially in the dorsal Cd. Therefore, we provide a detailed description morphology and distribution of striatal interneuron populations in a model as a valuable tool for studying neurodegenerative pathogenesis, progression and treatment strategies., (Copyright © 2023 Elsevier Ltd and Japan Neuroscience Society. All rights reserved.)

The incidence rates of light-induced retinopathies have increased significantly in the last decades because of continuous exposure to light from different electronic devices. Recent studies showed that exposure to blue light had been related to the pathogenesis of light-induced retinopathies. However, the pathophysiological mechanisms underlying changes induced by light exposure are not fully known yet. In the present study, the effects of exposure to light at different wavelengths with emission peaks in the blue light range (400-500 nm) on the localization of Calretinin-N18 (CaR-N18) and Calbindin-D28K (CaB-D28K) in adult zebrafish retina are studied using double immunofluorescence with confocal laser microscopy. CaB-D28K and CaR-N18 are two homologous cytosolic calcium-binding proteins (CaBPs) implicated in essential process regulation in central and peripheral nervous systems. CaB-D28K and CaR-N18 distributions are investigated to elucidate their potential role in maintaining retinal homeostasis under distinct light conditions and darkness. The results showed that light influences CaB-D28K and CaR-N18 distribution in the retina of adult zebrafish, suggesting that these CaBPs could be involved in the pathophysiology of retinal damage induced by the short-wavelength visible light spectrum.

The superior colliculus (SC) is a layered midbrain structure with functions that include polysensory and sensorimotor integration. Here, we describe the distribution of different immunohistochemically identified classes of neurons in the SC of adult marmoset monkeys (Callithrix jacchus). Neuronal nuclei (NeuN) staining was used to determine the overall neuronal density in the different SC layers. In addition, we studied the distribution of neurons expressing different calcium-binding proteins (calbindin [CB], parvalbumin [PV] and calretinin [CR]). Our results indicate that neuronal density in the SC decreases from superficial to deep layers. Although the neuronal density within the same layer varies little across the mediolateral axis, it tends to be lower at rostral levels, compared to caudal levels. Cells expressing different calcium-binding proteins display differential gradients of density according to depth. Both CB- and CR-expressing neurons show markedly higher densities in the stratum griseum superficiale (SGS), compared to the stratum opticum and intermediate and deep layers. However, CR-expressing neurons are twice as common as CB-expressing neurons outside the SGS. The distribution of PV-expressing cells follows a shallow density gradient from superficial to deep layers. When normalized relative to total neuronal density, the proportion of CR-expressing neurons increases between the superficial and intermediate layers, whereas that of CB-expressing neurons declines toward the deep layers. The proportion of PV-expressing neurons remains constant across layers. Our data provide layer-specific and accurate estimates of neuronal density, which may be important for the generation of biophysical models of how the primate SC transforms sensory inputs into motor signals., (© 2022 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

Calcium-binding proteins (CBPs) regulate neuronal function in midbrain dopamine (DA)-ergic neurons in mammals by buffering and sensing the intracellular Ca 2+ , and vesicular release. In birds, the equivalent set of neurons are important in song learning, directed singing, courtship, and energy balance, yet the status of CBPs in these neurons is unknown. Herein, for the first time, we probe the nature of CBPs, namely, Calbindin-, Calretinin-, Parvalbumin-, and Secretagogin-expressing DA neurons in the ventral tegmental area (VTA) and substantia nigra (SN) in the midbrain of zebra finch, Taeniopygia guttata. qRT-PCR analysis of ventral midbrain tissue fragment revealed higher Calbindin- and Calretinin-mRNA levels compared to Parvalbumin and Secretagogin. Application of immunofluorescence showed CBP-immunoreactive (-i) neurons in VTA (anterior [VTAa], mid [VTAm], caudal [VTAc]), SN (compacta [SNc], and reticulata [SNr]). Compared to VTAa, higher Calbindin- and Parvalbumin-immunoreactivity (-ir), and lower Calretinin-ir were observed in VTAm and VTAc. Secretagogin-ir was highly localized to VTAa. In SN, Calbindin- and Calretinin-ir were higher in SNc, SNr was Parvalbumin enriched, and Secretagogin-ir was not detected. Weak, moderate, and intense tyrosine hydroxylase (TH)-i VTA neurons were demarcated as subtypes 1, 2, and 3, respectively. While subtype 1 TH-i neurons were neither Calbindin- nor Calretinin-i, ∼80 and ∼65% subtype 2 and ∼30 and ∼45% subtype 3 TH-i neurons co-expressed Calbindin and Calretinin, respectively. All TH-i neuronal subtypes co-expressed Parvalbumin with reciprocal relationship with TH-ir. We suggest that the CBPs may determine VTA DA neuronal heterogeneity and differentially regulate their activity in T. guttata., (© 2022 Wiley Periodicals LLC.)

Derangements to the functioning of calcium-regulating organs have been associated with type 2 diabetes mellitus (T2DM), a condition preceded by pre-diabetes. Type 2 diabetes has shown to promote renal calcium wastage, intestinal calcium malabsorption and increased bone resorption. However, the changes to the functioning of calcium-regulating organs in pre-diabetes are not known. Subsequently, the effects of diet-induced pre-diabetes on the functioning of calcium-regulating organs in a rat model for pre-diabetes was investigated in this study. Male Sprague Dawley rats were separated into two groups (n=6, each group): non-pre-diabetic (NPD) group and a diet-induced pre-diabetic (DIPD) group for 20 weeks. After the experimental period, postprandial glucose and HOMA-IR were analysed in addition to plasma and urinary calcium concentrations. Gene expressions of intestinal vitamin D (VDR), intestinal calbindin-D9k, renal 1-alpha hydroxylase and renal transient receptor potential vanilloid 5 (TRPV5) expressions in addition to plasma osteocalcin and urinary deoxypyridinoline concentrations were analysed at week 20. The results demonstrated significantly increased concentrations of postprandial glucose, HOMA-IR and urinary calcium in addition to unchanged plasma calcium levels in the DIPD group by comparison to NPD. Renal TRPV5, renal 1-alpha hydroxylase, intestinal VDR and intestinal calbindin-D9k expressions were increased in the DIPD group by comparison to NPD. Furthermore, plasma osteocalcin levels were increased and urine deoxypyridinoline levels were decreased in the DIPD group by comparison to NPD. These observations may suggest that calcium-regulating organs compensate for the changes to calcium homeostasis by inducing increased renal calcium reabsorption, increased intestinal calcium absorption and decreased bone resorption followed by increased bone formation., Competing Interests: The co-author AK of this manuscript is the editor of the Frontiers Journal Thieme (Pre-diabetes and endocrine function) The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Naidoo, Ngubane and Khathi.)

Few brain regions have such wide-ranging inputs and outputs as the claustrum does, and fewer have posed equivalent challenges in defining their structural boundaries. We studied the distributions of three calcium-binding proteins-calretinin, parvalbumin, and calbindin-in the claustrum and dorsal endopiriform nucleus of the fruit bat, Carollia perspicillata. The proportionately large sizes of claustrum and dorsal endopiriform nucleus in Carollia brain afford unique access to these structures' intrinsic anatomy. Latexin immunoreactivity permits a separation of claustrum into core and shell subregions and an equivalent separation of dorsal endopiriform nucleus. Using latexin labeling, we found that the claustral shell in Carollia brain can be further subdivided into at least four distinct subregions. Calretinin and parvalbumin immunoreactivity reinforced the boundaries of the claustral core and its shell subregions with diametrically opposite distribution patterns. Calretinin, parvalbumin, and calbindin all colocalized with GAD67, indicating that these proteins label inhibitory neurons in both claustrum and dorsal endopiriform nucleus. Calretinin, however, also colocalized with latexin in a subset of neurons. Confocal microscopy revealed appositions that suggest synaptic contacts between cells labeled for each of the three calcium-binding proteins and latexin-immunoreactive somata in claustrum and dorsal endopiriform nucleus. Our results indicate significant subregional differences in the intrinsic inhibitory connectivity within and between claustrum and dorsal endopiriform nucleus. We conclude that the claustrum is structurally more complex than previously appreciated and that claustral and dorsal endopiriform nucleus subregions are differentially modulated by multiple inhibitory systems. These findings can also account for the excitability differences between claustrum and dorsal endopiriform nucleus described previously., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Distinguishing and characterising the different classes of neurons that make up a neural circuit has been a long-term goal for many neuroscientists. The enteric nervous system is a large but moderately simple part of the nervous system. Enteric neurons in laboratory animals have been extensively characterised morphologically, electrophysiologically, by projections and immunohistochemically. However, studies of human enteric nervous system are less advanced despite the potential availability of tissue from elective surgery (with appropriate ethics permits). Recent studies using single cell sequencing have confirmed and extended the classification of enteric neurons in mice and human, but it is not clear whether an encompassing classification has been achieved. We present preliminary data on a means to distinguish classes of myenteric neurons in specimens of human colon combining immunohistochemical, morphological, projection and size data on single cells. A method to apply multiple layers of antisera to specimens was developed, allowing up to 12 markers to be characterised in individual neurons. Applied to multi-axonal Dogiel type II neurons, this approach demonstrated that they constitute fewer than 5% of myenteric neurons, are nearly all immunoreactive for choline acetyltransferase and tachykinins. Many express the calcium-binding proteins calbindin and calretinin and they are larger than average myenteric cells. This methodology provides a complementary approach to single-cell mRNA profiling to provide a comprehensive account of the types of myenteric neurons in the human colon., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

EF-hand is a common motif in Ca 2+ -binding proteins, some of which present a conformational change upon Ca 2+ -binding, a relevant property for signal transduction. In the present work, we investigated the behavior of Calbindin D 9k , a modulator protein with a high affinity for Ca 2+ but structurally insensitive to its presence. Its non-canoncal N-terminal EF-hand was replaced by chimeric motifs, containing increasing structural elements from the sensor troponin C SCIII motif. We demonstrated that the loop and helix II were the necessary elements for a conformational change promoted by calcium in chimeric Calbindin D 9k . Fusion of the isolated chimeric motifs to an activity reporter gene showed the loop as the minimal element to promote a conformational change. The discrepancy between these results is discussed in the light of inter-motif interactions and helix I participation in modulating the Ca 2+ affinity and restricting motif conformation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Vitamin D is important for gonadal function in rodents, and improvement of vitamin D status in men with low sperm counts increases live birth rate. Vitamin D is a regulator of transcellular calcium transport in the intestine and kidney and may influence the dramatic changes in the luminal calcium concentration in epididymis. Here, we show spatial expression in the male reproductive tract of vitamin D receptor (VDR)-regulated factors involved in calcium transport: transient receptor potential vanilloid 5/6 , sodium/calcium exchanger 1, plasma membrane calcium ATPase 1, calbindin D9k, calcium-sensing receptor (CaSR), and parathyroid hormone-related peptide (PTHrP) in mouse and human testis and epididymis. Testicular Casr expression was lower in Vdr ablated mice compared with controls. Moreover, expression levels of Casr and Pthrp were strongly correlated in both testis and epididymis and Pthrp was suppressed by 1,25(OH)2D3 in a spermatogonial cell line. The expression of CaSR in epididymis may be of greater importance than in the gonad in mice as germ cell-specific Casr deficient mice had no major reproductive phenotype, and coincubation with a CaSR-agonist had no effect on human sperm-oocyte binding. In humans, seminal calcium concentration between 5 and 10 mM was associated with a higher fraction of motile and morphologically normal sperm cells, and the seminal calcium concentration was not associated with serum calcium levels. In conclusion, VDR regulates CaSR and PTHrP, and both factors may be involved in the regulation of calcium transport in the male reproductive tract with possible implications for sperm function and storage.

Inhibitory circuits in the basolateral nuclear complex of the amygdala (BNC) critical for controlling the acquisition, expression, and extinction of emotional responses are mediated by GABAergic interneurons (INs). Studies in rodents have demonstrated that separate IN subpopulations, identified by their expression of calcium-binding proteins and neuropeptides, play discrete roles in the intrinsic circuitry of the BNC. Far less is known about IN subpopulations in primates. In order to fill in this gap in our understanding of primate INs, the present investigation used dual-labeling immunohistochemistry for IN markers to identify subpopulations expressing cholecystokinin (CCK), calbindin (CB), calretinin (CR), and somatostatin (SOM) in somata and axon terminals in the monkey BNC. In general, colocalization patterns seen in somata and axon terminals were similar. It was found that there was virtually no colocalization of CB and CR, the two calcium-binding proteins investigated. Three subtypes of CCK-immunoreactive (CCK+) INs were identified on the basis of their expression of CR or CB: (1) CCK+/CR+; (2) CCK+/CB+); and (3) CCK+/CR-/CB-. Almost no colocalization of CCK with SOM was observed, but there was extensive colocalization of SOM and CB. CCK+, CR+, and CCK+/CR+ double-labeled axon terminals were seen surrounding pyramidal cell somata in basket-like plexuses, as well as in the neuropil. CB+, SOM+, and CB+/SOM+ terminals did not form baskets, suggesting that these IN subpopulations are mainly dendrite-targeting neurons. In general, the IN subpopulations in the monkey are not dissimilar to those seen in rodents but, unlike rodents, CB+ INs in the monkey are not basket cells., (Copyright © 2020 IBRO. Published by Elsevier Ltd. All rights reserved.)

The primate amygdaloid complex (AC) contains projection neurons as well as subsets of interneurons (IN), many of which express calcium-binding proteins, that through their local circuits control the activity of the projection neurons. The inhibitory parvalbumin (PV) and calretinin (CR)-positive (+) AC IN have a crucial role in the appearance of synchronized oscillations in local ensembles of projection neurons that mediate the consolidation and recall of fear memories. The GABAergic transmission of these subsets of IN is modulated by dopamine. To expand the knowledge regarding the cellular composition and distribution of IN in the human AC, we focused on two non-overlapping populations: the PV+ and CR+. We have analyzed the distribution of these IN throughout the AC from subjects without any neurological or psychiatric disorders and estimated their absolute number and density using stereological methods. We have also provided percentages of the IN with respect to the total AC neurons. The CR + IN were distributed throughout the AC, whereas the PV+ were only present in the basolateral nuclear group. The quantity of CR + IN was four times higher than that of PV+ and the percentages varied from less than 1% for PV + IN to 6-20% for CR+. The differences in quantity and distribution of CR+ and PV + IN could be related to their differential inhibitory properties and to the intrinsic and extrinsic connections of every amygdaloid region., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: We reported that the pancreas of the interferon-regulatory factor (IRF) 2 knock-out (KO) mouse represents an early phase of acute pancreatitis, including defective regulatory exocytosis, intracellular activation of trypsin, and disturbance of autophagy. The significantly upregulated and downregulated genes in the IRF2 KO pancreas have been reported. The catalogue of gene transcripts included two types of calcium-binding proteins (S100 calcium binding protein G [S100g] and Annexin A10 [Anxa10]), which were highly upregulated in the IRF2 KO pancreas. As the intracellular calcium signal plays a pivotal role in regulatory exocytosis and its disturbance is related to pancreatitis, we then evaluated the role of S100g and Anxa10 in acute pancreatitis., Method: We induced cerulein-pancreatitis in wild-type mice and examined the changes in the expression of these genes by qPCR and immunohistochemistry. We constructed S100g-overexpressing or Anxa10-overexpressing AR42J cells (AR42J-S100g, AR42J-Anxa10). We examined the changes in amylase secretion, intracellular calcium ([Ca 2+ ]i), and cell viability in these cells, when incubated with cholecystokinin (CCK)., Results: The expression of S100g and Anxa10 was increased in cerulean-induced pancreatitis. The acini were patchily stained for S100g and the cytosol of acini was evenly but weakly stained for Anxa10. Stimulation with 100pM CCK-8, decreased amylase secretion and inhibited the [Ca 2+ ]i increase in AR42J-S100g cells. These effects were weak in AR42J-Anxa10 cells. Cell viability was not changed by incubation with cerulein., Conclusion: In cerulean pancreatitis, the expression of S100g and Anxa10 was induced in the acini. S100g may work as a Ca 2+ buffer in acute pancreatitis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Bovine S100 G (calbindin D 9k , small Ca 2+ -binding protein of the EF-hand superfamily) is considered as a calcium buffer protein; i.e., the binding of Ca 2+ practically does not change its general conformation. A set of experimental approaches has been used to study structural properties of apo- and Ca 2+ -loaded forms of mouse S100 G (81.4% identity in amino acid sequence with bovine S100 G). This analysis revealed that, in contrast to bovine S100 G, the removal of calcium ions increases α-helices content of mouse S100 G protein and enhances its accessibility to digestion by α-chymotrypsin. Furthermore, mouse apo-S100 G is characterized by a decreased surface hydrophobicity and reduced tendency for oligomerization. Such behavior is typical of calcium sensor proteins. Apo-state of mouse S100 G still has rather compact structure, which can be cooperatively unfolded by temperature and GdnHCl. Computational analysis of amino acid sequences of S100 G proteins shows that these proteins could be in a disordered state upon a removal of the bound calcium ions. The experimental data show that, although mouse apo-S100 G is flexible compared to the Ca 2+ -loaded state, the apo-form is not completely disordered and preserves some cooperatively meting structure. The origin of the unexpectedly high stability of mouse S100 G can be rationalized by an exceptionally strong association of its N- and C-terminal parts containing the EF-hands I and II, respectively., Competing Interests: Declaration of Competing Interest We, Sergei E. Permyakov, Elena N. Yundina, Alexei S. Kazakov, Maria E. Permyakova, Vladimir N. Uversky, and Eugene A. Permyakov, authors of the manuscript entitled “Mouse S100 G protein exhibits properties characteristic of a calcium sensor”, wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Recently, it was reported that 25(OH)D 3 (25D3) has physiological bioactivity in certain tissues derived from Cyp27b1 knockout mice. To investigate the function of 25D3 in the kidney as an informational crossroad of various calciotropic substances, we employed the CRISPR-Cas9 system to knock out Cyp27b1 in the mouse renal distal tubular mDCT cell line. Unlike the previously reported mice in which Cyp27b1 was targeted systemically, Cyp27b1 knockout mDCT cells did not produce any measurable 1α,25(OH) 2 D 3 (1,25D3) after 25D3 administration. As was seen with treatment of Cyp27b1 knockout mDCT cells with ≥10 -8 M of 1,25D3, the administration of 10 -7 M of 25D3 translocated the vitamin D 3 receptor (VDR) into the nucleus and promoted the expression of the representative 1,25D3-responsive gene Cyp24a1. The exhaustive target gene profiles of 25D3 were similar to those of 1,25D3. Subsequently, we confirmed that 25D3 induced the expression of the calcium reabsorption-related gene calbindin-D9K, in a way similar to 1,25D3. We also found that 1,25D3 and 25D3 induced the expression of the megalin gene. A chromatin immunoprecipitation assay identified two vitamin D response elements in the upstream region of the megalin gene that seemed to contribute to its expression. Together, we surmise that the ability of 25D3 to stimulate VDR target genes may provide a novel perspective for its role in certain tissues., Competing Interests: Declaration of Competing Interest None, (Copyright © 2020. Published by Elsevier Ltd.)

17β-estradiol (E2) is considered to be an important instigator of prolactinoma, and can positively regulate the expression of calbindin-D9k (CaBP-9k) which contains an estrogen responsive element (ERE) via estrogen receptors (ERs). However, the detailed mechanism of E2 in promoting CaBP-9k expression and their roles in prolactinoma progression remain unclear. Here, we aimed to characterize it. The luciferase gene reporter assay with luc-ERE transfection showed that E2 treatment significantly enhanced the transcriptional level of CaBP-9k, whereas CaBP-9k activity was reduced when GH3 and MMQ cells were treated with AZD9496, an antagonist of ERα. E2 treatment increased the protein expressions of CaBP-9k and ERα but not ERβ, whereas this effect was also abolished when cells were treated with AZD9496. Besides, immunoprecipitation (IP) and immunofluorescence assays demonstrated that CaBP-9k could directly interact with ERα not ERβ, and Chromatin IP (ChIP) assay showed that ERα could bind to ERE of the CaBP-9k promoter. Moreover, cell counting kit-8 (CCK-8) and flow cytometry assays showed that E2 treatment significantly enhanced cell viability and inhibited cell apoptosis, but these effects were all abolished when ERα was down-regulated by short hairpin RNA (shRNA) or inhibited by AZD9496, as well as CaBP-9K suppression in both GH3 and MMQ cell lines. Taken together, these findings indicated that E2 stimulation promoted prolactin cell proliferation and inhibited cell apoptosis through ERα-induced CaBP-9k up-regulation, which then accelerated the advanced progression of prolactinoma., (© 2020 The Author(s).)

For precisely regulating intracellular Ca 2+ signals in a time- and space-dependent manner, cells make use of various components of the "Ca 2+ signaling toolkit," including Ca 2+ entry and Ca 2+ extrusion systems. A class of cytosolic Ca 2+ -binding proteins termed Ca 2+ buffers serves as modulators of such, mostly short-lived Ca 2+ signals. Prototypical Ca 2+ buffers include parvalbumins (α and β isoforms), calbindin-D9k, calbindin-D28k, and calretinin. Although initially considered to function as pure Ca 2+ buffers, that is, as intracellular Ca 2+ signal modulators controlling the shape (amplitude, decay, spread) of Ca 2+ signals, evidence has accumulated that calbindin-D28k and calretinin have additional Ca 2+ sensor functions. These other functions are brought about by direct interactions with target proteins, thereby modulating their targets' function/activity. Dysregulation of Ca 2+ buffer expression is associated with several neurologic/neurodevelopmental disorders including autism spectrum disorder (ASD) and schizophrenia. In some cases, the presence of these proteins is presumed to confer a neuroprotective effect, as evidenced in animal models of Parkinson's or Alzheimer's disease., (Copyright © 2020 Cold Spring Harbor Laboratory Press; all rights reserved.)

Intracellular calcium ion content is tightly regulated for the maintenance of cellular functions and cell survival. Calbindin-D 9k (CaBP-9k) is responsible for regulating the distribution of cytosolic free-calcium ions. In this study, we aimed to investigate the effect of CaBP-9k on cell survival in pancreatic beta cells. Six-month-old wildtype CaBP-9k, CaBP-28k, and CaBP-9k/28k knockout (KO) mice were used to compare the pathological phenotypes of calcium-binding protein-deleted mice. Subsequently, the endoplasmic reticulum (ER) stress reducer tauroursodeoxycholic acid (TUDCA) was administered to wildtype and CaBP-9k KO mice. In vitro assessment of the role of CaBP-9k was performed following CaBP-9k overexpression and treatment with the ER stress inducer thapsigargin. Six-month-old CaBP-9k KO mice showed reduced islet volume and up-regulation of cell death markers resulting from ER stress, which led to pancreatic beta cell death. TUDCA treatment recovered islet volume, serum insulin level, and abdominal fat storage by CaBP-9k ablation. CaBP-9k overexpression elevated insulin secretion and recovered thapsigargin-induced ER stress in the INS-1E cell line. The results of this study show that CaBP-9k can protect pancreatic beta cell survival from ER stress and contribute to glucose homeostasis, which can reduce the risk of type 1 diabetes and provide the molecular basis for calcium supplementation to diabetic patients.

Calbindin-D 9k (CaBP-9k), one of the major calcium-binding and calcium-buffering proteins, is important in the physiological functioning of organs. The neuroanatomical localization of CaBP-9k in the rodent brain has not been reported; thus, this study investigated the neuroanatomical distribution of CaBP-9k and the regulation of CaBP-9k expression on steroid hormones in the immature rat brain. To confirm the influence of steroid hormones on CaBP-9k expression, immature female rats were injected for 5 days with estrogen (E2), progesterone (P4), dexamethasone (DEX), and their antagonists (ICI 182, 780 and RU 486). The localization and expression of the CaBP-9k protein in brain regions were identified by immunofluorescence and western blot assays, respectively. We observed that CaBP-9k expression was especially strong in hypothalamus, cerebellum, and brain stem. In addition, CaBP-9k was colocalized with mature-, GABAergic, dopaminergic, and oxytocinergic neurons. We also observed that the CaBP-9k protein level was significantly increased by P4 and reversed by antagonist RU 486 treatment in immature rat brain. In summary, CaBP-9k positive cells have a wide distribution in the immature rat brain, and CaBP-9k expression is regulated by P4. We suggest that CaBP-9k expression regulated by steroid hormone may serve as an important regulator of cytosolic calcium concentration in the brain., (Copyright © 2019. Published by Elsevier Inc.)

Dexamethasone inhibits mucin secretion considering the primary option for treating acute asthma exacerbation. However, the mechanism underlying dexamethasone-induced decreased in mucosecretion is unclear. Recent studies have reported that dexamethasone exerts an inhibitory effect on mucosecretion in the lung by modulating the expression of calcium processing genes. However, the expression of the calcium processing genes in the trachea is not examined yet. Thus, the present study is the first to report the localization of calcium processing proteins such as transient receptor potential vanilloid-4 (Trpv4), transient receptor potential vanilloid-6 (Trpv6), calbindin-D 9k (CaBP-9k) and plasma membrane Ca 2+ -ATPase 1 (Pmca1) in the mouse trachea and their glucocorticoid-induced response. In this study, mice were subcutaneously injected with dexamethasone for 5 days, and their tracheal samples were collected by dividing the trachea into the cervical, and thoracic sections based on its anatomical structure. The localization of TRPV4, TRPV6, CaBP-9k, and PMCA1 proteins was detected in the tracheal epithelium, submucosal glands, cartilages and muscles. Dexamethasone treatment downregulated the mRNA expression of the four calcium processing genes and mucin producing genes. The dexamethasone-induced decrease in the secretion of mucosubstances in the trachea was determined by performing Alcian blue-periodic acid-Schiff staining. Thus, the findings of the present study suggest that glucocorticoids simultaneously can regulate the expression of calcium processing genes and tracheal mucosecretion.

This study explored the influence of triclosan (TCS) in the absence and presence of sodium fluoride (NaF) on estrogenic activity and thyroid function of adolescent female rats. The results indicated that the individual exposure to TCS evoked a significant decline in T 3 and T 4 but the levels of estradiol, FSH, and LH were significantly elevated beside marked up regulation of calbindin-D9k and estrogen α mRNA expression. On the other hand, the single exposure to NaF causes insignificant changes in thyroid hormones, but evoked a trend toward an increase in both estradiol and LH levels. No significant differences in the TSH level were recorded among the experimental groups. The joint exposure to TCS and NaF induced a significant improvement in thyroid and reproductive hormone levels. Overall, these findings revealed that exposure to TCS resulted in significant endocrine and reproductive alterations in immature female rats, while TCS + NaF coexposure resulted in lessening most effects., (© 2018 Wiley Periodicals, Inc.)

Significant alterations in maternal calcium (Ca 2+ ) and magnesium (Mg 2+ ) balance occur during lactation. Ca 2+ is the primary divalent cation mobilized into breast milk by demineralization of the skeleton and alterations in intestinal and renal Ca 2+ transport. Mg 2+ is also concentrated in breast milk, but the underlying mechanisms are not well understood. To determine the molecular alterations in Ca 2+ and Mg 2+ transport in the intestine and kidney during lactation, three groups of female mice consisting of either nonpregnant controls, lactating mice, or mice undergoing involution were examined. The fractional excretion of Ca 2+ , but not Mg 2+ , rose significantly during lactation. Renal 1-α hydroxylase and 24-OHase mRNA levels increased markedly, as did plasma 1,25 dihydroxyvitamin D levels. This was accompanied by significant increases in intestinal expression of Trpv6 and S100g in lactating mice. However, no alterations in the expression of cation-permeable claudin-2, claudin-12, or claudins-15 were found in the intestine. In the kidney, increased expression of Trpv5 and Calb1 was observed during lactation, while no changes in claudins involved in Ca 2+ and Mg 2+ transport (claudin-2, claudin-14, claudin-16, or claudin-19) were found. Consistent with the mRNA expression, expression of both calbindin-D 28K and transient receptor potential vanilloid 5 (TRPV5) proteins increased. Colonic Trpm6 expression increased during lactation, while renal Trpm6 remained unaltered. In conclusion, proteins involved in transcellular Ca 2+ and Mg 2+ transport pathways increase during lactation, while expression of paracellular transport proteins remained unchanged. Increased fractional Ca 2+ excretion can be explained by vitamin D-dependent intestinal hyperabsorption and bone demineralization, despite enhanced transcellular Ca 2+ uptake by the kidney., (Copyright © 2017 the American Physiological Society.)

Supplementation with interleukin (IL)-10, an important anti-inflammatory cytokine, has shown disappointing efficacy for inflammatory bowel diseases (IBD). IL-10 may down-regulate the expression of other anti-inflammatory mediators following colitis induction. We used a colitis model characterized by hapten-protein visualization, which indicates the site of hapten-protein formation after colitis induction for histological and gene expression analyses. Under IL-10 deficiency, following colitis induction inflammatory changes were reduced, and S100G expression was elevated. S100G was expressed in fibroblasts, and S100G expression was down-regulated by IL-10. S100G suppressed the production of monocyte chemotactic protein-1 (MCP-1) through the inhibition of NF-κB activation. Therefore, S100G, also known as Calbindin-D9k, may be an important anti-inflammatory mediator in fibroblasts following colitis induction, and down-regulation of S100G expression might be one reason for the insufficient performance of IL-10 supplementation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Uteri of mature Egyptian buffalo cows (5-10 years old) were collected at follicular (n=12) and luteal (n=16) phases of estrous cycle to investigate the expression of calbindin-D9k (CaPB-9k) and vitamin D receptor (VDR). This study was done using avidin-biotin immunohistochemistry method. In addition, blood levels of calcium (Ca), vitamin D3 (Vit D), estrogen (E2) and progesterone (P4) were measured. The immunohistochemical findings restricted the expressions of CaBP-9k and VDR to the luminal and glandular epithelia of the endometrium implicating the importance of CaBP-9K and VDR in the function of endometrial epithelium, especially the glandular one, in order to prepare a receptive uterus. On the other hand, the myometrium did not express CaBP-9k or VDR that denies the potential role of CaBP-9k and VDR in the uterine contractility during the estrous cycle of Egyptian buffalo. All of Ca, Vit D, and P4 blood levels significantly (P<0.05) increased during luteal phase however, blood level of E2 significantly (P<0.05) increased during follicular phase. The expressions of CaBP-9k and VDR in the uterus of Egyptian buffalo were significantly (P<0.05) higher during luteal (P4 dominant) phase than during the follicular (E2 dominant) phase indicating that P4 up-regulates the expressions of CaBP-9k and VDR. In view of these observations, this study represents the first characterization of CaBP-9K and VDR expression in the uterus of Egyptian buffalo and suggests the pivotal role of CaBP-9k and VDR in the uterine receptivity. Furthermore, it demonstrates the regulatory role of P4 for expressions of CaBP-9k and VDR in buffalo uterus., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Calbindin-D28k (CBD-28k) is a calcium binding protein located in the distal convoluted tubule (DCT) and plays an important role in active calcium transport in the kidney. Loop and thiazide diuretics affect renal Ca and Mg handling: both cause Mg wasting, but have opposite effects on Ca excretion as loop diuretics increase, but thiazides decrease, Ca excretion. To understand the role of CBD-28k in renal Ca and Mg handling in response to diuretics treatment, we investigated renal Ca and Mg excretion and gene expression of DCT Ca and Mg transport molecules in wild-type (WT) and CBD-28k knockout (KO) mice. Mice were treated with chlorothiazide (CTZ; 50 mg · kg(-1) · day(-1)) or furosemide (FSM; 30 mg · kg(-1) · day(-1)) for 3 days. To avoid volume depletion, salt was supplemented in the drinking water. Urine Ca excretion was reduced in WT, but not in KO mice, by CTZ. FSM induced similar hypercalciuria in both groups. DCT Ca transport molecules, including transient receptor potential vanilloid 5 (TRPV5), TRPV6, and CBD-9k, were upregulated by CTZ and FSM in WT, but not in KO mice. Urine Mg excretion was increased and transient receptor potential subfamily M, member 6 (TRPM6) was upregulated by both CTZ and FSM in WT and KO mice. In conclusion, CBD-28k plays an important role in gene expression of DCT Ca, but not Mg, transport molecules, which may be related to its being a Ca, but not a Mg, intracellular sensor. The lack of upregulation of DCT Ca transport molecules by thiazides in the KO mice indicates that the DCT Ca transport system is critical for Ca conservation by thiazides., (Copyright © 2016 the American Physiological Society.)

Background: Proton-pump inhibitor-induced hypomagnesemia (PPIH) is the most recognized side effect of proton-pump inhibitors (PPIs). Additionally, PPIH is associated with hypocalcemia and hypokalemia. It is hypothesized that PPIs reduce epithelial proton secretion and thereby increase the pH in the colon, which may explain the reduced absorption of and Mg2+ and Ca2+. Fermentation of dietary oligofructose-enriched inulin fibers by the microflora leads to acidification of the intestinal lumen and by this enhances mineral uptake. This study aimed, therefore, to improve mineral absorption by application of dietary inulin to counteract PPIH., Methods: Here, C57BL/J6 mice were supplemented with omeprazole and/or inulin. Subsequently, Mg2+ and Ca2+ homeostasis was assessed by means of serum, urine and fecal electrolyte measurements. Moreover, the mRNA levels of magnesiotropic and calciotropic genes were examined in the large intestine and kidney by real-time PCR., Results: Treatment with omeprazole significantly reduced serum Mg2+ and Ca2+ levels. However, concomitant addition of dietary inulin fibers normalized serum Ca2+ but not serum Mg2+ concentrations. Inulin abolished enhanced expression of Trpv6 and S100g in the colon by omeprazole. Additionally, intestinal and renal mRNA levels of the Trpm6 gene were reduced after inulin intake., Conclusions: This study suggests that dietary inulin counteracts reduced intestinal Ca2+ absorption upon PPI treatment. In contrast, inulin did not increase intestinal absorption of Mg2+ sufficiently to recover serum Mg2+. The clinical potential of dietary inulin treatment should be the subject of future studies.

Bisphenol A (BPA), an industrial chemical used in the manufacture of polycarbonate and epoxy resins, binds to the nuclear estrogen receptor with an affinity 4-5 orders of magnitude lower than that of estradiol. We reported previously that "high BPA" [100,000 and 300,000 µg/kg body weight (bw)/day], but not "low BPA" (2.5-2700 µg/kg bw/day), induced clear adverse effects in NCTR Sprague-Dawley rats gavaged daily from gestation day 6 through postnatal day (PND) 90. The "high BPA" effects partially overlapped those of ethinyl estradiol (EE2, 0.5 and 5.0 µg/kg bw/day). To evaluate further the potential of "low BPA" to induce biological effects, here we assessed the global genomic DNA methylation and gene expression in the prostate and female mammary glands, tissues identified previously as potential targets of BPA, and uterus, a sensitive estrogen-responsive tissue. Both doses of EE2 modulated gene expression, including of known estrogen-responsive genes, and PND 4 global gene expression data showed a partial overlap of the "high BPA" effects with those of EE2. The "low BPA" doses modulated the expression of several genes; however, the absence of a dose response reduces the likelihood that these changes were causally linked to the treatment. These results are consistent with the toxicity outcomes., (Published by Elsevier Ltd.)

The charge state of proteins in solution is a key biophysical parameter that modulates both long and short range macromolecular interactions. However, unlike in the case of many small molecules, the effective charges of complex biomolecules in solution cannot in general be predicted reliably from their chemical structures alone. Here we present an approach for quantifying the effective charges of solvated biomolecules from independent measurements of their electrophoretic mobilities and diffusion coefficients in free solution within a microfluidic device. We illustrate the potential of this approach by determining the effective charges of a charge-ladder family of mutants of the calcium binding protein calbindin D9k in solution under native conditions. Furthermore, we explore ion-binding under native conditions, and demonstrate the ability to detect the chelation of a single calcium ion through the change that ion binding imparts on the effective charge of calbindin D9k. Our findings highlight the difference between the dry sequence charge and the effective charge of proteins in solution, and open up a route towards rapid and quantitative charge measurements in small volumes in the condensed phase.

Ca(2+)-binding proteins are widely distributed throughout cells and play various important roles. Calbindin D9k is a member of the EF-hand Ca(2+)-binding protein family. In this study, we examined the binding of Ca(2+) to calbindin D9k in terms of the free energy of solvation, as obtained by 3D reference interaction site model theory, which describes the statistical mechanics of liquids. We also investigated the main structural biological factor using spatial decomposition analysis in which the solvation free energy values are decomposed into the residue. We found some characteristic residues that contribute to stabilization of the holo-structure (Ca(2+)-binding structure). These results indicated that, in the holo-structure, these residues are newly exposed to solvent. Subsequently, the gain in solvation free energy, involving a conformational change and exposure to solvent, forms the driving force for binding of the Ca(2+) ion to the EF-hand.

Optimizing peak bone mass in early life is one of key preventive strategies against osteoporosis. Fructus ligustri lucidi (FLL), the fruit of Ligustrum lucidum Ait., is a commonly prescribed herb in many kidney-tonifying traditional Chinese medicinal formulas to alleviate osteoporosis. Previously, FLL extracts have been shown to have osteoprotective effect in aged or ovariectomized rats. In the present study, we investigated the effects of FLL ethanol extract on bone mineral density (BMD) and mechanical properties in growing male rats and explored the underlying mechanisms. Male weaning Sprague-Dawley rats were randomized into four groups and orally administrated for 4 months an AIN-93G formula-based diet supplementing with different doses of FLL ethanol extract (0.40, 0.65, and 0.90 %) or vehicle control, respectively. Then calcium balance, serum level of Ca, P, 25(OH)2D3, 1,25(OH)2D3, osteocalcin (OCN), C-terminal telopeptide of type I collagen (CTX-I), and parathyroid hormone, bone microarchitecture, and calcium absorption-related genes expression in duodenum and kidney were analyzed. The results demonstrated that FLL ethanol extract increased BMD of growing rats and improved their bone microarchitecture and mechanical properties. FLL ethanol extract altered bone turnover, as evidenced by increasing a bone formation maker, OCN, and decreasing a bone resorption maker, CTX-I. Intriguingly, both Ca absorption and Ca retention rate were elevated by FLL ethanol extract treatment, possibly through the mechanisms of up-regulating the transcriptions of calcitropic genes in kidney (1α-hydroxylase) and duodenum (vitamin D receptor, calcium transporter calbindin-D9k, and transient receptor potential vanilloid 6). In conclusion, FLL ethanol extract increased bone mass gain and improved bone properties via modulating bone turnover and up-regulating calcium absorption-related gene expression in kidney and duodenum, which could then activate 1,25(OH)2D3-dependent calcium transport in male growing rats.

Malignant mesothelioma (MM) are highly aggressive asbestos-related neoplasms, which show strong chemotherapy resistance, and there is no effective cure for MM so far. Calretinin (CR) is widely used as a diagnostic marker for epithelioid and mixed (biphasic) mesothelioma; however, little is known about CR's putative functions in tumorigenesis. CR protects against asbestos-induced acute cytotoxicity mediated by the AKT/PI3K pathway, and furthermore, SV40 early region genes are able to upregulate CR in mesothelial cells. However, the precise role of CR in mesothelioma is still unknown. Downregulation of CR via lentiviral-mediated short-hairpin RNA significantly decreased the viability and proliferation of mesothelioma cells in vitro. The effect was strong in epithelioid-dominated cell lines (ZL55 and MSTO-211H). A weaker and delayed effect was observed in mesothelioma cells with prevalent sarcomatoid morphology (SPC111, SPC212 and ZL34). The specificity of the effect was confirmed by stable enhanced green fluorescent protein-CR expression in mesothelioma cell lines and subsequent downregulation. Depletion of CR led these cancer cell lines to enter apoptosis within 72 hr postinfection via strong activation of the intrinsic caspase 9-dependent pathway. Downregulation of CR in immortalized mesothelial cells LP9/TERT-1 strongly blocked proliferation and caused a G1 block without decreasing viability or activating apoptosis pathways. These results demonstrate that downregulation of CR had a strong effect on the viability of MM cells and that CR is essential for cells derived from MM. The authors anticipate these findings to reveal CR as a highly interesting new putative therapeutic target for mesothelioma treatment of especially the epithelioid, as well as of the mixed and sarcomatoid type., (© 2013 UICC.)

Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. The lifetime of in-cell NMR samples is however much shorter than that in culture media, presumably because of various stresses as well as the nutrient depletion in the anaerobic environment within the NMR tube. It is well known that Ca(2+)-bursts occur in HeLa cells under various stresses, hence the cytosolic Ca(2+) concentration can be regarded as a good indicator of the healthiness of cells in NMR tubes. In this study, aiming at monitoring the states of proteins resulting from the change of cytosolic Ca(2+) concentration during experiments, human calbindin D9k (P47M+C80) was used as the model protein and cultured HeLa cells as host cells. Time-resolved measurements of 2D (1)H-(15)N SOFAST-HMQC experiments of calbindin D9k (P47M+C80) in HeLa cells showed time-dependent changes in the cross-peak patterns in the spectra. Comparison with in vitro assignments revealed that calbindin D9k (P47M+C80) is initially in the Mg(2+)-bound state, and then gradually converted to the Ca(2+)-bound state. This conversion process initiates after NMR sample preparation. These results showed, for the first time, that cells inside the NMR tube were stressed, presumably because of cell precipitation, the lack of oxygen and nutrients, etc., thereby releasing Ca(2+) into cytosol during the measurements. The results demonstrated that in-cell NMR can monitor the state transitions of stimulated cells through the observation of proteins involved in the intracellular signalling systems. Our method provides a very useful tool for in situ monitoring of the "healthiness" of the cells in various in-cell NMR studies., (Copyright © 2013 Elsevier Inc. All rights reserved.)

It has remained difficult to ascribe a specific functional role to immobile or fixed intracellular calcium buffers in central neurons because the amount of these buffers is unknown. Here, we explicitly isolated the fixed buffer fraction by prolonged whole-cell patch-clamp dialysis and quantified its buffering capacity in murine hippocampal slices using confocal calcium imaging and the "added-buffer" approach. In dentate granule cells, the calcium binding ratio (κ) after complete washout of calbindin D28k (Cb), κfixed, displayed a substantial value of ∼100. In contrast, in CA1 oriens lacunosum moleculare (OLM) interneurons, which do not contain any known calcium-binding protein(s), κfixed amounted to only ∼30. Based on these values, a theoretical analysis of dendritic spread of calcium after local entry showed that fixed buffers, in the absence of mobile species, decrease intracellular calcium mobility 100- and 30-fold in granule cells and OLM cells, respectively, and thereby strongly slow calcium signals. Although the large κfixed alone strongly delays the spread of calcium in granule cells, this value optimizes the benefits of additionally expressing the mobile calcium binding protein Cb. With such high κfixed, Cb effectively increases the propagation velocity to levels seen in OLM cells and, contrary to expectation, does not affect the peak calcium concentration close to the source but sharpens the spatial and temporal calcium gradients. The data suggest that the amount of fixed buffers determines the temporal availability of calcium for calcium-binding partners and plays a pivotal role in setting the repertoire of cellular calcium signaling regimens.

Context: Up to 17% of all rectal suction biopsies performed in the evaluation of Hirschsprung disease are considered inadequate. In most instances, inadequate biopsies contain too little submucosa or are taken within the anal transition zone., Objectives: To examine the utility of calretinin stain in the workup of inadequate biopsies for patients with clinical suspicion of Hirschsprung disease., Design: A retrospective analysis was conducted of all rectal suction biopsies performed in the evaluation of Hirschsprung disease during the previous 12 years that were considered "inadequate." Seventeen cases were identified, and Hirschsprung disease status was determined by clinical or surgical follow-up. Immunohistochemistry for calretinin was performed for all cases containing columnar mucosa, which were evaluated without knowledge of clinical course., Results: All 12 patients without Hirschsprung disease had calretinin-positive nerve fibers in the lamina propria or muscularis mucosae, and all 5 patients with Hirschsprung disease had no calretinin staining of nerves., Conclusions: In this retrospective series, calretinin immunohistochemistry correctly predicted outcome in all instances. Although the gold standard for the diagnosis of Hirschsprung disease in rectal suction biopsies remains the evaluation of ganglion cells in a hematoxylin-eosin staining with sufficient submucosa, calretinin immunohistochemistry is quite helpful in triaging further workup based on clinical suspicion.

Enhancement of sound-evoked responses in auditory cortex (ACx) following administration of systemic nicotine is known to depend on activation of extracellular-signaling regulated kinase (ERK), but the nature of this enhancement is not clear. Here, we show that systemic nicotine increases the density of cells immunolabeled for phosphorylated (activated) ERK (P-ERK) in mouse primary ACx (A1). Cortical injection of dihydro-β-erythroidine reduced nicotine-induced P-ERK immunolabel, suggesting a role for nicotinic acetylcholine receptors located in A1 and containing α4 and β2 subunits. P-ERK expressing cells were distributed mainly in layers 2/3 and more sparsely in lower layers, with many cells exhibiting immunolabel within pyramidal-shaped somata and proximal apical dendrites. About one-third of P-ERK positive cells also expressed calbindin. In the thalamus, P-ERK immunopositive cells were found in the nonlemniscal medial geniculate (MG) and adjacent nuclei, but were absent in the lemniscal MG. Pairing broad spectrum acoustic stimulation (white noise) with systemic nicotine increased P-ERK immunopositive cell density in ACx as well as the total amount of P-ERK protein, particularly the phosphorylated form of ERK2. However, narrow spectrum (tone) stimulation paired with nicotine increased P-ERK immunolabel preferentially at a site within A1 where the paired frequency was characteristic frequency (CF), relative to a second site with a spectrally distant CF (two octaves above or below the paired frequency). Together, these results suggest that ERK is activated optimally where nicotinic signaling and sound-evoked neural activity converge., (Copyright © 2013 Wiley Periodicals, Inc.)

Transient receptor potential vanilloid subtype 1 (TRPV1) receptor is a pain-sensing, ligand-gated, non-selective cation channel expressed in peripheral sensory neurons. Prolonged activation of TRPV1 by capsaicin leads to cell swelling and formation of membrane blebs in rat dorsal root ganglion (DRG) neurons. Similar results were obtained in NIH3T3 fibroblast cells stably expressing TRPV1. Here, we assessed the contribution of Ca(2+) and Na(+) ions to TRPV1-mediated changes. Cell swelling was caused by a substantial influx of extracellular Na(+) via TRPV1 channels, causing concomitant transport of water. In the absence of extracellular Na(+), the membrane blebbing was completely inhibited, but Ca(2+) influx did not change under these conditions. Na(+) influx was modulated by the intracellular Ca(2+) concentration ([Ca(2+)]i). Elevation of [Ca(2+)]i by ionomycin sensitized/activated TRPV1 channels causing cell swelling in TRPV1-positive cells. In the absence of extracellular Ca(2+), capsaicin caused only little increase in [Ca(2+)]i indicating that the increase in [Ca(2+)]i observed after capsaicin application is derived essentially from extracellular Ca(2+) and not from internal Ca(2+) stores. In the absence of extracellular Ca(2+) also the process of cell swelling was considerably slower. Calretinin is a Ca(2+) buffer protein, which is expressed in a subset of TRPV1-positive neurons. Calretinin decreased the amplitude, but slowed down the decay of Ca(2+) signals evoked by ionomycin. Cells co-expressing TRPV1 and calretinin were less sensitive to TRPV1-mediated, capsaicin-induced volume increases. In TRPV1-expressing NIH3T3 cells, calretinin decreased the capsaicin-induced Ca(2+) and Na(+) influx. Swelling and formation of membrane blebs resulted in impaired plasma membrane integrity finally leading to cell death. Our results hint towards a mechanistic explanation for the apoptosis-independent capsaicin-evoked neuronal loss and additionally reveal a protective effect of calretinin; we propose that the Ca(2+)-buffering capacity of calretinin reduces the susceptibility of calretinin-expressing DRG neurons against cell swelling/death caused by overstimulation of TRPV1 channels. This article is part of a Special Issue entitled:12th European Symposium on Calcium., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Fear memory circuits in the brain function to allow animals and humans to recognize putative sources of danger and adopt an appropriate behavioral response; and research on animal models of fear have helped reveal the anatomical and neurochemical nature of these circuits. The nucleus (n.) incertus in the dorsal pontine tegmentum provides a strong GABAergic projection to forebrain 'fear centers' and is strongly activated by neurogenic stressors. In this study in adult male rats, we examined the effect of electrolytic lesions of n. incertus on different stages of the fear conditioning-extinction process and correlated the outcomes with anatomical data on the distribution of n. incertus-derived nerve fibers in areas implicated in fear circuits. In a contextual auditory fear conditioning paradigm, we compared freezing behavior in control (naïve) rats (n=23) and rats with sham- or electrolytic lesions of n. incertus (n=13/group). The effectiveness and extent of the lesions was assessed post-mortem using immunohistochemical markers for n. incertus neurons-calretinin and relaxin-3. There were no differences between the three experimental groups in the habituation, acquisition, or context conditioning phases; but n. incertus lesioned rats displayed a markedly slower, 'delayed' extinction of conditioned freezing responses compared to sham-lesion and control rats, but no differences in retrieval of extinguished fear. These and earlier findings suggest that n. incertus-related circuits normally promote extinction through inhibitory projections to the amygdala, which is involved in acquisition of extinction memories., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The subthalamic nucleus (STN) of the basal ganglia plays a key role in motor control, and STN efferents are known to mainly target the external segment of the globus pallidus (GPe), entopeduncular nucleus (Ep), and substantia nigra (SN) with some axon collaterals to the other regions. However, it remains to be clarified how each STN neuron projects axon fibers and collaterals to those target nuclei of the STN. Here we visualized the whole axonal arborization of single STN neurons in the rat brain by using a viral vector expressing membrane-targeted green fluorescent protein, and examined the distribution of axon boutons in those target nuclei. The vast majority (8-9) of 10 reconstructed STN neurons projected to the GPe, SN, caudate-putamen (CPu), and Ep, which received, on average ± SD, 457 ± 425, 400 ± 347, 126 ± 143, and 106 ± 100 axon boutons per STN neuron, respectively. Furthermore, the density of axon boutons in the GPe was highest among these nuclei. Although these target nuclei were divided into calbindin-rich and -poor portions, STN projection showed no exclusive preference for those portions. Since STN neurons mainly projected not only to the GPe, SN, and Ep but also to the CPu, the subthalamostriatal projection might serve as a positive feedback path for the striato-GPe-subthalamic disinhibitory pathway, or work as another route of cortical inputs to the striatum through the corticosubthalamostriatal disynaptic excitatory pathway., (Copyright © 2012 Wiley Periodicals, Inc.)

The core motor symptoms of Parkinson's disease (PD) are attributable to the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). Mitochondrial oxidant stress is widely viewed a major factor in PD pathogenesis. Previous work has shown that activity-dependent calcium entry through L-type channels elevates perinuclear mitochondrial oxidant stress in SNc dopaminergic neurons, providing a potential basis for their selective vulnerability. What is less clear is whether this physiological stress is present in dendrites and if Lewy bodies, the major neuropathological lesion found in PD brains, exacerbate it. To pursue these questions, mesencephalic dopaminergic neurons derived from C57BL/6 transgenic mice were studied in primary cultures, allowing for visualization of soma and dendrites simultaneously. Many of the key features of in vivo adult dopaminergic neurons were recapitulated in vitro. Activity-dependent calcium entry through L-type channels increased mitochondrial oxidant stress in dendrites. This stress progressively increased with distance from the soma. Examination of SNc dopaminergic neurons ex vivo in brain slices verified this pattern. Moreover, the formation of intracellular α-synuclein Lewy-body-like aggregates increased mitochondrial oxidant stress in perinuclear and dendritic compartments. This stress appeared to be extramitochondrial in origin, because scavengers of cytosolic reactive oxygen species or inhibition of NADPH oxidase attenuated it. These results show that physiological and proteostatic stress can be additive in the soma and dendrites of vulnerable dopaminergic neurons, providing new insight into the factors underlying PD pathogenesis.

The homeodomain transcription factor Pitx3 is critical for the survival of midbrain dopaminergic (mDA) neurons. Pitx3-deficient mice exhibit severe but selective developmental loss of mDA neurons, with accompanying locomotor deficits resembling those seen in Parkinson's disease (PD) models. Here, we identify specific mDA cell subpopulations that are consistently spared in adult Pitx3-hypomorphic (aphakia) mice, demonstrating that Pitx3 is not indiscriminately required by all mDA neurons for their survival. In aphakia mice, virtually all surviving mDA neurons in the substantia nigra (SN) and the majority of neurons in the adjacent ventral tegmental area (VTA) also express calbindin-D28k, a calcium-binding protein previously associated with resistance to injury in PD and in animal models. Cell-mapping studies in wild-type mice revealed that Pitx3 is primarily expressed in the ventral SN, a region particularly susceptible to MPTP and other dopaminergic neurotoxins. Furthermore, Pitx3-expressing SN cells are preferentially lost following MPTP treatment. Finally, SN mDA neurons in Pitx3 hemizygous mice show increased sensitivity when exposed to MPTP. Thus, SN mDA neurons are represented by at least two distinct subpopulations including MPTP-resistant Pitx3-autonomous, calbindin-positive neurons, and calbindin-negative Pitx-3-dependent cells that display elevated vulnerability to toxic injury, and probably correspond to the subpopulation that degenerates in PD. Impairment of Pitx3-dependent pathways therefore increases vulnerability of mDA neurons to toxic injury. Together, these data suggest a novel link between Pitx3 function and the selective pattern of mDA cell loss observed in PD., (© 2013 International Society for Neurochemistry.)

The present study evaluated the prebiotic effect of a standardised aqueous extract of Anoectochilus formosanus (SAEAF) and its effects on osteoporosis in ovariectomised (OVX) rats. The OVX rats were randomly divided into five groups and orally treated with water, SAEAF (200 and 400 mg/kg daily) and inulin (400 mg/kg daily) for 12 weeks. The sham group was orally treated with water. The SAEAF treatment enhanced the number of faecal bifidobacteria in OVX rats. The results of a Ca-balance experiment showed that SAEAF increased apparent Ca absorption and retention. The OVX rats were killed after SAEAF treatment lasting 12 weeks. The SAEAF decreased the caecal pH values and increased the caecal wall weight, caecal mucosa calbindin-D9k mRNA expression, free-Ca concentration and levels of SCFA in the caecum. The mineral content, density and biomechanical strength of bones were lower in OVX rats than the sham group, but these bone losses were prevented by SAEAF administration. Microtomography scanning showed that the SAEAF-treated rats had higher trabecular bone volume than the OVX rats. These results suggest that SAEAF prevented bone loss associated with ovarian hormone deficiency in the rats.

Accumulation of abnormally integrated, adult-born, hippocampal dentate granule cells (DGCs) is hypothesized to contribute to the development of temporal lobe epilepsy (TLE). DGCs have long been implicated in TLE, because they regulate excitatory signaling through the hippocampus and exhibit neuroplastic changes during epileptogenesis. Furthermore, DGCs are unusual in that they are continually generated throughout life, with aberrant integration of new cells underlying the majority of restructuring in the dentate during epileptogenesis. Although it is known that these abnormal networks promote abnormal neuronal firing and hyperexcitability, it has yet to be established whether they directly contribute to seizure generation. If abnormal DGCs do contribute, a reasonable prediction would be that the severity of epilepsy will be correlated with the number or load of abnormal DGCs. To test this prediction, we used a conditional, inducible transgenic mouse model to fate map adult-generated DGCs. Mossy cell loss, also implicated in epileptogenesis, was assessed as well. Transgenic mice rendered epileptic using the pilocarpine-status epilepticus model of epilepsy were monitored continuously by video/EEG for 4 weeks to determine seizure frequency and severity. Positive correlations were found between seizure frequency and (1) the percentage of hilar ectopic DGCs, (2) the amount of mossy fiber sprouting, and (3) the extent of mossy cell death. In addition, mossy fiber sprouting and mossy cell death were correlated with seizure severity. These studies provide correlative evidence in support of the hypothesis that abnormal DGCs contribute to the development of TLE and also support a role for mossy cell loss.

The field of neurogenesis has greatly benefited from stage-specific marker discoveries. However, such markers are not well defined in the olfactory epithelium (OE), where olfactory receptor neurons (ORNs) are constantly generated throughout lifetime. During OE neurogenesis, there is a lack of markers that label cells that are at the intermediate stage before they are fully mature. In this study, we show that during embryonic development calretinin is expressed transiently in the intermediate cells right before ORNs become mature. Calretinin is expressed between the end of beta-III tubulin (an immature neuronal marker) expression and the beginning of olfactory marker protein (OMP, a mature neuronal marker) expression in ORNs. Therefore, calretinin can serve as a marker of the intermediate ORNs. With this discovery, future studies can use calretinin as a tool to define these intermediate ORNs during olfactory neurogenesis., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Hyperpolarisation-activated (Ih) currents are considered important for dendritic integration, synaptic transmission, setting membrane potential and rhythmic action potential (AP) discharge in neurons of the central nervous system. Hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels underlie these currents and are composed of homo- and hetero-tetramers of HCN channel subunits (HCN1-4), which confer distinct biophysical properties on the channel. Despite understanding the structure-function relationships of HCN channels with different subunit stoichiometry, our knowledge of their expression in defined neuronal populations remains limited. Recently, we have shown that HCN subunit expression is a feature of a specific population of dorsal horn interneurons that exhibit high-frequency AP discharge. Here we expand on this observation and use neuroanatomical markers to first identify well-characterised neuronal populations in the lumbar spinal cord and hippocampus and subsequently determine whether HCN4 expression correlates with high-frequency AP discharge in these populations. In the spinal cord, HCN4 is expressed in several putative inhibitory interneuron populations including parvalbumin (PV)-expressing islet cells (84.1%; SD: ±2.87), in addition to all putative Renshaw cells and Ia inhibitory interneurons. Similarly, virtually all PV-expressing cells in the hippocampal CA1 subfield (93.5%; ±3.40) and the dentate gyrus (90.9%; ±6.38) also express HCN4. This HCN4 expression profile in inhibitory interneurons mirrors both the prevalence of Ih sub-threshold currents and high-frequency AP discharge. Our findings indicate that HCN4 subunits are expressed in several populations of spinal and hippocampal interneurons, which are known to express both Ih sub-threshold currents and exhibit high-frequency AP discharge. As HCN channel function plays a critical role in pain perception, learning and memory, and sleep as well as the pathogenesis of several neurological diseases, these findings provide important insights into the identity and neurochemical status of cells that could underlie such conditions., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

The gene encoding the WD repeat-containing protein 81 (WDR81) has recently been described as the disease locus in a consanguineous family that suffers from cerebellar ataxia, mental retardation, and quadrupedal locomotion syndrome (CAMRQ2). Adult mice from the N-ethyl-N-nitrosourea-induced mutant mouse line nur5 display tremor and an abnormal gait, as well as Purkinje cell degeneration and photoreceptor cell loss. We have used polymorphic marker mapping to demonstrate that affected nur5 mice carry a missense mutation, L1349P, in the Wdr81 gene. Moreover, homozygous nur5 mice that carry a wild-type Wdr81 transgene are rescued from the abnormal phenotype, indicating that Wdr81 is the causative gene in nur5. WDR81 is expressed in Purkinje cells and photoreceptor cells, among other CNS neurons, and like the human mutation, the nur5 modification lies in the predicted major facilitator superfamily domain of the WDR81 protein. Electron microscopy analysis revealed that a subset of mitochondria in Purkinje cell dendrites of the mutant animals displayed an aberrant, large spheroid-like structure. Moreover, immunoelectron microscopy and analysis of mitochondrial-enriched cerebellum fractions indicate that WDR81 is localized in mitochondria of Purkinje cell neurons. Because the nur5 mouse mutant demonstrates phenotypic similarities to the human disease, it provides a valuable genetic model for elucidating the pathogenic mechanism of the WDR81 mutation in CAMRQ2.

Hippocampal CA3 area generates temporally structured network activity such as sharp waves and gamma and theta oscillations. Parvalbumin-expressing basket cells, making GABAergic synapses onto cell bodies and proximal dendrites of pyramidal cells, control pyramidal cell activity and participate in network oscillations in slice preparations, but their roles in vivo remain to be tested. We have recorded the spike timing of parvalbumin-expressing basket cells in areas CA2/3 of anesthetized rats in relation to CA3 putative pyramidal cell firing and activity locally and in area CA1. During theta oscillations, CA2/3 basket cells fired on the same phase as putative pyramidal cells, but, surprisingly, significantly later than downstream CA1 basket cells. This indicates a distinct modulation of CA3 and CA1 pyramidal cells by basket cells, which receive different inputs. We observed unexpectedly large dendritic arborization of CA2/3 basket cells in stratum lacunosum moleculare (33% of length, 29% surface, and 24% synaptic input from a total of ∼35,000), different from the dendritic arborizations of CA1 basket cells. Area CA2/3 basket cells fired phase locked to both CA2/3 and CA1 gamma oscillations, and increased firing during CA1 sharp waves, thus supporting the role of CA3 networks in the generation of gamma oscillations and sharp waves. However, during ripples associated with sharp waves, firing of CA2/3 basket cells was phase locked only to local but not CA1 ripples, suggesting the independent generation of fast oscillations by basket cells in CA1 and CA2/3. The distinct spike timing of basket cells during oscillations in CA1 and CA2/3 suggests differences in synaptic inputs paralleled by differences in dendritic arborizations.

Preclinical studies indicate that selegiline (deprenyl), frequently used in some neurodegenerative diseases, exert protective effects on central nervous system neurons of individuals exposed to social isolation (SI). Furthermore, it has been suggested that SI produces neuronal dysfunction due in part to an excessive intracellular Ca(2+) overload. Since the main intracellular Ca(2+) buffering mechanism involves changes in the calcium-binding protein calbindin-D28k (CB), and that CB neuronal expression can increase in response to Ca(2+) transients, we hypothesized that chronic selegiline administration in early socially isolated animals could minimize cell CB expression as an indirect indicator of protective mechanism against Ca(2+) overload. In the present study male rats were weaned at postnatal day 21 (P21) and randomly assigned to social deprivation (SI) or control (SC) environments for 30 days (P21-51). SI animals were further subdivided in two experimental groups: socially deprived-saline (SI-SAL) and socially isolated-selegiline (SI-SEL) for additional 30 days (P52-82). Medial frontal CB immunoreactivity (CB-ir) neurons were quantitatively and qualitatively analyzed. The results obtained indicate that neocortical cells of adult rats submitted to early SI show a significant increase in the number of CB-ir neurons per cortical field, while selegiline treatment significantly reduces this parameter., (Copyright © 2012 ISDN. Published by Elsevier Ltd. All rights reserved.)