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Colocalization of calretinin and calbindin-D28k with oxytocin and vasopressin in rat supraoptic nucleus neurons: a quantitative study.

Recent electrophysiological experiments, in which purified calbindin-D28k (calbindin) and calretinin antibodies were diffused into these neurons, showed that Ca2+-dependent membrane potentials and firing patterns were profoundly and predictably affected by Ca2+-binding proteins (CaBPs). The present study used quantitative analyses of a dual-labeling immunofluorescence method to investigate the colocalization of the CaBPs, calbindin and calretinin in oxytocin (OT)- and (VP)-containing neurons of the supraoptic nucleus. Analyses of tissue immunostained with two different dilutions of each CaBP antibody used, revealed that 84% and 72% of the OT neurons were positive for calbindin immunoreactivity (-ir) at the higher and lower antibody concentrations, respectively. 52% and 50% of OT neurons were positive for calretinin-ir; thus, many OT neurons express both calbindin and calretinin. In contrast, only 25% and 18% of VP neurons showed calbindin-ir, and they were virtually devoid of calretinin-ir. These results provide evidence that CaBP expression in OT neurons is both greater and more diverse than in VP neurons, and are consistent with the hypothesis that Ca2+ buffering capacity contributes to the control of intrinsic firing patterns.

Analysis of Variance↗

Differential expression of calbindin and calmodulin in motoneurons after hypoglossal axotomy.

Axotomy induces a profound modification of Ca2+ homeostasis in injured neurons which may lead to neuronal death. Remarkably, after axotomy and resection of the hypoglossal nerve, 65-75% of the hypoglossal motoneurons survive in the long term and this suggests some adaptive mechanisms compensating the massive calcium influx. As potential components of this adaptation, we have examined calmodulin and calbindin-D28k by in situ hybridisation and immunohistochemistry in motoneurons of the rat after hypoglossal nerve transection. Neuronal calbindin mRNA and protein content was low in normal state, transiently increased to 200% of the basal expression at 8 days post-operation (dpo), then declined to normal again until 28 dpo. Calmodulin mRNA was highly expressed in normal hypoglossal motoneurons and remained constant after axotomy. Calmodulin protein immunoreactivity, however, was transiently decreased in axotomised motoneurons suggesting post-transcriptional modification. The upregulation of calbindin expression may facilitate the survival of injured motoneurons.

Animals↗

Calretinin immunoreactivity in the anterior olfactory nucleus of the rat.

Calretinin-immunoreactive structures in the anterior olfactory nucleus of the rat were studied using a polyclonal antibody, which does not cross-react with the highly homologous calcium-binding protein calbindin-D28k, and the avidin-biotin-peroxidase technique. Calretinin-immunopositive neurons were found in all regions of the anterior olfactory nucleus, with the highest number in the medial subdivision and dorsal transition area. The immunostained neurons, although morphologically heterogeneous, demonstrated typically small size. In addition to neuronal somata, calretinin-immunopositive fibres and terminals, some of them forming basket-like arrangements surrounding immunonegative neurons, were observed. Although calretinin and calbindin-D28k colocalize in several brain regions, and both proteins showed an extensive overlap in the anterior olfactory nucleus, immunostained semithin sections demonstrated that calretinin does not co-localize with calbindin-D28k in this nucleus.

Animals↗

Distribution of 5-hydroxytryptamine-immunoreactive boutons on immunohistochemically-identified Renshaw cells in cat and rat lumbar spinal cord.

A combination of anti-gephyrin- and anti-calbindin D28k-immunoreactivity was used to identify 129 and 171 Renshaw cells and their dendrites in cat and rat lumbar spinal cord, respectively. Using anti-5-hydroxytryptamine-immunoreactivity to label serotoninergic fibers and boutons, 1048 serotoninergic boutons were observed in close contact with the immunolabeled Renshaw cells, with an average of 4.4 and 2.8 close contacts on cat and rat Renshaw cells, respectively. Two-thirds of the observed appositions were formed on the somatic membrane.

Animals↗

Local circuit neurons showing calbindin D28k-immunoreactivity in the substantia gelatinosa of the medullary dorsal horn of the rat. An immunohistochemical study combined with intracellular staining in slice preparation.

Calbindin D28k (CB)-immunoreactive local circuit neurons in the substantia gelatinosa of the rat medullary dorsal horn were studied in slice preparations; intracellular injection of biocytin was followed by histochemical visualization of biocytin, then CB immunohistochemistry was performed. In six neurons so far examined, overall morphology, including those of dendritic fields, axonal fields, and arborization patterns, was common.

Animals↗

Calretinin and calbindin-D28k in dopaminergic neurons of the rat midbrain: a triple-labeling immunohistochemical study.

We used triple-labeling immunohistochemistry in rat midbrain sections to identify dopaminergic neurons that contain either one or both of the calcium-binding proteins, calretinin (CR) and calbindin-D28k (CB). Midbrain dopaminergic neurons were immunohistochemically labeled for tyrosine hydroxylase (TH), CR, and CB. In the substantia nigra pars compacta (SNC), TH+/CR+/CB+ cells were clustered in two regions: the dorsal tier of the rostral SNC and the medial part of the intermediate SNC. The ventral tier of the rostral SNC mainly comprised both TH+/CR+/CB- and TH+/CR-/CB- cells. The lateral part of the intermediate SNC and the caudal SNC primarily consisted of TH+/CR-/CB- cells. Throughout the extent of the SNC, approximately half of the TH+ neurons were stained for neither CR nor CB, while the remaining TH+ populations were labeled for CR and/or CB. Throughout the ventral tegmental area, TH+/CR+/CB+ cells, TH+/CR+/CB- cells, TH+/CR-/CB+ cells, and TH+/CR-/CB- cells were found generally scattered, though the TH+/CR-/CB- cells were dominant in number. In the substantia nigra pars lateralis, interfascicular nucleus, and caudal linear nucleus, more than half of the TH+ cells were stained for both CR and CB. In the retrorubral field, two-thirds of the TH+ neurons contained neither protein. The present findings suggest that the SNC can be divided into subcompartments based on the distribution of dopaminergic neurons that contain calcium-binding proteins. Furthermore, because CR and CB likely contribute to calcium homeostasis by buffering intracellular calcium concentrations, midbrain dopaminergic neurons containing one or both of these calcium-binding proteins may have a higher calcium-buffering capacity than those lacking the two proteins.

Animals↗

Effect of short-term ethanol exposure on the suprachiasmatic nucleus of hypothalamus: immunohystochemical study in mice.

Morphological changes of the suprachiasmatic nucleus (SCN) of the hypothalamus were investigated in mice exhibiting intoxication signs of stages 2 or 3 after a short application term of 6% ethanol. Alterations in glial cells and neurons were examined using anti-glial fibrillary acidic protein (GFAP) and anti-calbindin D28k monoclonal antibody, respectively. The results revealed that short-term ethanol exposure led to strong expression of GFAP-immunoreactivity (GFAP-IR) in the dorsomedial part of the SCN. Furthermore, GFAP-IR astrocytes showed an increase in number and hypertrophy with longer processes. However, calbindin D28k-IR neurons were apparently little changed in the SCN. It is concluded that neuroadaptive response of astrocytes could occur before the neurotoxic effects emerge on neurons on the SCN.

Animals↗

Age-related changes in calbindin-D28k, calretinin, and parvalbumin-immunoreactive neurons in the human cerebral cortex.

Calbindin-D(28k) (CB), calretinin (CRT), and parvalbumin (PV) are high-affinity cytosolic calcium (Ca(2+)) binding proteins (CBP) that have been found to regulate intracellular calcium concentrations in neurons through their buffering capacity and to protect neurons from insults that induce elevations of intracellular Ca(2+). In earlier studies we observed a substantial and neurochemically specific loss of CB from the human basal forebrain cholinergic neurons (BFCN) in the course of normal aging. In the present experiments we expanded our investigation of age-related changes in calcium binding proteins in the human brain by investigating the status of CB-, CRT-, and PV-positive neurons in 17 cortical areas. There was a trend toward a decrease in the number of CB-immunoreactive neurons in all areas studied. However, this trend reached significance in only 4 areas in which the loss of CB-positive neurons ranged between 20 and 46%. Immunoreactivity for CRT was also decreased in many areas and this difference reached significance in three regions (26-37%). Cortical neurons displaying PV immunoreactivity did not show an age-related change. Comparison with other neurochemically specific cortical neurons indicated a similar age-related loss of nonphosphorylated neurofilament and NADPH-d activity in only a few cortical areas. In contrast, neuronal acetylcholinesterase activity was increased in a few cortical areas. These observations indicate that loss of CBP-positive neurons occurs in restricted cortical regions and is not a specific change as other neurochemically specific neurons also display restricted age-related changes. Furthermore, the age-related changes in cortical CBP-positive neurons appear to be considerably smaller than similar changes in the BFCN. The age-related depletion of CBPs is likely to deprive neurons from the capacity to buffer intracellular calcium and thus to leave them vulnerable to pathological processes that can cause increased intracellular calcium and lead to their degeneration.

Acetylcholinesterase↗

Relationship between ataxin-1 nuclear inclusions and Purkinje cell specific proteins in SCA-1 transgenic mice.

Spinocerebellar ataxia-1 (SCA-1), like other polyglutamine diseases, is associated with aggregation of mutant protein ataxin-1 in the nuclei of susceptible neurons. The role of ataxin-1 aggregates in the pathogenesis of susceptible neurons, especially cerebellar Purkinje cells, is unknown. The present study was initiated to determine the temporal relationship between ataxin-1 aggregation and the sequence of specific biochemical changes in Purkinje cells in SCA-1 transgenic mice (TM). Earlier, we demonstrated that SCA-1 TM with no Purkinje cell loss and no alterations in home cage behavior show decreased expression of calcium-binding proteins calbindin-D28k (CaB) and parvalbumin (PV) in Purkinje cells. To determine if increased expression of mutant ataxin-1 in TM is also associated with earlier biochemical changes in Purkinje cells, both heterozygous and homozygous (B05 line of SCA-1) TM were used. The age of onset of ataxia in SCA-1 TM was at 12 weeks in heterozygotes and 6 weeks in homozygotes. In 6 week old heterozygous TM, Western blot analysis of growth associated protein 43 (GAP-43) and synaptophysin revealed no significant alterations as compared with the age-matched nontransgenic mice (nTM), whereas CaB was significantly reduced. beta-III-Tubulin was used as a specific Purkinje cell marker protein, immunohistochemical localization showed strong beta-III-tubulin immunoreactivity (IR) in Purkinje cells in 6 week old heterozygous TM, whereas CaB and PV IR were markedly reduced in the same neurons (double immunofluorescence staining). Most Purkinje cells from heterozygous (12 weeks old) and homozygous (6 weeks old) TM contained ataxin-1 nuclear inclusions (NIs). Cells with and without visible NIs revealed reduced PV and CaB IR; however, the changes were overtly more severe in cells with visible NIs. In contrast, the same cells were strongly immunoreactive to beta-III-tubulin. CaB, which is also present in the nucleus, colocalized with ataxin-1 and ubiquitin positive NIs. Further, RT-PCR analysis of CaB mRNA in the cerebellum in 6 week old heterozygous TM demonstrated a significant decrease in mRNA in comparison with the aged-matched nTM. These data suggest that there are selective alterations in the expression of CaB and PV in Purkinje cells which possibly occur earlier than ataxin-1 aggregation. Further, we speculate that ataxin-1 aggregates may not be toxic in general; however, they may deplete specific proteins essential for Purkinje cell viability in SCA-1 TM.

Animals↗

Interneuronal survival and calbindin-D28k expression following motoneuron degeneration.

Degeneration of both motoneurons and interneurons has been previously observed in amyotrophic lateral sclerosis. It is unclear whether interneuronal loss is due to an intrinsic neuronal defect or if it occurs secondary to loss of their target motoneurons. We have examined the target dependence of interneurons, their survival and alterations in the expression of the calcium binding protein, calbindin-D28k (CB), in the ventral horn of the rat lumbar cord after extensive motoneuron degeneration was induced by unilateral rhizotomy of spinal nerves L2-L6 at postnatal day 3 (P3). Counts of Nissl-stained cells at P21 revealed no significant interneuronal death despite loss of 80% of their target motoneurons. At P6, some motoneurons transiently expressed CB on the operated side compared to the control side. Since most of these cells are destined to die, this transiently increased CB expression may represent an abortive attempt by the axotomised motoneurons to buffer the neurotoxic consequences of high intracellular calcium. In contrast, there was a time-dependent decrease in CB expression in ventral horn interneurons, with only 35% of putative Renshaw cells expressing CB by P21. These results indicate that neonatal interneurons are capable of surviving the loss of their motoneuron targets, but alter their phenotype as indicated by functional alterations in calcium-binding proteins.

Amyotrophic Lateral Sclerosis↗

Intracerebroventricular kainic acid administration to neonatal rats alters interneuron development in the hippocampus.

The effects of neonatal exposure to excitotoxins on the development of interneurons have not been well characterized, but may be relevant to the pathogenesis of neuropsychiatric disorders. In this study, the excitotoxin, kainic acid (KA) was administered to rats at postnatal day 7 (P7) by intracerebroventricular (i.c.v.) infusion. At P14, P25, P40 and P60, Nissl staining and immunohistochemical studies with the interneuron markers, glutamic acid decarboxylase (GAD-67), calbindin-D28k (CB) and parvalbumin (PV) were performed in the hippocampus. In control animals, the total number of interneurons, as well as the number of interneurons stained with GAD-67, CB and PV, was nearly constant from P14 through P60. In KA-treated rats, Nissl staining, GAD-67 staining, and CB staining revealed a progressive decline in the overall number of interneurons in the CA1 and CA3 subfields from P14 to P60. In contrast, PV staining in KA-treated rats showed initial decreases in the number of interneurons in the CA1 and CA3 subfields at P14 followed by increases that approached control levels by P60. These results suggest that, in general, early exposure to the excitotoxin KA decreases the number of hippocampal interneurons, but has a more variable effect on the specific population of interneurons labeled by PV. The functional impact of these changes may be relevant to the pathogenesis of neuropsychiatric disorders, such as schizophrenia.

Aging↗

Calbindin-D28k is regulated by adrenal steroids in hypothalamic tissue during prenatal development.

Regulation of calbindin-D28k (CALB) in the medial basal hypothalamus (MBH) from male and female fetuses was examined by Western analysis. Control fetal males displayed significantly higher MBH CALB levels compared to females at gestational day 20. Whereas, in general, the lowest CALB levels were recorded in male and female fetuses from long-term prenatally stressed or fetuses from adrenalectomized pregnant rats. These data indicate that corticosterone regulates MBH CALB expression during prenatal development and CALB may be implicated in modulating the sexual differentiation of neural structures within the MBH during perinatal development.

Adrenal Cortex Hormones↗

Differential expression of calretinin, calbindin D28K and parvalbumin in the developing human cerebellum.

Three calcium-binding proteins, calretinin, calbindin D28K and parvalbumin, were immunohistochemically localized in the human cerebellum at different developmental stages. Cells positive for calretinin were not detected during early development of the cerebellum until 21 weeks of gestation at which stage weak staining was found in Purkinje and basket cells of the cortex and in neurons of the dentate nucleus. Both the number of positive cells and the intensity of immunoreactivities were found to increase as the cerebellum became more mature. Calbindin D28K immunoreactivity was, however, detected early in development at 14 weeks of gestation. Positive cells were found in Purkinje, basket, stellate and granule cells of the cerebellar cortex and in neurons of fastigial, globose, emboliform and dentate nuclei. The number of positive cells and the staining intensity for calbindin in both the cerebellar cortex and deep nuclei decreased at more advanced developmental stages. At 21-31 weeks of gestation, positive staining was restricted to Purkinje and basket cells of the cortex. Parvalbumin immunoreactivity was also observed early in development at 14 weeks of gestation. Positivity was found in Purkinje, basket and stellate cells of the cerebellar cortex and in neurons of all the deep nuclei, with the highest number of positive cells in the fastigial nucleus followed by emboliform, globose and dentate nuclei. As the cerebellum became more mature, both the number of positive cells and the staining intensity for parvalbumin decreased in the cortex and deep nuclei. The results of the present study showed that among the three calcium-binding proteins examined, strong immunoreactivities for calbindin D28K and parvalbumin were found inthe human cerebellum early in development at 14 weeks of gestation, but there was a decrease in both the intensity and number of positive cells at more advanced stages. In contrast, calretinin positive cells were not detected until 21 weeks of gestation and the immunoreactivity increased as the cerebellum became more mature. A possible correlation between the developmentally regulated expression of the calcium-binding proteins and expression of different neurotransmitters during development is discussed.

Abortion, Induced↗

Calbindin-D28k- and astroglial protein-immunoreactivities, and ultrastructural differentiation in the prenatal rat cerebral cortex and hippocampus are affected by maternal adrenalectomy.

Maternal adrenal steroid hormones have been proven to be crucial for lung and adrenal prenatal maturation. These hormones mediate the effects of prenatal stress crossing the placenta and influencing the development of the hypothalamus-pituitary-adrenal axis of fetuses. In the present study, we have compared the prenatal development of fetuses from adrenalectomized mothers (ADX group) and from sham-operated mothers. We have used immunohistochemistry for calcium binding-protein Calbindin-D28k, astroglial proteins vimentin and glial fibrillary acidic protein (GFAP), and the ultrastructural differentiation of the cerebral cortex and hippocampus to measure putative differences. The ontogeny of the Calbindin-D28k immunoreactivity was delayed, as transient Calbindin-positive neuronal populations in the ADX group disappeared later during development as compared to that of control animals both in cerebral cortex and hippocampus; cell counts revealed that ADX animals had a significantly higher number of Calbindin-positive cells than controls in the cerebral cortex, while that number was lower in ADX fetuses' hippocampus. Cerebral cortex of ADX animals also had a scattered distribution of stained cells compared with controls, while the hippocampi of the ADX animals had an impaired migration of marginal zone interneurons. No GFAP immunoreactivity was found in the studied prenatal stages. Instead, vimentin-immunoreactivity appeared more profusely distributed throughout the cerebral cortex, in the ADX group than in control animals. At the ultrastructural level, no remarkable differences were found before E20, when a higher undifferentiation in the ADX group, in both cerebral cortex and hippocampus, was evident. The results show for the first time the vulnerability of the prenatal rat brain to maternal adrenalectomy and the necessity of maternal glucocorticoids for encephalic development.

Adrenalectomy↗

Phytoestrogens alter hypothalamic calbindin-D28k levels during prenatal development.

Calbindin-D28K (CALB) in the medial basal hypothalamic (MBH) and preoptic area (POA) of male and female fetuses from pregnant rats fed different phytoestrogens diets during gestation was examined by Western analysis. In animals fed a phytoestrogen containing diet (Phyto-200), males displayed significantly higher CALB levels compared to females. Whereas, in animals fed a phytoestrogen-free diet (Phyto-free), females exhibited significantly higher CALB levels compared to Phyto-200 female values. The present data have far reaching implications where phytoestrogen content in diets apparently influence MBH-POA CALB levels prenatally. The altered CALB levels may in turn modify sexually dimorphic brain structures during neuronal development by buffering Ca2+ that is associated with programmed cell death.

Animals↗

Distribution of NADPH-diaphorase in the superior colliculus of Cebus monkeys, and co-localization with calcium-binding proteins.

We examined the distribution of the enzyme dihydronicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) in the superior colliculus (SC) of the New World monkey Cebus apella, and the co-localization of this enzyme with the calcium-binding proteins (CaBPs) calbindin-D28K, parvalbumin and calretinin. Despite the intensely labeled neuropil, rare NADPH-d-positive cells were observed in the stratum griseum superficiale (SGS). Most of the labeled cells in the SC were found in the intermediate layers, with a great number also in the deeper layers. This pattern is very similar to that described in the opossum (Didelphis marsupialis) and in the cat, and different from the pattern found in the rat, which shows labeled cells mainly in the SGS. Cells doubly stained for NADPH-d and CaBPs were observed throughout the SC, although in a small number. Of the NADPH-d-positive cells, 20.3% were doubly labeled for NADPH-d and parvalbumin, 10.2% revealed co-localization with calretinin, and 5.6% with calbindin. The low number of double-stained cells for NADPH-d and the CaBPs indicates that these molecules must participate in different functional circuits within the SC.

Animals↗

Androgen-dependent modulation of calbindin-D28K in hypothalamic tissue during prenatal development.

We examined the influence of androgens on fetal medial basal hypothalamic and preoptic area (MBH-POA) calbindin-D28K levels (via Western analysis) by treating pregnant rats with testosterone or flutamide, (an androgen receptor blocker). MBH-POA calbindin-D28K levels were significantly decreased in flutamide-treated male fetuses, whereas, MBH-POA calbindin-D28K levels were significantly increased in testosterone-treated female fetuses compared to controls. These results suggest that MBH-POA calbindin-D28K is modulated during prenatal development by androgens.

Anal Canal↗

Single-cell RT-PCR detects shifts in mRNA expression profiles of basal forebrain neurons during aging.

The medial septum and nucleus of the diagonal band (MS/nDB) contain cholinergic and GABAergic neuronal populations that have been identified based on immunohistochemical staining and/or electrophysiological properties. We explored the molecular diversity of MS/nDB neurons using single-cell reverse transcription-polymerase chain reaction (scRT-PCR) to assess gene expression profiles during aging in individual neurons acutely isolated from young (2-4 months) and aged (26-27 months) F344 rats. Neuronal gene expression profiles were characterized by detection of mRNAs for choline acetyltransferase (ChAT, cholinergic) and glutamate decarboxylase (GAD67, GABAergic), as well as mRNAs for calcium binding proteins (CaBPs) calbindin-D28k, calretinin and parvalbumin. Four major neuronal populations were identified: ChAT-positive (ChAT+) cells, GAD-positive (GAD+) cells, ChAT+/GAD+ cells and ChAT negative/GAD negative (ChAT-/GAD-) cells. With age, the percentage of cells expressing ChAT mRNA decreased from 53% in young to 40%, and the expression of GAD67 mRNA was reduced from 56 to 35% of the cells tested. The percentage of cells with detectable levels of both ChAT and GAD67 mRNA was reduced from 24% in young to 9% in aged. Concomitantly, the percentage of ChAT-/GAD- cells increased from 15 to 34% with age. Of the CaBPs, calretinin expression was observed most frequently in this study, and its detection decreased from 33 to 22% of the cells with age. Observations concerning the CaBPs were confirmed using in situ hybridization. These results suggest a shift in the mRNA expression profiles of MS/nDB neuronal populations during aging and exemplify the molecular diversity of cholinergic and GABAergic cells.

Aging↗