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PubMed1992

Effects of sodium butyrate on 1,25-dihydroxyvitamin D3 receptor activity in primary chick kidney cells.

The genomic effects of the steroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by high affinity nuclear associated specific receptors that belong to the superfamily of ligand induced transcription factors. The carboxylic acid, sodium butyrate--a potent inhibitor of histone deacetylase--is known to modulate gene expression in a variety of systems. Specific binding of 1,25(OH)2D3 to its receptor was examined in primary chick kidney cells, the chick macrophage cell line HD-11, and other mammalian cell lines such as ROS 17/2.8, HT-29 and CV-1 cells, that were all cultured in the presence or absence of 1 mM sodium butyrate. Treatment with n-butyrate resulted in significant (4.0-4.5-fold) increases in 1,25(OH)2D3 receptor binding without changing binding affinity only in the primary cultures of chick renal epithelial cells and the chick macrophage cell line but not in the other heterologous receptor-positive cell lines. The maximum increase in receptor binding was evident at 1 mM butyrate concentration. This effect reached a maximum at 15 h treatment, beyond which there was slow attenuation in increased binding until 24 h. The butyrate induced increases in receptor activity was associated with increases in the 1,25(OH)2D3-mediated induction of calbindin-D28K protein only in primary chick kidney cultures but not in the macrophage cell line (HD-11). Similarly, calbindin-D28K promoter activity was enhanced only in butyrate-treated primary chick kidney cultures, transfected with chimeric plasmids containing the 5' flanking sequence of the calbindin-D28K promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene but not in HD-11 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Development of direct GABAergic projections from the zona incerta to the somatosensory cortex of the rat.

The postnatal development of direct thalamocortical projections from the zona incerta of the ventral thalamus to the whisker representation area of the rat primary somatosensory cortex was investigated. Cytoarchitectonic analysis based on Nissl staining, cytochrome oxidase histochemistry and immunohistochemistry for glutamic acid decarboxylase, GABA, parvalbumin and calbindin D28K revealed that the zona incerta can be clearly distinguished from surrounding diencephalic structures from the day of birth. Moreover, four distinct anatomical subdivisions of this nucleus were identified: the rostral, dorsal, ventral and caudal. Of these, the ventral subdivision is by far the most conspicuous, containing the highest density of neurons, and the highest levels of cytochrome oxidase, glutamate decarboxylase, GABA, parvalbumin and calbindin D28K. In contrast, the dorsal, rostral and caudal subdivisions contain fewer cells, lower levels of glutamic acid decarboxylase and GABA and very few parvalbumin-positive and calbindin-positive neurons. Small injections of rhodamine coated microspheres or Fluoro-gold in the primary somatosensory cortex of animals at different stages of development revealed the existence of retrogradely labeled neurons in the rostral and dorsal subdivisions of the zona incerta from postnatal day 1. At this age, retrogradely labeled cells were also found in the ventral lateral, ventral posterior medial, posterior medial, centrolateral, ventral medial and magnocellular subdivision of the medial geniculate nuclei of the dorsal thalamus. The density of the incertocortical projection reaches its maximum between the first and second postnatal weeks, decreasing subsequently, until an adult pattern of labeling is achieved. Tracer injections combined with immunohistochemistry revealed that the majority of the incertocortical projection derives from GABAergic neurons, implying a potentially inhibitory role for the incertocortical projection. These results demonstrate that the rat trigeminal system contains parallel thalamocortical pathways of opposite polarity, emerging from both the dorsal (glutamatergic, excitatory) and ventral (GABAergic, inhibitory) thalamus since the day of birth. As such, these findings suggest that, contrary to the classical notion, not only the dorsal but also the ventral thalamus may play a special role in both cortical maturation and function.

Animals↗
PubMed1994

Immunocytochemical properties of rat renal afferent neurons in dorsal root ganglia: a quantitative study.

Immunocytochemical properties of dorsal root ganglion neurons innervating the kidney were studied with retrograde tracing of Fluorogold or Fast Blue dyes applied to the cut renal nerves in the rat. The proportions and sizes of renal afferent neurons labelled with a variety of markers were quantified in T9-L1 dorsal root ganglia from five rats. Compared with the overall size distribution in these ganglia, renal afferent neurons were mainly small with a few medium-sized neurons. The majority (79%) of renal afferent dorsal root ganglion neuronal somata were unlabelled by an anti-neurofilament antibody, RT79, and classified as neurofilament-poor with probable C-fibres. These had an approximately normal distribution of cell sizes. Only 21% were RT79-positive and classified as neurofilament-rich with probable A-fibres, and even these were small to medium sized cells, consistent with them being mostly A delta-fibre neurons. Percentages of renal afferent neurons showing labelling were as follows: peripherin-like immunoreactivity: 69%; calcitonin-gene related peptide: 93%; substance P: 37%; the lectins soybean agglutinin: 57% and peanut agglutinin: 68%; Calbindin D28k-like immunoreactivity: 21% (only weak labelling); carbonic anhydrase like immunoreactivity: 0%. There were differences between probable C-fibre and probable A-fibre neurons, classified according to their labelling with RT97. The percentages of RT97-negative and RT97-positive neurons respectively labelled with the other markers were as follows: peripherin-like immunoreactivity: 82%, 25%; calcitonin gene-related peptide-like immunoreactivity: 99%, 79%; substance P-like immunoreactivity: 43%, 0%; soybean agglutinin: 69%, 24%; peanut agglutinin: 76%, 47%; calbindin-like immunoreactivity: 26%, 0%. Thus, the biggest differences between the probable A- and C-fibre renal afferent neurons were in their peripherin, substance P and calbindin contents. Thus, renal afferent neurons in the dorsal root ganglion are not homogeneous and it is suggested the differences may relate to the known different afferent receptor types within the kidney. It is suggested that the low proportion of neurons with substance P-like immunoreactivity in the renal afferent innervation compared to that of other viscera may relate to the role of the renal vasculature in urine formation.

Animals↗
PubMed1995

Cytoskeletal and calcium-binding proteins in the mammalian organ of Corti: cell type-specific proteins displaying longitudinal and radial gradients.

Whole mounts and tissue sections of the organ of Corti from two representative mammalian species, the Mongolian gerbil (Meriones unguiculatus) and the guinea pig (Cavea porcellus) were probed with antibodies to cytoskeletal and calcium-binding proteins (actin, tubulin, including post-translational modifications, spectrin, fimbrin, calmodulin, parvalbumin, calbindin, S-100 and calretinin). All of the proteins tested were expressed in both species. New findings include the following. Actin is present in large accumulations in cell bodies of the Deiters cells under the outer hair cells (OHC), as well as in the filament networks previously described. These accumulations are more prominent in the apical turns. Tubulin is present in sensory cells in the tyrosinated (more dynamic) form, while tubulin in the supporting cells is post-translationally modified, indicating greater stability. Fimbrin, present in the stereocilia of both IHCs and OHCs, is similar to the isoform of fimbrin found in the epithelial cells of the intestine (fimbrin-I), which implies that actin bundling by fimbrin is reduced in the presence of increased calcium. Parvalbumin appears to be an IHC-specific calcium-binding protein in the gerbil as well as in the guinea pig; labeling displays a longitudinal gradient, with hair cells at the apex staining intensely and hair cells at the base staining weakly. Calbindin displays a similar longitudinal gradient, with staining intense in the IHCs and OHCs at the apex and weak to absent in the base. In the middle turns of the guinea pig cochlea, OHCs in the first row near the pillar cells lose immunoreactivity to calbindin before those in the second and third rows. Calmodulin is found throughout the whole cochlea in the IHCs and OHCs in the stereocilia, cuticular plate, and cell body. Calretinin is present in IHCs and Deiters cells in both species, as well as the tectal cell (modified Hensen cell) in the gerbil. S-100 is a supporting cell-specific calcium-binding protein which has not been localized in the sensory cells of these two species. The supporting cells containing S-100 include the inner border, inner phalangeal, pillar, Deiters, tectal (in gerbil) and Hensen cells, where labeling displays a longitudinal gradient decreasing in intensity towards the apex (opposite to what has been seen with labeling for other proteins in the cochlea).

Actins↗
PubMed1996

Distribution of neuronal populations containing neurofilament protein and calcium-binding proteins in the canine neocortex: regional analysis and cell typology.

Neurophysiological experiments in carnivores have revealed the existence of a large number of cortical regions and an organization of sensory systems quite similar to that found in primates. However, the cyto- and chemoarchitecture of the cerebral cortex is relatively poorly known in carnivores. We analyzed the distribution and typology of classes of neurons containing neurofilament protein or the calcium-binding proteins parvalbumin, calbindin, and calretinin in six neocortical regions of the dog. In all these areas, neurofilament protein was present in a subpopulation of medium-to-large size pyramidal neurons predominantly distributed in layers III and V. Parvalbumin was present in a large population of morphologically diverse interneurons. Small ovoid and multipolar neurons were observed throughout the cortical layers, but predominated in layers II and IV. Layers III and V-VI were characterized by the presence of larger and intensely immunoreactive neurons with bitufted or multipolar morphology, and layers V-VI also contained large multipolar neurons. Calbindin was observed in small round and multipolar interneurons in layer II, and typical double bouquet cells in layer III. Layers IV-VI contained isolated double bouquet cells and large multipolar neurons. A few calbindin-immunoreactive pyramidal neurons were also observed in layer V. Calretinin was localized in bipolar and double bouquet cells in layers II and upper III. The lower part of layer III and layers IV-VI contained rare calretinin-immunoreactive neurons. In some areas, layer III displayed a few large isolated multipolar neurons and pyramidal neurons containing calretinin. In addition, the results show that there is a substantial degree of variability in the distribution of these proteins among cortical regions, and that although they are found in morphologically comparable neuronal types in dog, monkeys, and humans, many differences exist in their regional distribution patterns between carnivores and primates.

Animals↗
PubMed2005

Analysis of neuronal subpopulations in mice over-expressing suppressor of cytokine signaling-2.

Developing an understanding of factors that regulate development of the nervous system is important if we hope to be able to repair the nervous system after injury or disease. Suppressor of cytokine signaling-2 (SOCS2) is an intracellular regulator of cytokine signaling that blocks the inhibitory effects of growth hormone on neuronal differentiation and promotes neurogenesis. Here we examine the effect of SOCS2 over-expression on brain development by assessing density and soma size of different neuronal populations in the somatosensory cortex and striatum of SOCS2 transgenic mice compared with wildtype C57BL/6 mice. There were no significant differences in brain weight, cortical thickness or striatal area between mice of either genotype. Analysis of NeuN positive neuronal cell density showed a modest but significant 9% increase across layers 2-6 of SOCS2 transgenic cortex, while cortical interneuron subpopulations were variably affected. In the cortex, parvalbumin and somatostatin expressing neuron densities were unaffected, while calretinin and calbindin positive neuronal densities increased by 48% and 45% respectively. There was no apparent difference in glial fibrillary acidic protein positive astrocyte numbers in layers 1 or 6b of cortex. Furthermore, soma sizes of calretinin and calbindin positive cortical neurons were significantly smaller than wildtype, although there was no difference in size of Cresyl Violet-stained layer 5 projection neurons nor of parvalbumin or somatostatin positive cortical neurons. Additionally, synaptic density and dendritic branching were found to be increased in SOCS2 transgenic cortex. These effects on calretinin and calbindin positive cortical neurons and cortical neuronal circuitry were not observed in the striatum of SOCS2-Tg brains. However, striatal cholinergic interneurons were significantly smaller in SOCS2-Tg brains. At embryonic day 14.5, proliferation and apoptosis in the developing telencephalon were similar in each genotype. Therefore, over-expression of SOCS2 variably affects different cortical regions and neuronal populations, with the predominant effect appearing to be on interneurons and neuronal connectivity in the cortex.

Animals↗
PubMed2000

Phytoestrogens decrease brain calcium-binding proteins but do not alter hypothalamic androgen metabolizing enzymes in adult male rats.

Phytoestrogen [plant estrogenic-like molecule(s)] research has grown rapidly in recent years due to their potential health benefits. However, little is known about phytoestrogen's effects on the CNS. Androgen metabolizing enzymes are known to regulate neuroendocrine functions and reproductive behaviors, while calcium-binding proteins are associated with protecting against neurodegenerative diseases. Therefore, we examined aromatase and 5alpha-reductase enzyme activities in the medial basal hypothalamic and preoptic area (mbh-poa) and characterized mbh-poa and amygdala (amy) calbindin and calretinin levels (via Western analysis) from animals fed a phytoestrogen-free (P-free) vs. a phytoestrogen-containing diet [(P-600); that had 600 microg/g of phytoestrogens]. After approximately 5 weeks on the diets, the male rats were killed at 105 days. P-600 plasma phytoestrogen levels were 78-fold higher than the P-free values and the mbh-poa phytoestrogen content was 8-fold higher than the P-free group, demonstrating the passage of phytoestrogens into brain. In general, brain aromatase or 5alpha-reductase activity levels were not significantly altered by the experimental diets. However, independent of brain site (i.e., mbh-poa or amy) the abundance of calbindin from male P-600 rats was significantly lower than P-free animals. Conversely, for calretinin there were no significant alterations in the mbh-poa tissue site, while in the amy a similar pattern of expression was seen to that of the calbindin results. These data suggest that consumption of phytoestrogens via a soy diet for a relatively short interval can significantly: (1) elevate plasma and brain phytoestrogens levels and (2) decrease brain calcium-binding proteins without altering brain androgen metabolizing enzymes.

Androgens↗
PubMed2001

Localization of neurotransmitters and calcium binding proteins to neurons of salamander and mudpuppy retinas.

We wished to identify the different types of retinal neurons on the basis of their content of neuroactive substances in both larval tiger salamander and mudpuppy retinas, favored species for electrophysiological investigation. Sections and wholemounts of retinas were labeled by immunocytochemical methods to demonstrate three calcium binding protein species and the common neurotransmitters, glycine, GABA and acetylcholine. Double immunostained sections and single labeled wholemount retinas were examined by confocal microscopy. Immunostaining patterns appeared to be the same in salamander and mudpuppy. Double and single cones, horizontal cells, some amacrine cells and ganglion cells were strongly calbindin-immunoreactive (IR). Calbindin-IR horizontal cells colocalized GABA. Many bipolar cells, horizontal cells, some amacrine cells and ganglion cells were strongly calretinin-IR. One type of horizontal cell and an infrequently occurring amacrine cell were parvalbumin-IR. Acetylcholine as visualized by ChAT-immunoreactivity was seen in a mirror-symmetric pair of amacrine cells that colocalized GABA and glycine. Glycine and GABA colocalized with calretinin, calbindin and occasionally with parvalbumin in amacrine cells.

Acetylcholine↗
PubMed1998

Postnatal expression pattern of calcium-binding proteins in organotypic thalamic cultures and in the dorsal thalamus in vivo.

The present study describes the postnatal expression of calbindin, calretinin and parvalbumin and glutamic acid decarboxylase (GAD) and microtubule-associated protein 2 (MAP2) in organotypic monocultures of rat dorsal thalamus compared to the thalamus in vivo. Cultures were maintained for up to 7 weeks. Cortex-conditioned medium improved the survival of thalamic cultures. MAP2-immunoreactive material was present in somata and dendrites of small and large-sized neurons throughout the cultures. Parvalbumin immunoreactivity was present in larger multipolar or bitufted neurons along the edge of a culture. These neurons also displayed strong parvalbumin mRNA and GAD mRNA expression, and GABA immunoreactivity. They likely corresponded to cells of the nucleus reticularis thalami. Parvalbumin mRNA, but neither parvalbumin protein nor GAD mRNA, was expressed in neurons with large somata within the explant. They likely represented relay cells. GAD mRNA, but not parvalbumin mRNA, was expressed in small neurons within the explants. Small neurons also displayed calbindin- and calretinin-immunoreactivity. The small neurons likely represented local circuit neurons. The time course of expression of the calcium-binding proteins revealed that all were present at birth with the predicted molecular weights. A low, but constant parvalbumin expression was observed in vitro without the developmental increase seen in vivo, which most likely represented parvalbumin from afferent sources. In contrast, the explantation transiently downregulated the calretinin and calbindin expression, but the neurons recovered the expression after 14 and 21 days, respectively. In conclusion, thalamic monocultures older than three weeks represent a stable neuronal network containing well differentiated neurons of the nucleus reticularis thalami, relay cells and local circuit neurons.

Aging↗
PubMed1998

Calcium-binding proteins in primate cerebellum.

Single and double antigen localization procedures were used to study the distribution of the calcium-binding proteins calretinin, calbindin and parvalbumin in the cerebellum of the squirrel monkey (Saimiri sciureus). The immunostaining experiments have revealed that each of the three calcium-binding proteins occurred, either alone or in various combinations, in many neuronal types of the monkey cerebellum, including the Purkinje cells. Immunoreactivity for calbindin was detected in virtually all Purkinje cells, whereas immunoreactivity for calretinin and parvalbumin was encountered only in some subpopulations of Purkinje cells. In the vermal region, parvalbumin immunostaining appeared in the form of typical weak and strong alternating parasagittal bands. Calretinin immunoreactivity was found in virtually all neurons and fiber systems related to the granular layer, including the monodendritic cells, the granule cells and their parallel fibers, the Golgi and Lugaro cells and the mossy fibers. The Golgi cells also displayed calbindin and parvalbumin immunoreactivity. Parvalbumin was found to labeled both the climbing and mossy fibers, as well as the basket and stellate cells lying in the molecular layer. These results reveal that virtually all the different neuronal types in the primate cerebellum contain at least one of three calcium-binding proteins investigated in the present study. Furthermore, calretinin appears to be a particularly reliable molecular maker for all the neuronal elements associated with the granular layer in the primate cerebellum.

Animals↗
PubMed2001

The spiny rat Proechimys guyannensis as model of resistance to epilepsy: chemical characterization of hippocampal cell populations and pilocarpine-induced changes.

At variance with pilocarpine-induced epilepsy in the laboratory rat, pilocarpine administration to the tropical rodent Proechimys guyannensis (casiragua) elicited an acute seizure that did not develop in long-lasting status epilepticus and was not followed by spontaneous seizures up to 30 days, when the hippocampus was investigated in treated and control animals. Nissl staining revealed in Proechimys a highly developed hippocampus, with thick hippocampal commissures and continuity of the rostral dentate gyri at the midline. Immunohistochemistry was used to study calbindin, parvalbumin, calretinin, GABA, glutamic acid decarboxylase, and nitric oxide synthase expression. The latter was also investigated with NADPH-diaphorase histochemistry. Cell counts and densitometric evaluation with image analysis were performed. Differences, such as low calbindin immunoreactivity confined to some pyramidal cells, were found in the normal Proechimys hippocampus compared to the laboratory rat. In pilocarpine-treated casiraguas, stereological cell counts in Nissl-stained sections did not reveal significant neuronal loss in hippocampal subfields, where the examined markers exhibited instead striking changes. Calbindin was induced in pyramidal and granule cells and interneuron subsets. The number of parvalbumin- or nitric oxide synthase-containing interneurons and their staining intensity were significantly increased. Glutamic acid decarboxylase(67)-immunoreactive interneurons increased markedly in the hilus and decreased in the CA1 pyramidal layer. The number and staining intensity of calretinin-immunoreactive pyramidal cells and interneurons were significantly reduced. These findings provide the first description of the Proechimys hippocampus and reveal marked long-term variations in protein expression after an epileptic insult, which could reflect adaptive changes in functional hippocampal circuits implicated in resistance to limbic epilepsy.

Animals↗
PubMed2002

Time course and manner of Purkinje neuron death following a single ethanol exposure on postnatal day 4 in the developing rat.

The present study was designed to evaluate the time course and manner of Purkinje cell death following a single ethanol dose delivered intragastrically on postnatal day (PN) 4 to rat pups. Analysis included immunolabeling of Purkinje cells with antibody specific for calbindin D28k and counting of Purkinje cells in each lobule of a mid-vermal slice. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling analysis and immunodetection for cleaved (activated) caspase-3 enzyme was used to identify apoptosis, with calbindin D28k co-immunolabeling to identify apoptotic Purkinje cells. Finally, immunodetection for cytochrome c, again with co-labeling using calbindin D28k antibody, identified intracellular release of cytochrome c from the mitochondria into the cytoplasm of Purkinje cells. The data demonstrate that a single dose of ethanol results in a significant and extensive, lobular dependent loss of Purkinje cells within 24 h after administration. Extensive loss in the early developing lobules (I-III, VIII-X) and less to no loss in the later developing lobules (IV-VII) is consistent with prior literature reports on the ethanol-induced effects on Purkinje cells at this age. Clear and consistent evidence of apoptotic Purkinje cells was identified and the pattern was transient in nature. Finally, cytochrome c is released from the mitochondria of Purkinje cells in a time course consistent with the activation of the mitochondrial pathway of apoptosis. These data support the hypothesis that ethanol-induced loss of Purkinje cells involves apoptotic mechanisms. Furthermore, the initiation of apoptosis by ethanol is consistent with ethanol-induced interruptions of Purkinje cell neurotrophic support leading to activation of the mitochondrial pathway of apoptosis.

Alcohol-Induced Disorders, Nervous System↗
PubMed2002

Immunohistochemical localization of calcium-binding proteins in the brainstem vestibular nuclei of the jaundiced Gunn rat.

Vestibular gaze and postural abnormalities are major sequelae of neonatal hyperbilirubinemia. The sites and cellular effects of bilirubin toxicity in the brainstem vestibular pathway are not easily detected. Since altered intracellular calcium homeostasis may play a role in neuronal cell death, we hypothesized that altered expression of calcium-binding proteins may occur in brainstem vestibular nuclei of the classic animal model of bilirubin neurotoxicity. The expression of the calcium-binding proteins calbindin-D28k and parvalbumin in the brainstem vestibular pathways and cerebellum of homozygous recessive jaundiced (jj) Gunn rats was examined by light microscopy and immunohistochemistry at 18 days postnatally and compared to the findings obtained from age-matched non-jaundiced heterozygous (Nj) littermate controls. Jaundiced animals exhibited decreased parvalbumin immunoreactivity specifically in synaptic inputs to superior, medial, and inferior vestibular nuclei, and to oculomotor and trochlear nuclei, whereas the neurons retained their normal immunoreactivity. Jaundiced animals also demonstrated a decrease in calbindin expression in the lateral vestibular nuclei and a paucity of calbindin-immunoreactive synaptic endings on the somata of Deiters' neurons. The involved regions are related to the control of the vestibulo-ocular and vestibulospinal reflexes. Decreased expression of calcium-binding proteins in brainstem vestibular neurons may relate to the vestibulo-ocular and vestibulospinal dysfunction seen with clinical kernicterus, and may provide a sensitive new way to assess bilirubin toxicity in the vestibular system.

Animals↗
PubMed2003

Auditory peripheral influences on calcium binding protein immunoreactivity in the cochlear nucleus during aging in the C57BL/6J mouse.

The C57BL/6J (C57) mouse was selected as a suitable model for early presbyacusis to determine if there were correlations between peripheral pathology (spiral ganglion loss, inner and outer hair cell loss) and calcium binding immunoreactivity in the cochlear nucleus during aging. The quantitative stereological method, the optical fractionator, was used for determining the total number of neurons and calcium binding immunopositive neurons (calbindin, parvalbumin and calretinin) during aging in the posteroventral- and dorsal cochlear nucleus (PVCN and DCN) in C57 mice. Comparing 30-month-old to 1-month-old C57 mice, a percent increase in parvalbumin and calbindin immunoreactivity was evident in both the PVCN and DCN. Correlations were made between peripheral pathology (spiral ganglion and inner and outer hair cell loss) and calcium binding protein expression. Significant correlations between cochlear pathology and the percentage of parvalbumin and calretinin immunoreactive neurons were demonstrated in the DCN. Moreover, significant correlations were found between cochlear pathology and parvalbumin and calbindin in the PVCN. In summary, the findings imply that degenerative changes in the auditory periphery can modulate neuronal homeostasis by increasing calcium binding proteins in the PVCN and DCN during aging. Taken together, these findings suggest a role for calcium binding proteins in protecting against age-induced calcium toxicity.

Aging↗
PubMedDate not supplied

Calretinin in the cat retina: colocalizations with other calcium-binding proteins, GABA and glycine.

Immunocytochemical techniques were used to determine the distribution of the calcium-binding protein calretinin in the cat retina. Comparisons were made with parvalbumin and calbindin as well as with the inhibitory neurotransmitters GABA and glycine. Calretinin immunoreactivity was seen in horizontal cells and multiple subpopulations of amacrine and ganglion cells. Cone outer segments were also stained. Calbindin immunoreactivity was present in cone photoreceptors, horizontal cells, at least two subtypes of cone bipolar cell, numerous amacrine cells, and cells residing in the ganglion cell layer. Heavy staining for parvalbumin was found in both A- and B-type horizontal cells, distinct subpopulations of amacrine and ganglion cells, and a small population of cone photoreceptor cells. To confirm the identity of cone photoreceptors, comparisons were made with retinas stained for opsins specific for red/green or blue cones (Szél et al., 1986). The localization of parvalbumin corresponded with that of blue-type cones only whereas calretinin and calbindin staining showed the same distribution as both red/green and blue cones. Double-label immunofluorescence studies revealed colocalization of all three of the calcium-binding proteins in a number of neurons including horizontal cells and AII amacrine cells. To assess a possible transmitter-specific relationship for calretinin, double-label studies were carried out with GABA and glycine. However, the staining patterns for each of these inhibitory amino acids differed substantially from that of calretinin. The possibility remains that calretinin and other calcium-binding proteins may play a role in neurotransmission through interactions with receptors or second-messenger agents.

Animals↗
PubMed2003

Patterns of calcium-binding proteins support parallel and hierarchical organization of human auditory areas.

The human primary auditory cortex (AI) is surrounded by several other auditory areas, which can be identified by cyto-, myelo- and chemoarchitectonic criteria. We report here on the pattern of calcium-binding protein immunoreactivity within these areas. The supratemporal regions of four normal human brains (eight hemispheres) were processed histologically, and serial sections were stained for parvalbumin, calretinin or calbindin. Each calcium-binding protein yielded a specific pattern of labelling, which differed between auditory areas. In AI, defined as area TC [see C. von Economo and L. Horn (1930) Z. Ges. Neurol. Psychiatr.,130, 678-757], parvalbumin labelling was dark in layer IV; several parvalbumin-positive multipolar neurons were distributed in layers III and IV. Calbindin yielded dark labelling in layers I-III and V; it revealed numerous multipolar and pyramidal neurons in layers II and III. Calretinin labelling was lighter than that of parvalbumin or calbindin in AI; calretinin-positive bipolar and bitufted neurons were present in supragranular layers. In non-primary auditory areas, the intensity of labelling tended to become progressively lighter while moving away from AI, with qualitative differences between the cytoarchitectonically defined areas. In analogy to non-human primates, our results suggest differences in intrinsic organization between auditory areas that are compatible with parallel and hierarchical processing of auditory information.

Aged↗
PubMed1995

Identification of metal-binding sites in rat brain calcium-binding protein.

Calbindin D28K binds 3 mol of terbium per mol of protein. To determine which of six EF-hand structures in the protein are responsible for terbium binding, we constructed three mutant forms of this protein, one lacking EF-hand 2 (RCaBP delta 2), the other lacking EF-hands 2 and 6 (RCaBP delta 2,6), and the third containing only EF-hands 3 and 4 (RCaBP delta 1,2,5,6), and examined their binding properties by fluorescence techniques. Full-length calbindin D28K and RCaBP delta 2 and RCaBP delta 2,6 bound 3 mol of terbium per mol of protein with high affinity. Thus, EF-hand domains 2 and 6 are not essential for calcium binding to the proteins, and an absence of EF-hands 2 and/or 6 does not alter the pattern of terbium binding to the protein. Using resonance energy transfer from tryptophan residues, one of the high affinity terbium-binding sites (site A) had a greater affinity than the other two sites (sites B and C) of each protein. Site A was filled before the other two sites. Calcium competition experiments showed that a greater amount of calcium was required to displace terbium from site A than from sites B or C. Energy transfer experiments from terbium to holmium showed that two of the terbium-binding sites are in close proximity while the third site is distant from the other two sites. To determine whether EF-hand 3 or 4 was responsible for binding of terbium, we examined the terbium binding properties of a delta 1,2,5,6 RCaBP construct. The truncated protein RCaBP delta 1,2,5,6 contained a single terbium-binding site. Analysis of the terbium binding to RCaBP delta 1,2,5,6 construct showed that site 4 bound terbium, whereas site 3 did not. Analysis of the terbium binding characteristics of the proteins suggests that EF-hands 1, 4, and 5 of rat brain calbindin D28K are responsible for terbium binding.

Amino Acid Sequence↗
PubMed2005

Characterization of the rhesus monkey superior olivary complex by calcium binding proteins and synaptophysin.

This study was performed in order to characterize the main nuclei of the rhesus monkey superior olivary complex by means of antibodies against the calcium binding proteins parvalbumin, calbindin and calretinin and the synaptic vesicle protein synaptophysin. These markers revealed the neuronal morphology and organization of nuclei located within the rhesus monkey superior olivary complex. The architectural details included the distribution of axonal terminals on neurons. The medial superior olivary nucleus was present as a column of neurons. No clear segregation of calretinin-positive terminals was noticed on the medial and lateral dendritic fields of these neurons. The lateral superior olivary nucleus was characterized by a distinct nuclear shape. Calretinin-, parvalbumin- or calbindin-positive terminals contacted somata and dendrites. The medial nucleus of trapezoid body could be clearly differentiated as a distinct region in the rhesus monkey superior olivary complex. Somata of that nucleus showed calbindin- and parvalbumin-labelling whereas somatic calyces of Held were reavealed by calretinin and synaptophysin labelling. The results are discussed with respect to the processing of acoustic information in primate species and their ability to hear high and low frequencies, which is reflected by anatomical correlates.

Animals↗