Calcium‐binding proteins: Differential expression in the rat olfactory cortex after neonatal olfactory bulbectomy

Calbindin, parvalbumin, and calretinin, members of EF‐hand calcium‐binding proteins, play important roles in buffering intracellular calcium ions. These proteins are localized in distinct populations of cells in the olfactory bulb (the primary sensory relay in the olfactory system) and its major synaptic target, the primary olfactory cortex (POC). In the present study, the postnatal expression of these calcium‐binding proteins in layer III of POC was quantitatively examined 30 days after neonatal bulbectomy, a manipulation known to cause cell death and neurotransmitter changes. The numbers of both calbindin and parvalbumin‐immunoreactive profiles showed significant increases (68% and 163%, respectively), while calretinin‐immunoreactive profiles exhibited a 46% reduction. The data demonstrate that the expression of these calcium‐binding proteins is regulated in part by the afferent input from the olfactory bulb. Furthermore, the resultant increase in calbindin and parvalbumin expression may provide neuroprotective support necessitated by possible alterations in intracellular calcium ions and other neurochemical factors that accompany neonatal bulb removal. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 207–217, 1999     

Postnatal developmental expression of calbindin, calretinin and parvalbumin in mouse main olfactory bulb

The distribution of calbindin, calretinin and parvalbumin during the development of the mouse main olfactory bulb (MOB) was studied using immunohistochemistry techniques. The results are as follows:(1) calbindin-immunoreactive profiles were mainly located in the glomerular layer, and few large calbindin-immunoreactive cells were found in the subependymal layer of postnatal day 10 (P10) to postnatal day 40 (P40) mice; (2) no calbindin was detected in the mitral cell layer at any stage; (3) calretinin-immunoreactive profiles were present in all layers of the main olfactory bulb at all stages, especially in the olfactory nerve layer, glomerular layer and granule cell layer; (4) parvalbumin-immunoreactive profiles were mainly located in the external plexiform layer (except for P10 mice); (5) weakly stained parvalbumin-immunoreactive profiles were present in the glomerular layer at all stages; and (6) no parvalbumin was detected in the mitral cell layer at any stage.     

Antibody Recognition of Calcium-Binding Proteins Depends on Their Calcium-Binding Status

Abstract: Previous studies have revealed changes in immunohistochemical stains for calcium-binding proteins after manipulations that influence intracellular calcium. Cases have been revealed in which these changes in immunoreactivity were not correlated with changes in protein amounts. The present experiments examined whether these effects might be explained by changes in antiserum recognition due to calcium-induced changes in protein conformation. Calretinin, calbindin D28k, and parvalbumin incubated in high calcium were recognized by antisera better than when they were incubated in low calcium. Using a calbindin D28k antibody, it was shown that this effect occurs within physiological calcium concentrations. Formalin fixation of the proteins in the presence of calcium resulted in greater antibody recognition than did fixation of proteins in calcium-free states. The calretinin antiserum appeared to recognize a portion of the molecule previously shown to undergo calcium-dependent conformational changes. A calcium-insensitive antiserum was made to a different fragment of calretinin. These results indicate that some antibodies to calcium-binding proteins preferentially recognize particular calcium-induced protein conformations. Given the potential for wide fluctuations in neuronal calcium, the present results indicate that quantitative estimates of intracellular calcium-binding proteins obtained from immunohistochemical studies of neurons must be interpreted with caution.     

Calcium-binding proteins in the retina of a calbindin-null mutant mouse

Calcium-binding proteins are abundantly expressed in many neurons of mammalian retinae. Their physiological roles are, however, largely unknown. This is particularly true for calcium-modulating proteins (“calcium buffers”) such as calbindin D28k. Here, we have studied retinae of wildtype (+/+) and calbindin-null mutant (–/–) mice by using immunocytochemical methods. Although calbindin immunoreactivity was completely absent in the calbindin (–/–) retinae, those cells that express the protein in wildtype retinae, such as horizontal cells, were still present and appeared normal. This was verified by immunostaining horizontal cells for various neurofilament proteins. In order to assess whether other calcium-binding proteins are upregulated in the mutant mouse and may thus compensate for the loss of calbindin, mouse retinae were also immunolabeled for parvalbumin, calretinin, and a calmodulin-like protein (CALP). In no instance could a change in the expression pattern of these proteins be detected by immunocytochemical methods. Thus, our results show that calbindin is not required for the maintenance of the light-microscopic structure of the differentiated retina and suggest roles for this protein in retinal function.     

Use of method of protein engineering in studying calcium-binding proteins

Major results of the use of protein engineering methods in studies of calcium-binding proteins with the highest affinity for calcium and known three-dimensional structure (parvalbumin, calmodulin, troponin C, calbindin, recoverin, alpha-lactalbumin, and others) are presented. Specific features of recombinant calcium-binding proteins are discussed. Experiments with genetic introduction of fluorescent probes, tryptophan and tyrosine, into proteins are overviewed. Effects of mutations in different parts of protein molecules (calcium-binding loops, hydrophobic core, and others) on their structure and properties and attempts of creation of artificial calcium-binding sites are discussed.     

Immunohistochemical distribution of calbindin D-28k and parvalbumin in the head of the caudate nucleus and substantia nigra of the cat

The heterogeneous anatomy of both the dorsal striatum at the level of the head of the caudate nucleus and of the substantia nigra of cats was analyzed immunohistochemically using two calcium-binding proteins, namely, calbindin D-28k and parvalbumin. The striatal histochemical markers nicotinamide-adenine dinucleotide phosphate diaphorase and acetylcholinesterase were revealed in sections adjacent to those used for the immunohistochemical procedure. The distribution of both the calbindin D-28k and the parvalbumin immunoreactivities is heterogeneous in dorsal, ventral, lateral, and medial areas of the head of the caudate nucleus and is in register with the striosome/matrix pattern displayed by the histochemical markers. These calcium-binding proteins preferentially are located in the matrix compartment of the rostral caudate nucleus. Moreover, in some areas of the rostral two-thirds of the substantia nigra, calbindin D-28k and parvalbumin immunoreactivities appear to follow a complementary pattern that is quite different from the mesencephalic distribution of these two calcium-binding proteins. © 1994 Wiley-Liss, Inc.     

Expression of Calcium Binding Proteins in Mouse Type II Taste Cells

It is well established that calcium is a critical signaling molecule in the transduction of taste stimuli within the peripheral taste system. However, little is known about the regulation and termination of these calcium signals in the taste system. The authors used Western blot, immunocytochemical, and RT-PCR analyses to evaluate the expression of multiple calcium binding proteins in mouse circumvallate taste papillae, including parvalbumin, calbindin D28k, calretinin, neurocalcin, NCS-1 (or frequenin), and CaBP. They found that all of the calcium binding proteins they tested were expressed in mouse circumvallate taste cells with the exception of NCS-1. The authors correlated the expression patterns of these calcium binding proteins with a marker for type II cells and found that neurocalcin was expressed in 80% of type II cells, whereas parvalbumin was found in less than 10% of the type II cells. Calretinin, calbindin, and CaBP were expressed in about half of the type II cells. These data reveal that multiple calcium binding proteins are highly expressed in taste cells and have distinct expression patterns that likely reflect their different roles within taste receptor cells.     

Distribution of AMPA glutamate receptor GluR1 subunit-immunoreactive neurons and their co-localization with calcium-binding proteins and GABA in the mouse visual cortex

The neuronal localization of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor (GluR) subunits is vital as they play key roles in the regulation of calcium permeability. We have examined the distribution of the calcium permeable AMPA glutamate receptor subunit GluR1 in the mouse visual cortex immunocytochemically. We compared this distribution to that of the calcium-binding proteins calbindin D28K, calretinin, and parvalbumin, and of GABA. The highest density of GluR1-immunoreactive (IR) neurons was found in layers II/III. Enucleation appeared to have no effect on the distribution of GluR1-IR neurons. The labeled neurons varied in morphology; the majority were round or oval and no pyramidal cells were labeled by the antibody. Two-color immunofluorescence revealed that 26.27%, 10.65%, and 40.31% of the GluR1-IR cells also contained, respectively, calbindin D28K, calretinin, and parvalbumin. 20.74% of the GluR1-IR neurons also expressed GABA. These results indicate that many neurons that express calcium-permeable GluR1 also express calcium binding proteins. They also demonstrate that one fifth of the GluR1-IR neurons in the mouse visual cortex are GABAergic interneurons.     

Alterations in the localization of calbindin D28K-, calretinin-, and parvalbumin-immunoreactive neurons of rabbit retinal ganglion cell layer from ischemia and reperfusion

Calcium-binding proteins are thought to play important roles in calcium buffering. The present study investigated the effects of ischemia and reperfusion on calbindin D28K, calretinin, and parvalbumin immunoreactivity in the ganglion cell layer of the rabbit. Rabbits were administered ischemic damage by increasing the intraocular pressure. After 60 and 90 min of ischemia, reperfusion (7 d) was allowed to occur. The b-wave of the electroretinogram (ERG) was reduced by more than 50% and almost 80% in retina given ischemia for 60 and 90 min, respectively. The oscillatory potential (OPs) wave was reduced approximately 50% at 60 min ischemia and 70% at 90 min ischemia. In both normal and ischemic-treated retina, calcium-binding protein immunoreactivity was seen in many cells in the ganglion cell layer. In eyes subjected to 60 min ischemia, there was a decrease of the density of calbindin D28K- (8.29%), calretinin- (14.44%), and parvalbumin- (26.83%) immunoreactive (IR) cells compared to the control retina. In eyes subjected to 90 min ischemia, there was a higher decrease of the density of calbindin D28K- (18.48%), calretinin- (33.59%), and parvalbumin- (54.26%) IR cells than at 60 min. Some calcium-binding protein-IR neurons, especially calretinin-IR neurons, showed aggregations that were abnormally packed together in retina subjected to ischemia for 90 min. The results show that calbindin D28K-, calretinin-, and parvalbumin-IR cells in the ganglion cell layer are susceptible to ischemic damage and reperfusion. The degree of reduction varied among different calcium-binding proteins and ischemic damage times. These results suggest that calbindin D28K-containing neurons are less susceptible to ischemic damage than calretinin- and parvalbumin-containing neurons in the ganglion cell layer of rabbit retina.     

Distribution of Calretinin, Calbindin D28k, and Parvalbumin in Subcellular Fractions of Rat Cerebellum: Effects of Calcium

Abstract: The distribution of calretinin, calbindin D28k, and parvalbumin was examined in subcellular fractions prepared from rat cerebellum and analyzed by immunoblot. Calretinin was also quantified by radioimmunoassay. As expected, all three soluble, EF-hand calcium-binding proteins were predominantly localized in the cytosolic fraction. Calretinin and calbindin D28k were also detected in membrane fractions. Calretinin was more abundant in synaptic membrane than in microsomal fractions. The cerebellar microsomal fraction contained the greatest concentration of membrane-associated calbindin D28k. The association of calretinin and calbindin D28k with membrane fractions was decreased in samples prepared or incubated in low calcium. Quantification of calretinin in subcellular fractions of rat cerebellum revealed a greater amount of calretinin in cytosolic fractions prepared or incubated in low calcium and reduced amounts of calretinin in all membrane fractions incubated in low calcium with the exception of the mitochondrial fraction. These results imply that calretinin and calbindin D28k might have physiological target molecules that are associated with, or are components of, brain membranes.     

Primary culture of embryonic rat olfactory receptor neurons

Embryonic cells are very robust in surviving dissection and culturing protocols and easily adapt to their in vitro environment. Despite these advantages, research in the olfactory field on cultured embryonic olfactory neurons is sparse. In this study, two primary rat olfactory explant cultures of different embryonic d (E17 and E20) were established, comprising epithelium and bulb. The functionality of these neurons was tested by measuring intracellular calcium responses to cAMP-inducing agents forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) with fluorescence microscopy. For E17, the responsive cell fraction increased over time, from an initial 3% at the 1 d in vitro (DIV) to a maximum of 19% at 11 DIV. The response of E20 neurons fluctuated over time around a more or less stable 13%. A logistic regression analysis indicated a significant difference between both embryonic d in the response to FSK + IBMX. In addition, of these functional neurons, 23.3% of E17 and 54.3% of E20 cultures were responsive to the odorant isoamyl acetate.     

Developmental and Functional Studies of Parvalbumin and Calbindin D28K in Hypothalamic Neurons Grown in Serum-Free Medium

The Ca2+-binding proteins parvalbumin (Mr = 12K) and calbindin D28K [previously designated vitamin D-dependent Ca2+-binding protein (Mr = 28K)] are neuronal markers, but their functional roles in mammalian brain are unknown. The expression of these two proteins was studied by immunocytochemical methods in serum-free cultures of hypothalamic cells from 16-day-old fetal mice. Parvalbumin is first detected in all immature neurons, but during differentiation, the number of parvalbumin-immunoreactive neurons greatly declines to a level reminiscent of that observed in vivo, where only a subpopulation of neurons stains for parvalbumin. In contrast, calbindin D28K was expressed throughout the period investigated only in a distinct subpopulation of neurons. Depolarization of fully differentiated hypothalamic neurons in culture resulted in a dramatic decrease of parvalbumin immunoreactivity but not of calbindin D28K immunoreactivity. The parvalbumin staining was restored on repolarization. Because the anti-parvalbumin serum seems to recognize only the metal-bound form of parvalbumin, the loss of immunoreactivity may signal a release of Ca2+ from intracellular parvalbumin during depolarization of the cells. We suggest that parvalbumin might be involved in Ca2+-dependent processes associated with neurotransmitter release.     

Guidepost neurons for the lateral olfactory tract: Expression of metabotropic glutamate receptor 1 and innervation by glutamatergic olfactory bulb axons

The guidepost neurons for the lateral olfactory tract, which are called lot cells, are the earliest‐generated neurons in the neocortex. They migrate tangentially and ventrally further down this tract, and provide scaffolding for the olfactory bulb axons projecting into this pathway. The molecular profiles of the lot cells are largely uncharacterized. We found that lot cells specifically express metabotropic glutamate receptor subtype‐1 at a very early stage of development. This receptor is functionally competent and responds to a metabotropic glutamate receptor agonist with a transient increase in the intracellular calcium ion concentration. When the glutamatergic olfactory bulb axons were electrically stimulated, lot cells responded to the stimulation with a calcium increase mainly via ionotropic glutamate receptors, suggesting potential neurotransmission between the axons and lot cells during early development. Together with the finding that lot cells themselves are glutamatergic excitatory neurons, our results provide another notable example of precocious interactions between the projecting axons and their intermediate targets. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

The sorting behaviour of olfactory and vomeronasal axons during regeneration

Summary In order to begin to understand how primary olfactory and vomeronasal organ (VNO) axons target specific regions of the olfactory bulb, we examined the sorting behaviour of these axons following neonatal unilateral olfactory bulbectomy. Bulbectomy induced widespread ipsilateral death of the primary olfactory and VNO neurons. After 4 weeks, many new sensory axons had re-grown into the cranial cavity and established a prominent plexus with evidence of dense tufts that were similar in gross appearance to glomeruli. Axons expressing the cell adhesion molecule OCAM, which normally innervate the ventrolateral and rostral halves of the main and accessory olfactory bulbs, respectively, sorted out and segregated from those axons not expressing this molecule within the plexus. In addition, VNO axons formed large discrete bundles that segregated from main olfactory axons within the plexus. Thus, VNO and primary olfactory axons as well as discrete subpopulations of both are able to sort out and remain segregated in the absence of the olfactory bulb. Sorting and convergence of axons therefore occur independently of the olfactory bulb and are probably attributable either to inherent properties of the axons themselves or to interactions between the axons and accompanying glial ensheathing cells.     

Acid-sensing ion channels in mouse olfactory bulb M/T neurons

The olfactory bulb contains the first synaptic relay in the olfactory pathway, the sensory system in which odorants are detected enabling these chemical stimuli to be transformed into electrical signals and, ultimately, the perception of odor. Acid-sensing ion channels (ASICs), a family of proton-gated cation channels, are widely expressed in neurons of the central nervous system. However, no direct electrophysiological and pharmacological characterizations of ASICs in olfactory bulb neurons have been described. Using a combination of whole-cell patch-clamp recordings and biochemical and molecular biological analyses, we demonstrated that functional ASICs exist in mouse olfactory bulb mitral/tufted (M/T) neurons and mainly consist of homomeric ASIC1a and heteromeric ASIC1a/2a channels. ASIC activation depolarized cultured M/T neurons and increased their intracellular calcium concentration. Thus, ASIC activation may play an important role in normal olfactory function.     

Immunocytochemical localization of calcium-binding proteins, calbindin D28K-, calretinin-, and parvalbumin-containing neurons in the dog visual cortex

Although the dog is widely used to analyze the function of the brain, it is not known whether the distribution of calcium-binding proteins reflects a specific pattern in the visual cortex. The distribution of neurons containing calcium-binding proteins, calbindin D28K, calretinin, and parvalbumin in adult dog visual cortex were studied using immunocytochemistry. We also compared this labeling to that of gamma-aminobutyric acid (GABA). Calbindin D28K-immunoreactive (IR) neurons were predominantly located in layer II/III. Calretinin- and parvalbumin-IR neurons were located throughout the layers with the highest density in layers II/III and IV. The large majority of calbindin D28K-IR neurons were multipolar stellate cells. The majority of the calretinin-IR neurons were vertical fusiform cells with long processes traveling perpendicular to the pial surface. And the large majority of parvalbumin-IR neurons were multipolar stellate and round/oval cells. More than 90% of the calretinin- and parvalbumin-IR neurons were double-labeled with GABA, while approximately 66% of the calbindin D28K-IR neurons contained GABA. This study elucidates the neurochemical structure of calcium-binding proteins. These data will be informative in appreciating the functional significance of different laminar distributions of calcium-binding proteins between species and the differential vulnerability of calcium-binding proteins-containing neurons, with regard to calcium-dependent excitotoxic procedures.     

Calcium-binding Proteins Calbindin D28K, Calretinin, and Parvalbumin Immunoreactivity in the Rabbit Visual Cortex

The distribution and morphology of neurons containing three calcium-binding proteins, calbindin D28K, calretinin, and parvalbumin in the adult rabbit visual cortex were studied. The calcium-binding proteins were identified using antibody immunocytochemistry. Calbindin D28K-immunoreactive (IR) neurons were located throughout the cortical layers with the highest density in layer V. However, calbindin D28K-IR neurons were rarely encountered in layer I. Calretinin-IR neurons were mainly located in layers II and III. Considerably lower densities of calretinin-IR neurons were observed in the other layers. Parvalbumin-IR neurons were predominantly located in layers III, IV, V, and VI. In layers I and II, parvalbumin-IR neurons were only rarely seen. The majority of the calbindin D28K-IR neurons were stellate, round or oval cells with multipolar dendrites. The majority of calretinin-IR neurons were vertical fusiform cells with long processes traveling perpendicularly to the pial surface. The morphology of the majority of parvalbumin-IR neurons was similar to that of calbindin D28K: stellate, round or oval with multipolar dendrites. These results indicate that these three different calcium-binding proteins are contained in specific layers and cells in the rabbit visual cortex.