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PubMed1999

Coexpression of neurocalcin with other calcium-binding proteins in the rat main olfactory bulb.

The distribution patterns of four calcium-binding proteins (CaBPs)-calbindin D-28k (CB), calretinin (CR), neurocalcin (NC), and parvalbumin (PV)-in the rat main olfactory bulb were compared, and the degrees ofcolocalization of NC with the other CaBPs were determined by using double immunocytochemical techniques. All investigated CaBPs were detected in groups of periglomerular cells and Van Gehuchten cells, whereas other cell types expressed some of the investigated proteins but not all four. Double-labeling techniques demonstrated the colocalization of NC with CB, CR, or PV in periglomerular cells, whereas each neurochemical group constituted entirely segregated populations in the remaining neuronal types. This is evident in granule cells that demonstrated large but segregated populations immunoreactive to either NC or CR. This study provides a further biochemical characterization of interneuronal types in the rat main olfactory bulb. On the basis of the distinct calcium-binding affinities, each neurochemically defined population may have different responses to calcium influx that would result in the existence of distinct functional subgroups within morphologically defined neuronal types. The expression of the investigated CaBPs in periglomerular cells with both single and colocalized patterns suggests that the local circuits in the glomerular layer are constituted by a complex network of elements with particular calcium requirements.

Animals↗
PubMed1998

Neurocalcin-immunoreactive cells in the rat hippocampus are GABAergic interneurons.

Neurocalcin (NC) is a recently described calcium-binding protein isolated and characterized from bovine brain. NC belongs to the neural calcium-sensor proteins defined by the photoreceptor cell-specific protein recoverin that have been proposed to be involved in the regulation of calcium-dependent phosphorylation in signal transduction pathways. We analyzed the distribution and morphology of the NC-immunoreactive (IR) neurons in the rat dorsal hippocampus and the coexistence of NC with GABA and different neurochemical markers which label perisomatic inhibitory cells [parvalbumin (PV) and cholecystokinin (CCK)], mid-proximal dendritic inhibitory cells [calbindin D28k (CB)], distal dendritic inhibitory cells [somatostatin (SOM) and neuropeptide Y (NPY)], and interneurons specialized to innervate other interneurons [calretinin (CR) and vasoactive intestinal polypeptide (VIP)]. NC-IR cells were present in all layers of the dentate gyrus and hippocampal fields. In the dentate gyrus, NC-IR cells were concentrated in the granule cell layer, especially in the hilar border, whereas in the CA fields they were most frequently found in the stratum radiatum. NC-IR cells were morphologically heterogeneous and exhibited distinctive features of non-principal cells. In the dentate gyrus, pyramidal-like, multipolar and fusiform (horizontal and vertical) cells were found. In the CA3 region most NC-IR cells were multipolar, but vertical and horizontal fusiform cells also appeared. In the CA1 region, where NC-IR cells showed most frequently vertically arranged dendrites, multipolar, bitufted and fusiform (vertical and horizontal) cells could be distinguished. All the NC-IR cells were found to be GABA-IR in all hippocampal layers and regions, and they represented about 19% of the GABA-positive cells. NC/CB, NC/CR and NC/VIP double-labeled cells were found in all hippocampal regions, and represented 29%, 24% and 18% of the NC-IR cells, respectively. NC and CCK did not coexist in the dentate gyrus; however, 9% of the NC-IR cells in the CA fields also contained CCK. No coexistence of NC with PV, SOM or NPY was found in any hippocampal region. We conclude that NC is exclusively expressed by interneurons in the rat hippocampus. NC-IR cells are a morphologically and neurochemically heterogeneous subset of GABAergic non-principal cells, which, on the basis of the known termination pattern of the colocalizing markers, are also functionally heterogeneous and are mainly involved in feed-forward dendritic inhibition in the commissural-associational and Schaffer collateral termination zones (CB containing cells), in innervation of other interneurons (CR- and VIP-containing cells), and in perisomatic inhibition (CCK-containing cells). NC is never present in perisomatic inhibitory PV-containing cells, or in feed-back distal dendritic inhibitory SOM/NPY-containing cells.

Animals↗
PubMed2000

Neurocalcin-immunoreactive neurons in the mammalian dorsal root ganglia, including humans.

Neurocalcin (NC) is a recently characterized EF-hand calcium-binding protein present in a discrete population of sensory neurons and their peripheral mechanoreceptors, but its presence in peripheral nervous system neurons other than in the rat is still unknown. The present study was designed to investigate the occurrence of NC in the dorsal root ganglia (DRG) of several mammalian species (horse, buffalo, cow, sheep, pig, dog, and rat), including humans. DRG were fixed, embedded in paraffin, and processed for immunohistochemistry using a polyclonal antibody against NC. The size of the immunoreactive neurons was measured. In all species examined, NC immunoreactivity (IR) was restricted to neurons but the percentage, as well as the size of the immunoreactive neurons, varied among different species. As a rule, small neurons (diameter <20 microm) lack NC IR. In some species (pig, dog, buffalo, cow), only the largest neurons showed IR, whereas in others (sheep, horse, rat, and humans) they covered the entire range of neuron sizes. The pattern of immunostaining was cytoplasmic, although in some species (cow and buffalo), it formed a peripheral "ring." The present results demonstrate that mammalian DRG contain a subpopulation of NC-positive neurons, which varies from one species to another. Based on the neuron size, the possible function of the NC-containing neurons is discussed.

Adult↗
PubMed2001

Calcium-dependent association of annexin VI, protein kinase C alpha, and neurocalcin alpha on the raft fraction derived from the synaptic plasma membrane of rat brain.

A membrane microdomain enriched in cholesterol and sphingolipids or so called "raft" region was found to contain many signal transducing proteins such as GPI-anchored proteins, trimeric G proteins and protein tyrosine kinases. Because brain-derived raft contains two calmodulin-binding proteins, GAP-43 and NAP-22 as the major protein components, the raft domain is assumed to be important in the Ca(2+)-signaling. In this study, we analyzed protein components showing Ca(2+)-dependent binding to the raft of synaptic plasma membrane from rat brain. SDS-PAGE analysis of the protein components in the EGTA eluate from the raft prepared in the presence of Ca(2+)-ions showed the elution of 80 kDa, 68 kDa, 22 kDa, and 21 kDa proteins. These proteins were identified as protein kinase C alpha (80 kDa) and annexin VI (68 kDa) from the partial amino-acid sequencing, and neurocalcin alpha (22 kDa) and calmodulin (21 kDa) with western blotting and electrophoretic mobilities in the presence or absence of Ca(2+) ions. Further immunoblotting experiments showed the Ca(2+)-dependent association of conventional, but not non-conventional, subtypes of PKC to the raft.

Animals↗
PubMed2006

Analysis of the interacting partners of the neuronal calcium-binding proteins L-CaBP1, hippocalcin, NCS-1 and neurocalcin delta.

Intracellular Ca2+ signals are transduced by the binding of Ca2+ to sensor proteins, which subsequently modify the activity of their target proteins. Identification of these target proteins is, therefore, important for an understanding of cellular signalling processes. We have investigated the binding partners of four EF-hand Ca2+-binding proteins. Three proteins of the neuronal calcium sensor (NCS) family, hippocalcin, NCS-1 and neurocalcin delta were prepared as N-terminally tagged GST fusion proteins, and the less closely related protein L-CaBP1 was prepared in both N- and C-terminally tagged forms, the latter requiring generation of a new vector. Immobilised fusion proteins were used to purify binding partners from bovine brain cytosol and membrane extracts in the presence of 1 microM free Ca2+. Bound proteins were eluted with Ca2+-free and high-salt buffers and eluted proteins were identified by MALDI-MS and Western blotting. New protein targets detected included ARF1, Ca2+-dependent activator protein for secretion 1, cyclic nucleotide 3',5'-phosphodiesterase, the vacuolar ATPase, AP1 and AP2 complexes and the type I TGF-beta receptor. While certain of these interactions occurred with more than one of the Ca2+-binding proteins, others were found to be specific targets for particular Ca2+ sensors, and many of these did not overlap with known calmodulin-binding proteins. These findings provide new clues to the functional roles of the neuronal calcium sensor proteins.

Animals↗
PubMed2004

Neurocalcin-immunoreactive neurons in the petrosal ganglion innervate the taste bud.

The distribution and origin of neurocalcin-immunoreactive (NC-ir) nerve fibers in the taste bud and carotid body were examined by an immunofluorescence method. In the circumvallate papilla of the tongue, NC-ir nerve fibers made subepithelial nerve plexuses and occasionally penetrated the taste bud. However, the carotid body was devoid of ir nerve fibers. In the petrosal ganglion, 32% of neurons were immunoreactive for NC. Such neurons were mostly medium-sized to large, and scattered throughout the ganglion. In the superior cervical and intralingual ganglia, numerous ir varicose fibers surrounded postsynaptic neurons. However, NC-ir could not be detected in cell bodies of these neurons. The retrograde tracing method indicated that NC-ir petrosal neurons innervated taste buds in the circumvallate papilla. NC-ir neurons may have a gustatory function in the petrosal ganglion.

Animals↗
PubMed2000

Neurocalcin-immunoreactive primary sensory neurons in the trigeminal ganglion provide myelinated innervation to the tooth pulp and periodontal ligament.

The distribution of neurocalcin-immunoreactive (NC-ir) primary sensory neurons was examined in the trigeminal ganglion (TG), mesencephalic trigeminal tract nucleus (Mes5) and intraoral structures. NC-ir primary sensory neurons were located in the TG but not the Mes5. The coexpression study demonstrated that virtually all NC-ir TG neurons exhibited S100-immunoreactivity (-ir). In the tooth pulp, NC-ir nerve fibers were observed in the subodontoblastic and odontoblastic layers. Immunoelectron microscopic and retrograde tracing methods revealed that myelinated pulpal axons derived from the TG mostly exhibited the ir. In the periodontal ligament, bush-like endings showed NC-ir. These endings were morphologically identical to Ruffini-like endings. The present study suggests that NC-ir trigeminal primary sensory neurons have their cell bodies in the TG. Their peripheral axons are probably myelinated. Such neurons include pulpal nociceptors and low-threshold mechanoreceptors.

Animals↗
PubMed1998

Neurocalcin immunoreactivity in the rat main olfactory bulb.

The morphological characteristics and distribution of neurocalcin (NC)-immunoreactive elements were studied in the rat main olfactory bulb (OB) using a polyclonal antibody and the avidin-biotin immunoperoxidase method. NC-positive elements were abundant in the glomerular layer (GL), where numerous immunostained external tufted cells and periglomerular cells were detected. Other less abundant NC-immunolabeled populations included middle and internal tufted cells, Van Gehuchten cells, horizontal cells, vertical cells of Cajal, deep short-axon cells and granule cells. This study demonstrates the presence of NC immunoreactivity in subsets of different neuronal types in the rat main OB. This calcium-binding protein has been found in interneurons, and no evidence of immunoreactivity to NC is detected in projecting neurons. Despite the large population of labeled external tufted cells, most of them belong according to morphological criteria to the local circuit group and some others to those with interbulbar and/or intrabulbar connections. The identification of neuronal subpopulations expressing NC provides a further characterization and shows the existence of biochemical differences within morphologically identical neurons. Thus, this marker may be a useful tool in unravelling the circuitries of the rodent OB in both normal and experimental conditions. The exact physiological function of NC in the olfactory system remains unknown. On the basis of similarities to recoverin, it could be involved in mechanisms responsible for sensory adaptation. Additionally, its calcium-binding abilities may contribute to improve the temporal precision of stimuli transmission, or be concerned with general calcium-related events occurring in specific interneuronal groups.

Animals↗
PubMed2001

Decreased neurocalcin immunoreactivity in sympathetic and parasympathetic neurons of the major pelvic ganglion in aged rats.

In the rat the majority of sympathetic and parasympathetic postganglionic neurons that innervate the pelvic viscera are located together in the major pelvic ganglia. We have ascertained that it is only the sympathetic population of this ganglion that exhibits age-associated attrition. Recent immunohistochemical investigations of the distribution of calcium binding proteins in this ganglion in young adult and aged rats have demonstrated that calbindin-D28k is only present in the sympathetic neurons and that the number of calbindin-immunoreactive sympathetic neurons of the aged ganglion was dramatically reduced. In the present study we have investigated the distribution of neurocalcin (NC) alpha isoform in the major pelvic ganglion. In young adults 98.7% of sympathetic neurons (identified by anti-tyrosine hydroxylase immunostaining) and 98% of parasympathetic neurons (identified by anti-nitric oxide synthase immunostaining) contained NC immunoreactivity and these figures are reduced to 68 and 45.5% in the aged group. Thus, unlike calbindin-D28k, NC is not confined to the sympathetic neuron population in the major pelvic ganglion and decreases significantly in old age in both neuronal populations. The likely effects are to impair intracellular calcium-dependent signalling in neurons of the major pelvic ganglion, possibly compounding the effects of the previously reported decrease in calbindin-D28k in the sympathetic population.

Aging↗
PubMed2001

Calretinin-, neurocalcin-, and parvalbumin-immunoreactive elements in the olfactory bulb of the hedgehog (Erinaceus europaeus).

The distribution pattern and morphology of calretinin-, neurocalcin-, and parvalbumin-immunoreactive neurons were studied in the main and accessory olfactory bulbs of the hedgehog. The detection of these markers was carried out by using monoclonal or polyclonal antibodies and the avidin-biotin-immunoperoxidase method. Specific neuronal populations were positive for these calcium-binding proteins in the hedgehog olfactory bulb, revealing both similarities to and differences from the data reported in the olfactory bulb of rodent species. The distribution pattern of each calcium-binding protein studied in the accessory olfactory bulb was highly similar to that described in other macrosmatic species. However, in the main olfactory bulb, the markers analyzed were expressed in similar interneuronal populations as they are in the rodent olfactory bulb, whereas cell groups categorized as projecting neurons demonstrated striking differences in the expression of these calcium-binding proteins. These results suggest that the expression of calcium-binding proteins in a given brain region is not a constant feature among species despite a similar organization but that different factors could influence their expression. Thus, the accessory olfactory system involved in the processing of specific and similar olfactory cues among species demonstrates a more constant organization among species. By contrast, the functionally important role of the main olfactory system in the hedgehog is accompanied by a more complex organization, which is reflected in an increased diversity of calcium-buffering systems.

Animals↗
PubMed2005

Effects of neurotrophin-3 gene mutation in the expression of neurocalcin.

Neurocalcin (NC) is a neuron-specific "EF-hand" calcium-binding protein present in a non-fully characterized subpopulation of dorsal root ganglion (DRG) neurons, some kinds of mechanoreceptors and proprioceptors, and in motor end-plates. In the present study we have characterized NC expression in spinal sensory and motor neurons, and their endings in newborn mouse. Because the neurotrophic factor neurotrophin-3 (NT-3) appears to plays a major role in the development and maturation of sensory and motor neuronal populations, we have studied NC immunoreactivity in newborn NT-3 null mutant. In NT-3 deficient animals the overall number of NC-immunoreactive DRG neurons was reduced by as much as 70% including all large neurons, but subpopulations of NC expressing small and intermediate-sized neurons survived. As expected no muscle spindles were found in NT-3 mutant mice while they were present and normally innervated by NC-positive nerve fibers in wild-type animals. On the other hand, NC immunoreactivity was dramatically decreased in motoneurons of the spinal cord, ventral root nerves and motor end-plates in the absence of NT-3. The present results demonstrate that NC-containing DRG neurons include all proprioceptive, and a subset of mechanoreceptive and proprioceptive. Furthermore, they strongly suggest that NT-3 is involved in the maturation of motor end-plates.

Animals↗
PubMed1996

Cloning and characterization of aplycalcin and Aplysia neurocalcin, two new members of the calmodulin superfamily of small calcium-binding proteins.

We report here the sequences of two new proteins from Aplysia, aplycalcin and Aplysia neurocalcin. These proteins belong to a family of calcium-binding proteins, found primarily in vertebrate brain and retina, that have been proposed to play a role in calcium-dependent regulation of enzymes in signal transduction pathways. Like other members of this family, the Aplysia proteins have consensus sequences for myristoylation, bind calcium, and translocate from cytosol to membrane when the calcium level is raised above the resting intracellular concentration. Both proteins are relatively enriched in Aplysia nervous system, but are also found to a significant degree in other tissues. The expression of mRNA for these proteins in Aplysia nervous tissue is regulated during development, roughly paralleling the reported emergence of several forms of synaptic plasticity. The messages are present at low levels in stage 11, show a large increase by late stage 12, and decline to a plateau of approximately 30% of the peak value afterward. On the basis of the properties of these proteins and by analogy with proposed functions of some of the retinal homologues, we suggest that these proteins may play a role in mediating calcium-dependent processes in neuronal function. The presence of both proteins in other tissues may suggest analogous roles for the proteins in other cell types.

Animals↗
PubMed2000

The sequence and associated null phenotype of a C. elegans neurocalcin-like gene.

The neuronal calcium sensor (NCS) proteins belong to a subfamily of the EF-hand calcium binding proteins. These proteins are primarily expressed in the nervous system and currently include more than 20 members across species [Nakayama et al., J Mol Evol 34:416-448, 1992]. Two homologues of the ncs genes, Ce-ncs-1 and Ce-ncs-2, have recently been identified in the nematode C. elegans. Here we report the cDNA sequence of a third C. elegans ncs homologue, Ce-ncs-3. We demonstrate that a null mutation in this gene caused by a large deletion in the locus does not confer a visible phenotype in C. elegans. This, in addition to the strong homology between Ce-NCS-3 and the other C. elegans NCS proteins, may indicate functional redundancy between the three genes.

Alleles↗
PubMed1997

Calcium-binding proteins in the periglomerular region of typical and typical olfactory glomeruli.

The distribution of chemically identified neuronal populations was studied in the glomerular layer of the rat olfactory bulb using calcium-binding protein immunocytochemistry combined with acetylcholinesterase histochemistry. Four calcium-binding proteins (calbindin D-28k, parvalbumin, calretinin, and neurocalcin) were analyzed in the periglomerular region of two different glomerular subsets; typical and atypical glomeruli. Atypical glomeruli were clearly distinguishable from typical ones by their dense network of acetylcholinesterase-positive centrifugal fibers. Each calcium-binding protein studied showed a specific distribution pattern in the rat olfactory bulb. Calbindin D-28k-, calretinin-, and neurocalcin-immunoreactive neurons were specially abundant in the glomerular layer. These three calcium-binding proteins had their main expressions in neuronal subpopulations directly involved in the glomerular circuitries of the rat olfactory bulb. Specific populations of periglomerular cells were stained for calbindin D-28k, parvalbumin, calretinin, or neurocalcin, whereas external tufted cells were only immunoreactive to neurocalcin. Both neuronal types, periglomerular cells and external tufted cells, were found in the periglomerular region of both glomerular subsets. Nevertheless, a homogeneous distribution of calbindin D-28k- or calretinin-immunopositive periglomerular cells were found between typical and atypical glomeruli, whereas the neurocalcin-immunostained external tufted cells were statistically more abundant in typical glomeruli than atypical ones (P < 0.001). These data suggest that some neuronal subpopulations are related with general properties of the glomerular physiology, and they have a homogeneous distribution in different subsets of glomeruli, whereas other chemically identified populations are related with a finer tuning of the olfactory processing, and they are segregately distributed in relation to particular glomerular subsets. In addition, this work adds new differences in the cellular composition of typical and atypical glomeruli.

Acetylcholinesterase↗
PubMed2001

A novel calcium-regulated membrane guanylate cyclase transduction system in the olfactory neuroepithelium.

This report defines the identity of a calcium-regulated membrane guanylate cyclase transduction system in the cilia of olfactory sensory neurons, which is the site of odorant transduction. The membrane fraction of the neuroepithelial layer of the rat exhibited Ca(2+)-dependent guanylate cyclase activity, which was eliminated by the addition of EGTA. This indicated that the cyclase did not represent a rod outer segment guanylate cyclase (ROS-GC), which is inhibited by free Ca(2+). This interpretation was supported by studies with the Ca(2+) binding proteins, GCAPs (guanylate cyclase activating proteins), which stimulate photoreceptor ROS-GC in the absence of Ca(2+). They did not stimulate the olfactory neuroepithelial membrane guanylate cyclase. The olfactory neuroepithelium contained a Ca(2+) binding protein, neurocalcin, which stimulated the cyclase in a Ca(2+)-dependent fashion. The cyclase was cloned from the neuroepithelium and was found to be identical in structure to that of the previously cloned cyclase termed GC-D. The cyclase was expressed in a heterologous cell system, and was reconstituted with its Ca(2+)-dependent activity in the presence of recombinant neurocalcin. The reconstituted cyclase mimicked the native enzyme. Immunocytochemical studies showed that the guanylate cyclase coexists with neurocalcin in the apical region of the cilia. Deletion analysis showed that the neurocalcin-regulated domain resides at the C-terminal region of the cyclase. The findings establish the biochemical, molecular, and functional identity of a novel Ca(2+)-dependent membrane guanylate cyclase transduction system in the cilia of the olfactory epithelium, suggesting a mechanism of the olfactory neuroepithelial guanylate cyclase regulation fundamentally distinct from the phototransduction-linked ROS-GC.

Animals↗
PubMed1999

Mapping functional domains of the guanylate cyclase regulator protein, GCAP-2.

Guanylate cyclase regulator protein (GCAP)-2 is a Ca2+-binding protein that regulates photoreceptor outer segment membrane guanylate cyclase (RetGC) in a Ca2+-sensitive manner. GCAP-2 activates RetGC at free Ca2+ concentrations below 100 nM, characteristic of light-adapted photoreceptors, and inhibits RetGC when free Ca2+ concentrations are above the 500 nM level, characteristic of dark-adapted photoreceptors. We have mapped functional domains in GCAP-2 by using deletion mutants and chimeric proteins in which parts of GCAP-2 were substituted with corresponding fragments of other closely related recoverin-like proteins that do not regulate RetGC. We find that in addition to the EF-hand Ca2+-binding centers there are three regions that contain GCAP-2-specific sequences essential for regulation of RetGC. 1) The region between Phe78 and Asp113 determines whether GCAP-2 activates outer segment RetGC in low or high Ca2+ concentrations. Substitution of this domain with the corresponding region from neurocalcin causes a paradoxical behavior of the chimeric proteins. They activate RetGC only at high and not at low Ca2+ concentrations. 2) The amino acid sequence of GCAP-2 between Lys29 and Phe48 that includes the EF-hand-related motif EF-1 is essential both for activation of RetGC at low Ca2+ and inhibition at high Ca2+ concentrations. Most of the remaining N-terminal region can be substituted with recoverin or neurocalcin sequences without loss of GCAP-2 function. 3) Region Val171-Asn189, adjacent to the C-terminal EF-4 contributes to activation of RetGC, but it is not essential for the ability of Ca2+-loaded GCAP-2 to inhibit RetGC. Other regions of the molecule can be substituted with the corresponding fragments from neurocalcin or recoverin, or even partially deleted without preventing GCAP-2 from regulating RetGC. Substitution of these three domains in GCAP-2 with corresponding neurocalcin sequences also affects activation of individual recombinant RetGC-1 and RetGC-2 expressed in HEK293 cells.

Amino Acid Sequence↗
PubMed2001

Calcium-dependent regulation of interactions of caldesmon with calcium-binding proteins found in growth cones of chick forebrain neurons.

1. This study was undertaken to determine if caldesmon, calmodulin, S100beta, and neurocalcin delta were present in chick forebrain neurons, and if so, to investigate the interactions of these proteins in the presence of different concentrations of calcium. 2. Immunocytochemistry was used to determine the presence and localization of these proteins in cultured forebrain neurons. Western blotting, gel electrophoresis in the presence of different concentrations of calcium, chemical cross-linking, and affinity chromatography were used to investigate the interactions of these proteins with each other. 3. Our data show that caldesmon and three calcium-binding proteins (S100beta, calmodulin, and neurocalcin 3) are localized in growth cones and neurites of chick forebrain neurons in culture. In the presence of different concentration of calcium, these calcium-binding proteins have different affinities to caldesmon and to each other. S100beta binds with greater affinity than calmodulin to caldesmon, and its ability to bind to caldesmon is regulated by neurocalcin delta. 4. These findings suggest a specific calcium-dependent regulatory pathway for modulating actomyosin during growth cone motility.

Animals↗