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PubMed2005

Calcium-modulated S100 protein-phospholipid interactions. An NMR study of calbindin D9k and DPC.

The cellular functions of several S100 proteins involve specific interactions with phospholipids and the cell membrane. The interactions between calbindin D(9k) (S100D) and the detergent dodecyl phosphocholine (DPC) were studied using NMR spectroscopy. In the absence of Ca(2+), the protein associates with DPC micelles. The micelle-associated state has intact helical secondary structures but no apparent tertiary fold. At neutral pH, Ca(2+)-loaded calbindin D(9k) does not associate with DPC micelles. However, a specific interaction is observed with individual DPC molecules at a site close to the linker between the two EF-hands. Binding to this site occurs only when Ca(2+) is bound to the protein. A reduction in pH in the absence of Ca(2+) increases the stability of the micelle-associated state. This along with the corresponding reduction in Ca(2+) affinity causes a transition to the micelle-associated state also in the presence of Ca(2+) when the pH is lowered. Site-specific analysis of the data indicates that calbindin D(9k) has a core of three tightly packed helices (A, B, and D), with a dynamic fourth helix (C) more loosely associated. Evidence is presented that the Ca(2+)-binding characteristics of the two EF-hands are distinctly different in a micelle environment. The role of calbindin D(9k) in the cell is discussed, along with the broader implications for the function of the S100 protein family.

Amino Acid Sequence↗
PubMed2000

Caveolae from canine airway smooth muscle contain the necessary components for a role in Ca(2+) handling.

To explain that bronchial smooth muscle undergoes sustained agonist-induced contractions in a Ca(2+)-free medium, we hypothesized that caveolae in the plasma membrane (PM) contain protected Ca(2+). We isolated caveolae from canine tracheal smooth muscle by detergent treatment of PM-derived microsomes. Detergent-resistant membranes were enriched in caveolin-1, a specific marker for caveolae as well as for L-type Ca(2+) channels and Ca(2+) binding proteins (calsequestrin and calreticulin) as determined by Western blotting. Also, the PM Ca(2+) pump was present but not connexin 43 (a noncaveolae PM protein), the sarcoplasmic reticulum (SR) Ca(2+) pump, or the type 1 inositol 1,4, 5-trisphosphate receptor, supporting the idea that SR-derived membranes were not present. Antibodies to caveolin coimmunoprecipitated caveolin with calsequestrin or calreticulin. Thus some of the cellular calsequestrin and calreticulin associated with caveolin on the cytoplasmic face of each caveola. Immunohistochemistry of tracheal smooth muscle crysosections confirmed the localization of caveolin and the PM Ca(2+) pump to the cell periphery, whereas the SR Ca(2+) pump was located deeper in the cell. The presence of L-type Ca(2+) channels, the PM Ca(2+) pump, and the Ca(2+) bindng proteins calsequestrin and calreticulin in caveolin-enriched membranes supports caveola involvement in airway smooth muscle Ca(2+) handling.

Animals↗
PubMed1994

Neurochemical compartmentalization of the globus pallidus in the rat: an immunocytochemical study of calcium-binding proteins.

The globus pallidus external segment forms a major target center of the mammalian striatum which is characterized by neurochemically distinct compartments. The present study was undertaken to determine if a corresponding compartmentalization exists within the globus pallidus external segment in the rat. Immunocytochemical examination of the calcium-binding proteins parvalbumin and calbindin D28kDa, which are present in neurons of the striatal matrix compartment, was employed. The results indicate three neurochemically distinct compartments within the globus pallidus external segment: 1) an area in the medial aspect of the entire length of the globus pallidus that contains dense immunoreactivity for calbindin D28kDa; 2) a narrow rim at the striatopallidal junction in the rostral two-thirds of the globus palidus that contains calbindin D28kDa immunoreactivity designated as the "border zone" of the globus pallidus; and 3) an area between these two zones showing very poor immunoreactivity for calbindin D28kDa but containing parvalbumin immunoreactive neurons. The calbindin D28kDa immunoreactive border zone corresponds to the area of the globus pallidus where striatal inputs converge extensively, whereas the rest of the nucleus is involved in segregated, topographically organized pathways. Parvalbumin-containing neurons are involved in the propagation of striatal output related to striosomal and sensorimotor aspects of basal ganglia function. The present results also indicate that calbindin D28kDa immunoreactivity is completely absent from striosomal neurons and is therefore a useful marker for striatal compartments.

Animals↗
PubMed2003

Ionic currents underlying rhythmic bursting of ventral mossy cells in the developing mouse dentate gyrus.

The electrophysiological properties of mossy cells were examined in developing mouse hippocampal slices using whole-cell patch-clamp techniques, with particular reference to the dorsoventral difference. Dorsal mossy cells exhibited a higher spontaneous excitatory postsynaptic potential (EPSP) frequency and larger maximal EPSP amplitude than ventral mossy cells. On the other hand, the blockade of synaptic inputs with glutamatergic and GABAergic antagonists disclosed a remarkable dorsoventral difference in the intrinsic activity: none (0/27) of the dorsal mossy cells showed intrinsic bursting, whereas the majority (35/47) of the ventral mossy cells exhibited intrinsic rhythmic bursting. To characterize the ionic currents underlying the rhythmic bursting of mossy cells, we used somatic voltage-clamp recordings in the subthreshold voltage range. Ventral bursting cells possessed both hyperpolarization-activated current (Ih) and persistent sodium current (INaP), whereas dorsal and ventral nonbursting cells possessed Ih but no INaP. Blockade of Ih with cesium did not affect the intrinsic bursting of ventral mossy cells. In contrast, the blockade of INaP with tetrodotoxin or phenytoin established a stable subthreshold membrane potential in ventral bursting cells. The current-voltage curve of ventral bursting cells showed a region of tetrodotoxin-sensitive negative slope conductance between -55 mV and a spike threshold ( approximately -45 mV). On the other hand, no subthreshold calcium conductances played a significant role in the intrinsic bursting of ventral mossy cells. These observations demonstrate the heterogeneous electrophysiological properties of hilar mossy cells, and suggest that the subthreshold INaP plays a major role in the intrinsic rhythmic bursting of ventral mossy cells.

2-Amino-5-phosphonovalerate↗
PubMed2000

Temporal lobe epilepsy with and without psychosis: exploration of hippocampal pathology including that in subpopulations of neurons defined by their content of immunoreactive calcium-binding proteins.

We have investigated relationships between hippocampal/temporal lobe neuropathology and psychosis in subjects with temporal lobe epilepsy, paying particular attention to possible differences in density of hippocampal neurons immunoreactive for calcium-binding proteins. There was a trend for a greater prevalence of left handedness in the psychotic (n = 6) than the non-psychotic (n = 26) cases (P = 0.0504). Psychotic cases also differed from non-psychotic ones in having: (1) more focal lesions outside the hippocampus (P = 0.006); (2) less severe CAI neuron loss (P = 0.015); and (3) a trend, after Bonferroni correction, for a higher density of calbindin-immunoreactive neurons in the CA4 (P = 0.022). An additional finding was that dentate granule cell dispersion was significantly associated with the presence of a reduced density of calretinin-immunoreactive neurons in CA4 (P = 0.002) and with a more severe loss of CA4 neurons visible with Nissl stain (P = 0.003). Thus, cases of temporal lobe epilepsy with psychosis were distinguishable on the basis of a higher density of calbindin-reactive neurons in CA4 as well as on more general aspects of their pathology.

Adolescent↗
PubMed2003

Thiazide-induced hypocalciuria is accompanied by a decreased expression of Ca2+ transport proteins in kidney.

INTRODUCTION: Thiazide diuretics have the unique characteristic of increasing renal Na+ excretion, while decreasing Ca2+ excretion. However, the molecular mechanism responsible for this thiazide-induced hypocalciuria remains unclear. The present study investigates the effect of thiazides on the expression of the proteins involved in active Ca2+ transport as well as the role of extracellular volume (ECV) status. METHODS: Hydrochlorothiazide (HCTZ), 12 mg/24 hours, was administered during 7 days to Wistar rats by osmotic minipumps. In addition, ECV contraction was either prevented by Na+ repletion or induced by a low-salt diet. Expression levels of the proteins involved in active Ca2+ transport [i.e., epithelial Ca2+ channel (TRPV5/ECaC1), calbindin-D28K, Na+/Ca2+ exchanger (NCX1)], as well as the thiazide-sensitive Na+ Cl- cotransporter (NCC) were determined by real-time quantitative polymerase chain reaction (PCR) and semiquantitative immunohistochemistry. RESULTS: HCTZ significantly reduced urinary Ca2+ excretion (22%+/- 5% relative to controls). Hematocrit was significantly increased, confirming ECV contraction. In addition, Na+ depletion virtually abolished Ca2+ excretion (8%+/- 1%), while Na+ repletion during HCTZ treatment prevented both ECV contraction and hypocalciuria. HCTZ significantly decreased mRNA expression of TRPV5 (71%+/- 6%), calbindin-D28K (53%+/- 6%), NCX1 (51%+/- 8%) and NCC (50%+/- 11%), regardless of ECV status or calciuresis. Immunohistochemistry revealed reduced TRPV5 (43%+/- 2%), calbindin-D28K (59%+/- 1%) and NCC (56%+/- 4%) abundance. Furthermore, during HCTZ treatment, the subset of tubules coexpressing NCC and calbindin-D28K was significantly reduced (43%+/- 5%) and a disturbed cellular localization of NCC was observed. CONCLUSION: These data suggest that ECV contraction is a critical determinant of the thiazide-induced hypocalciuria, which is accompanied by a decreased expression of Ca2+ transport proteins.

Animals↗
PubMed2000

Ca(2+)- and H(+)-dependent conformational changes of calbindin D(28k).

Calbindin D(28k) is a member of a large family of intracellular Ca(2+) binding proteins characterized by EF-hand structural motifs. Some of these proteins are classified as Ca(2+)-sensor proteins, since they are involved in transducing intracellular Ca(2+) signals by exposing a hydrophobic patch on the protein surface in response to Ca(2+) binding. The hydrophobic patch serves as an interaction site for target enzymes. Other members of this group are classified as Ca(2+)-buffering proteins, because they remain closed after Ca(2+) binding and participate in Ca(2+) buffering and transport functions. ANS (8-anilinonaphthalene-1-sulfonic acid) binding and affinity chromatography on a hydrophobic column suggested that both the Ca(2+)-free and Ca(2+)-loaded form of calbindin D(28k) have exposed hydrophobic surfaces. Since exposure of hydrophobic surface is unfavorable in the aqueous intracellular milieu, calbindin D(28k) most likely interacts with other cellular components in vivo. A Ca(2+)-induced conformational change was readily detected by several optical spectroscopic methods. Thus, calbindin D(28k) shares some of the properties of Ca(2+)-sensor proteins. However, the Ca(2+)-induced change in exposed hydrophobic surface was considerably less pronounced than that in calmodulin. The data also shows that calbindin D(28k) undergoes a rapid and reversible conformational change in response to a H(+) concentration increase within the physiological pH range. The pH-dependent conformational change was shown to reside mainly in EF-hands 1-3. Urea-induced unfolding of the protein at pH 6, 7, and 8 showed that the stability of calbindin D(28k) was increased in response to H(+) in the range examined. The results suggest that calbindin D(28k) may interact with targets in a Ca(2+)- and H(+)-dependent manner.

Anilino Naphthalenesulfonates↗
PubMed1993

Quantitative measurement of calretinin and beta-actin mRNA [correction of mRNAIN] in rat brain micropunches without prior isolation of RNA.

A microdissection technique for quantitation of neurochemicals in discrete brain nuclei has been applied to quantitative measurement of mRNA. The method permits quantitation of low abundance mRNA from submilligram amounts of tissue (10-500 micrograms protein). Discrete nuclei and other regions of the brain are solubilized in concentrated guanidine thiocyanate solution, mRNA is directly hybridized with riboprobes, and detected with a ribonuclease protection assay. This method eliminates the necessity for RNA isolation from solid tissue. No assumptions regarding RNA recovery are necessary since tissue specimens are solubilized, hybridized and treated with ribonuclease in a single tube. We have determined the mRNA levels of calretinin, a predominantly neuron-specific calcium binding protein in microdissected nuclei and other regions of rat brain. For interassay comparison, measurement of sample protein and beta-actin mRNA permits normalization and quantitation in terms of these internal controls. The quantity of calretinin mRNA ranged from 281 +/- 35 fg/micrograms protein in the thalamic paraventricular nucleus to 2.3 +/- 0.5 fg/micrograms protein for the cerebral cortex. The calretinin/beta-actin ratios ranged from 79.9 +/- 9.3% to 1.3 +/- 0.1%, respectively. The combination of microdissection techniques with a lysate RNase protection assay: (1) establishes this technique as quantitative for detection of high and low abundance mRNAs from microdissected brain specimens; (2) bypasses the inefficiencies and uncertainties associated with isolating RNA; and (3) enables large numbers of determinations from discrete brain nuclei to be analyzed in 2 to 3 days.

Actins↗
PubMed2001

Unipolar brush cells form a glutamatergic projection system within the mouse cerebellar cortex.

Unipolar brush cells (UBCs) of the mammalian vestibulocerebellum receive mossy fiber projections primarily from the vestibular ganglion and vestibular nuclei. Recently, the axons of UBCs have been shown to generate an extensive system of cortex-intrinsic mossy fibers, which resemble traditional cerebellar mossy fiber afferents and synapse with granule cell dendrites and other UBCs. However, the neurotransmitter used by the UBC axon is still unknown. In this study, we used long-term organotypic slice cultures of the isolated nodulus (lobule X) from postnatal day 8 mouse cerebella to identify the neurotransmitter and receptors at synapses of the UBC axon terminals, relying on the notion that, in these cultures, all of the cortex-extrinsic fibers had degenerated during the first few days in vitro. Quantification of glutamate immunogold labeling showed that the UBC axon terminals have the same high gold-particle density as the glutamatergic parallel fiber varicosities. Furthermore, UBCs identified by calretinin immunoreactivity expressed the glutamate receptor subunits GluR2/3, NMDAR1, and mGluR2/3, like they do in the mature mouse cerebellum in situ. Evoked excitatory postsynaptic currents (EPSCs), spontaneous EPSCs, and burst discharges were demonstrated in UBCs and granule cells by patch-clamp recording. Both the evoked and spontaneous EPSCs were blocked by ionotropic glutamate receptor antagonists CNQX and D-AP5. We conclude that neurotransmission at the UBC axon terminals is glutamatergic. Thus, UBCs provide a powerful network of feedforward excitation within the granular layer, which may amplify vestibular signals and synchronize activity in clusters of functionally related granule cells which project vertically to patches of Purkinje cells.

2-Amino-5-phosphonovalerate↗
PubMed1996

The expression of calretinin in transfected PC12 cells provides no protection against Ca(2+)-overload or trophic factor deprivation.

To address the question whether calretinin (CR) may protect cells against Ca2+ overload or trophic factor deprivation, PC12 cells were transfected with plasmids containing a CR coding region under control of a cytomegalovirus promoter. Nerve growth factor (NGF) treatment induced differentiation, increased transfection efficiency (at least 10-fold) and activated the CR gene (as found by RNase protection method and immunohistochemistry). Exogenous CR expression was identified either in living cells by fluorescence of green fluorescent protein (when the CR coding region was fused to this protein) or in fixed cells by CR immunoreactivity. Undifferentiated and NGF-differentiated populations of transfected cells were incubated in the presence of a Ca(2+)-ionophore or in media deprived of serum or NGF. Expression of exogenous CR in undifferentiated or NGF-treated cells (due to transfection) or endogenous CR (due to gene activation by NGF) did not render PC12 cells more resistant to insults such as Ca(2+)-overload and trophic factor deprivation.

Animals↗
PubMed1995

Localization of Ca(2+)-dependent conformational changes of calretinin by limited tryptic proteolysis.

Calretinin is an EF-hand Ca(2+)-binding protein expressed predominantly in some neurons. We have found that the tryptic digestion pattern of rat recombinant calretinin depends on Ca2+ concentration as determined by SDS/PAGE, amino-acid-sequence analysis and electrospray-ionization MS. Ca(2+)-saturated calretinin was cleaved between amino acids 60 and 61 to yield two fragments, which accumulated during cleavage. Small amounts of the larger fragment (amino acid residues 61-271) were further cleaved from the C-terminal end. Ca(2+)-free calretinin was also cleaved between residues 60 and 61; however, under the latter conditions the fragment 61-271 was further cleaved from the N-terminal end. Native rat calretinin was cleaved by trypsin in a similar Ca(2+)-dependent fashion. All identified fragments of recombinant calretinin bound 45Ca2+ on nitrocellulose filters, although to a different extent. The 61-271 fragment was released by EGTA from an octyl-agarose column in a manner similar to intact calretinin, while fragment 61-233 was not eluted by EGTA. These observations show that there are trypsin cleavage sites in calretinin that are available regardless of Ca2+ binding, other sites that are completely protected against trypsin on Ca(2+)-binding and sites which become partially available on Ca(2+)-binding. Together these data show that calretinin changes its conformation on Ca2+ binding and identify the regions which are exposed in apo and Ca(2+)-bound form.

Animals↗
PubMed2004

Spatiotemporal patterning of IP3-mediated Ca2+ signals in Xenopus oocytes by Ca2+-binding proteins.

Ca(2+)-binding proteins (CaBPs) are expressed in a highly specific manner across many different cell types, yet the physiological basis underlying their selective distribution patterns remains unclear. We used confocal line-scan microscopy together with photo-release of IP(3) in Xenopus oocytes to investigate the actions of mobile cytosolic CaBPs on the spatiotemporal properties of IP(3)-evoked Ca(2+) signals. Parvalbumin (PV), a CaBP with slow Ca(2+)-binding kinetics, shortened the duration of IP(3)-evoked Ca(2+) signals and 'balkanized' global responses into discrete localized events (puffs). In contrast, calretinin (CR), a presumed fast buffer, prolonged Ca(2+) responses and promoted 'globalization' of spatially uniform Ca(2+) signals at high [IP(3)]. Oocytes loaded with CR or PV showed Ca(2+) puffs following photolysis flashes that were subthreshold in controls, and the spatiotemporal properties of these localized events were differentially modulated by PV and CR. In comparison to results we previously obtained with exogenous Ca(2+) buffers, PV closely mimicked the actions of the slow buffer EGTA, whereas CR showed important differences from the fast buffer BAPTA. Most notably, puffs were never observed after loading BAPTA, and this exogenous buffer did not show the marked sensitization of IP(3) action evident with CR. The ability of Ca(2+) buffers and CaBPs with differing kinetics to fine-tune both global and local intracellular Ca(2+) signals is likely to have significant physiological implications.

Animals↗
PubMed2003

Selective neuronal vulnerability following mild focal brain ischemia in the mouse.

The evolution of cellular damage over time and the selective vulnerability of different neuronal subtypes was characterized in the striatum following 30-minute middle cerebral artery occlusion and reperfusion in the mouse. Using autoradiography we found an increase in the density of [3H]PK11195 binding sites--likely reflecting microglial activation--in the lesion border at 3 days and in the whole striatum from 10 days to 6 weeks. This was accompanied by a distinct loss of [3H]flumazenil and [3H]CGP39653 binding sites from 10 days up to 6 weeks reflecting neuronal loss. Brain ischemia resulted in a substantial loss of medium spiny projection neurons as seen at three days by Nissl staining, TUNEL and immunocytochemistry using antibodies against microtubule-associated protein (MAP2), NeuN, mu-opioid receptors, substance P, L-enkephalin, neurokinin B, choline acetyltransferase, parvalbumin, calretinin and somatostatin. Both patch and matrix compartments were involved in ischemic damage. In contrast, the numbers of cholinergic, GABAergic, and somatostatin-containing interneurons in the ischemic striatum were not different from those in the contralateral hemisphere at 3 and 14 days. A low density of glutamate receptors, the ability to sequester calcium by calcium-binding proteins and other hitherto unidentified factors may explain this relative resistance of interneurons to acute ischemia.

2-Amino-5-phosphonovalerate↗
PubMed2006

Influence of the "open field" exposure on calbindin D28K, calretinin, and parvalbumin containing cells in the rat midbrain - developmental study.

The aim of our study was to analyze the influence of the open field (OF) exposure on: 1. Distribution of c-Fos positive nuclei in: ventral tegmental area, substantia nigra, periaqueductal gray. 2. Appearance of calbindin-D28k, calretinin and parvalbumin in midbrain neurons that are engaged in the stress response. 3. Changes of c-Fos and calcium-binding proteins expression during maturation. The material consisted of Wistar rats of age between 0 and 90 days. The OF exposure was applied throughout 10 min and 90 min before the death of the animals. The brain sections were double stained using the antibodies against c-Fos, CB, CR or PV. Our results showed that in all studied nuclei age-related increase of c-Fos expression (without changing of its distribution properties) was found. PV didn't show any co-localization with c-Fos in neurons of studied regions at any ages, however some PV-immunoreactive (PV-ir) basket-like structures around c-Fos-immunoreactive (c-Fos-ir) neurons were observed. In the youngest group of rats c-Fos-ir cells and cells immunoreactive for CB and CR constituted separate neuronal populations. During maturation increases in the level of their co-localization with c-Fos was observed. We may conclude that in adult rat midbrain structures CB-immunoreactive (CB-ir) and CR-immunoreactive (CR-ir) cells (probably projection neurons) are mainly activated in the stress response following OF exposure. In the contrary PV-ir cells has only an indirect (modulatory) influence upon the c-Fos-ir cells.

Animals↗
PubMed2004

Developmental expression of the Ca2+-binding proteins calretinin and parvalbumin at the calyx of Held of rats and mice.

Ca(2+)-binding proteins of the EF-hand family are widely expressed in the CNS, and contribute to intracellular Ca(2+) buffering in neurons. In nerve terminals, Ca(2+)-binding proteins are likely to regulate transmitter release probability and synaptic short-term-plasticity. Here, we investigated the developmental expression pattern of calretinin and parvalbumin at a large excitatory synapse, the calyx of Held in the medial nucleus of the trapezoid body (MNTB) of rats and mice. We used two-colour immunofluorescence imaging with primary antibodies detecting one of the Ca(2+)-binding proteins, and a presynaptic marker protein, Rab-3A. Calretinin was found in nerve terminals of the calyx of Held, but not in postsynaptic principal cells. The presynaptic density of Calretinin staining, and the degree of colocalization with Rab-3A increased during postnatal development (P6-P31). Surprisingly, not all calyces of Held expressed calretinin. In rats, calretinin-containing calyces were irregularly interspersed with calretinin-negative calyces, whereas in mice, calretinin-positive calyces were preferentially located in the lateral portion of the MNTB. The percentage of calretinin-positive calyces increased during development, to about 75% and 20% at P30 in rats and in mice, respectively. Parvalbumin was present in the presynaptic calyces of Held and in the nerve fibres entering the MNTB, as well as in the somata of the MNTB principal neurons. An up-regulation of calretinin and parvalbumin in calyces of Held probably increases the presynaptic Ca(2+) buffering strength during postnatal development, but the unexpected heterogeneity of calretinin expression might cause differences in Ca(2+) signalling and transmitter release probability between calyces of Held.

Age Factors↗
PubMed2001

Differential immunoreactivity for alpha-actinin-2, an N-methyl-D-aspartate-receptor/actin binding protein, in hippocampal interneurons.

Recent studies have demonstrated that hippocampal interneurons possess distinct cytoskeletal and cell-signaling proteins in comparison to hippocampal principal cells; however, little is known about the differences in the actin cytoskeleton between these two populations. This study examined the immunoreactivity of alpha-actinin-2, an actin binding/N-methyl-D-aspartate-receptor linking protein, in the rat hippocampal formation using double-labelling immunofluorescence. Alpha-actinin-2 immunoreactivity is seen throughout the hippocampus with heavy labeling observed in the dendrites of granule cells, in CA2 pyramidal cells and in presumed interneuronal somata throughout the dentate gyrus and CA1. All the cells with heavy somatic alpha-actinin-2 immunoreactivity in the dentate gyrus and CA1 were GABAergic interneurons labeled by glutamate decarboxylase (99%). Examination of the neurochemical marker content of the alpha-actinin-2 immunoreactive interneurons revealed that the majority of this population was neuropeptide-Y-positive and a minority was positive for calretinin. Fluid percussion head trauma did not result in significant alterations of alpha-actinin-2 immunoreactivity in hippocampal interneurons. The developmental profile of alpha-actinin-2 immunoreactivity showed the presence of alpha-actinin-2 in the hippocampus at P1, labeling of interneurons by P7 and the adult staining pattern seen by P21. This study demonstrates that principal cells and interneurons are differentially immunoreactive for alpha-actinin-2, and that alpha-actinin-2 staining is restricted to a subpopulation of interneurons. Each of the three classes of cytoskeletal elements have been shown to be differentially expressed in hippocampal interneurons and principal cells, suggesting that the cytoskeleton is a defining feature of neuronal populations. Additionally, the limited expression of alpha-actinin-2 could have important functional implications in N-methyl-D-aspartate receptor localization and modulation.

Actinin↗
PubMed2004

Immunohistochemical staining for calretinin is useful for differentiating schwannomas from neurofibromas.

We studied 25 cases of schwannoma and 42 cases of neurofibroma immunohistochemically with antibodies to calretinin and S-100 protein to explore the potential usefulness of calretinin in differentiating schwannomas from neurofibromas. Of 25 schwannomas, 24 (96%) showed moderate to strong staining for calretinin, with the extent of staining ranging from focal to diffuse. In contrast, only 3 (7%) of 42 neurofibromas displayed focal weak to moderate staining with calretinin. All 42 cases of neurofibromas and all 25 cases of schwannomas showed diffuse moderate to strong staining with S-100 protein. Calretinin also labeled mast cells, whose presence was confirmed further by staining for c-kit, which commonly was present in both tumor types in a scattered individual cell pattern easily differentiated from the clustered pattern of neoplastic spindle cells. Taken together, these results indicate that calretinin is detected in almost all schwannomas and in only a small percentage of neurofibromas, suggesting it is a useful marker for differentiating schwannomas from neurofibromas. Although mast cells present in these 2 neoplasms also react with calretinin, the pattern of staining can be distinguished easily from that of neoplastic cells.

Calbindin 2↗
PubMed1993

Characterization of calretinin-immunoreactive structures in the striatum of the rat.

Previous observations have shown that the striatum contains a population of neurones that display immunoreactivity for calretinin. In order to morphologically characterize these neurones, sections of the rat striatum were immunostained to reveal calretinin and examined at both light and electron microscopic levels. The striatum contained a small population of calretinin-immunoreactive neurones, which were of medium-size (9-17 microns) and possessed few aspiny, infrequently branching dendrites which tapered to become very thin processes in their most distal portions. Although the calretinin-immunoreactive neurones were homogeneously distributed in the frontal plane, there was a marked rostrocaudal gradient with a much greater density of cells in the rostral than in the caudal parts of the striatum. At the ultrastructural level, calretinin-immunoreactive neurones were seen to possess an indented nucleus and to receive synaptic input from at least three types of boutons. In addition to the calretinin-immunoreactive neurones, the striatum also contained axons and terminal boutons that displayed immunoreactivity for calretinin. At least two types of immunoreactive terminals were identified, those forming symmetrical synaptic specialisations and those forming asymmetrical synaptic specialisations. Approximately 50% formed asymmetrical contacts with spines and 30% formed symmetrical synaptic contact with dendritic shafts. In an attempt to further chemically characterize the calretinin-containing neurones, double pre-embedding immunocytochemistry for calretinin and parvalbumin or choline acetyltransferase was carried out and calretinin immunocytochemistry was combined with histochemistry for NADPH-diaphorase. Analysis of these double-stained sections revealed that the population of calretinin-immunoreactive neurones was distinct from the populations of neurones containing parvalbumin, choline acetyltransferase or NADPH-diaphorase. It is concluded that: (1) on the basis of distribution, morphology, chemistry, ultrastructure and afferent synaptic input, the calretinin-immunoreactive neurones are distinct from the major classes of neurones that have been previously recognised in the striatum; (2) calretinin-immunoreactive terminals are heterogeneous and are probably derived from local calretinin-containing neurones and possibly other sources.

Animals↗