Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Calbindin 2”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 397 records · Page 22Linked to original sources
PubMed1998

Olfactory bulb development is altered in small-eye (Sey) mice.

Small-eye (Sey) is a spontaneous, semidominant murine mutation that results from a point mutation in the Pax-6 gene. Both the eyes and the olfactory system fail to develop in homozygotes and these animals die neonatally. Heterozygotes (Sey/+) have different degrees of eye abnormalities including decreased lens size and cataracts. In the present study, we examined whether one mutated allele of Pax-6 also affects olfactory system development. By 42 days of age, main olfactory bulb volume was significantly decreased in Sey/+ animals compared with wild-type littermates, and this effect was even more dramatic in 70-day-old animals. In contrast, there was no effect on accessory olfactory bulb, olfactory epithelial, or vomeronasal organ development at any age in Sey/+ animals, demonstrating the specificity of the effect. In the main olfactory bulb, the largest differences in laminar volume were found in the glomerular and granule cell layers. These layers contain the olfactory bulb interneurons, and a subpopulation of these cells were found to be Pax-6 immunoreactive. Examination of the neurochemical consequences of this mutation showed that the number of both tyrosine hydroxylase (TH)- and gamma-aminobutyric acid (GABA)-immunoreactive profiles were dramatically decreased in Sey/+ animals as compared with controls. In contrast, neither calretinin nor calbindin immunoreactivity was affected by this mutation. Dual-labeling immunohistochemistry showed that nearly all TH-immunoreactive cells and a subpopulation of GABA-immunoreactive cells coexpressed Pax-6. However, calretinin- and calbindin-immunoreactive cells were not Pax-6 immunopositive. These data indicate that two normal alleles of Pax-6 are required for normal olfactory bulb development and, as part of this effect, this gene may be involved in the development of specific neurotransmitter systems.

Aging↗
PubMed1995

Structure of the gene encoding mouse reticulocalbin, a novel endoplasmic reticulum-resident Ca(2+)-binding protein with multiple EF-hand motifs.

Reticulocalbin, an endoplasmic reticulum (ER)-resident Ca(2+)-binding protein, is a member of the EF-hand Ca(2+)-binding protein superfamily [Ozawa, M. and Muramatsu, T. (1993) J. Biol. Chem. 268, 699-705]. Reticulocalbin has six repeats of a domain containing the EF-hand motif. In addition, the protein has an amino-terminal leader sequence which serves as a signal for transfer of the protein into the lumen of the ER, and a His-Asp-Glu-Leu sequence at its carboxy terminus which functions as a signal to retain the protein in the ER. In this paper, we describe the genomic structure of this unique Ca(2+)-binding protein. Southern blot analysis of mouse genomic DNA revealed that there is a single copy of the reticulocalbin gene per haploid genome. The gene spans over 13 kilobase pairs and encodes six separate exons. Thus, reticulocalbin differs from the cytosolic Ca(2+)-binding protein calbindin D28 which also has six EF-hand motif domains, but the gene for which is divided into 11 exons. While there is some correlation between exon division and protein domain structure, these relationships are not as clear as they are in other genes. Comparison of the gene organization of reticulocalbin with that of other EF-hand proteins revealed that reticulocalbin diverged very early from other members of the EF-hand protein super-family.

Amino Acid Sequence↗
PubMed2002

S-nitrosation of Ca(2+)-loaded and Ca(2+)-free recombinant calbindin D(28K) from human brain.

Calbindin D(28K) is noted for its abundance and specific distribution in mammalian brain and sensory neurons. It can bind three to five Ca(2+) ions and may act as a Ca(2+) buffer to maintain intracellular Ca(2+) homeostasis, but its exact role is still unknown. In the present study, mass spectrometric analysis reveals that the five cysteine residues in recombinant human brain calbindin D(28K) (rHCaBP) are derivatized with N-ethylmaleimide, consistent with the determination of 5.3 +/- 0.4 and 4.7 +/- 0.4 free thiols in the protein using the thiol-specific reagents 5,5'-dithiobis(2-nitrobenzoic acid) and 5-(octyldithio)-2-nitrobenzoic acid, respectively. The results of UV-vis and circular dichroism absorption, intrinsic fluorescence, and mass spectrometry measurements indicate that both Ca(2+)-loaded (holo) and Ca(2+)-free (apo) rHCaBP are S-nitrosated by S-nitrosocysteine (CysNO). The number of cysteine residues S-nitrosated in holorHCaBP and aporHCaBP are 2.6 +/- 0.05 and 3.4 +/- 0.09, respectively, as determined by the Saville assay. HolorHCaBP also undergoes S-nitrosation at one to three cysteine residues when exposed to S-nitrosoglutathione (GSNO), and Cys100 was found to be an S-nitrosation site by peptide mass mapping. Treatment of holorHCaBP with free NO resulted in a mass increase of 59 +/- 2 Da, corresponding to two NO adducts. Since up to four cysteine residues can be S-nitrosated in rHCaBP, it is proposed that the protein may act as a NO buffer or reservoir in the brain in a manner similar to serum albumin in blood. It is significant in this context that rHCaBP is found coexistent with nitric oxide synthase in cerebellum and that S-nitrosation varies with Ca(2+) binding, with S-nitrosation occurring to a greater extent in aporHCaBP than in the holoprotein. Furthermore, exposure of rHCaBP to either CysNO or GSNO also leads to rapid S-thiolation of Cys187. We demonstrate here for the first time that intrinsic protein fluorescence is a sensitive probe of protein S-nitrosation. This is due to efficient Förster energy transfer (R(0) approximately 17 A) between tryptophan donors and S-nitrosothiol acceptors.

Binding Sites↗
PubMed1992

Three-dimensional solution structure of Ca(2+)-loaded porcine calbindin D9k determined by nuclear magnetic resonance spectroscopy.

The three-dimensional solution structure of native, intact porcine calbindin D9k has been determined by distance geometry and restrained molecular dynamics calculations using distance and dihedral angle constraints obtained from 1H NMR spectroscopy. The protein has a well-defined global fold consisting of four helices oriented in a pairwise antiparallel manner such that two pairs of helix-loop-helix motifs (EF-hands) are joined by a linker segment. The two EF-hands are further coupled through a short beta-type interaction between the two Ca(2+)-binding loops. Overall, the structure is very similar to that of the highly homologous native, minor A form of bovine calbindin D9k determined by X-ray crystallography [Szebenyi, D. M. E., & Moffat, K. (1986) J. Biol. Chem. 261, 8761-8776]. A model structure built from the bovine calbindin D9k crystal structure shows several deviations larger than 2 A from the experimental distance constraints for the porcine protein. These structural differences are efficiently removed by subjecting the model structure to the experimental distance and dihedral angle constraints in a restrained molecular dynamics protocol, thereby generating a model that is very similar to the refined distance geometry derived structures. The N-terminal residues of the intact protein that are absent in the minor A form appear to be highly flexible and do not influence the structure of other regions of the protein. This result is important because it validates the conclusions drawn from the wide range of studies that have been carried out on minor A forms rather than the intact calbindin D9k.

Amino Acid Sequence↗
PubMed2001

Structure of intraglomerular dendritic tufts of mitral cells and their contacts with olfactory nerve terminals and calbindin-immunoreactive type 2 periglomerular neurons.

Intraglomerular dendritic tufts of Golgi-impregnated and biotinylated dextran amine (BDA)-labeled mitral cells in the rat main olfactory bulb were analyzed in detail. In particular, the relationships of BDA-labeled tufts with olfactory nerve (ON) terminals and processes of calbindin D-28K-immunoreactive (CB-IR) cells were investigated with confocal laser-scanning light microscopic (CLSM) and electron microscopic (EM) analyses. CB-IR cells were type 2 periglomerular cells that restricted their processes in the ON-free (non-ON) zone of the glomerulus and received few synapses from ON terminals. The mitral tufts varied in complexity, but individual branches were rather simple, smooth processes that bore some branchlets and spines and extended more or less in a straight line or a gentle curve rather than winding tortuously within glomeruli as though they did not consider the compartmental organization, which consisted of ON and non-ON zones that interdigitated in a complex manner with one another. Conventional EM analysis revealed that both thin and thick, presumed proximal branches of mitral/tufted cell dendritic tufts received asymmetrical synapses from ON terminals. Correlated CLSM-EM analysis confirmed direct contacts between the BDA- and CB-labeled processes detected in the CLSM examinations, and synapses were recognized at some of those sites. Furthermore, ON terminals and CB-IR processes were distributed on both proximal and distal dendritic branches in a more or less mosaic pattern. These findings revealed that, on the mitral dendritic tufts, ON terminals and processes of type 2 periglomerular neurons were not clearly segregated proximodistally but, rather, were arranged in a mosaic pattern, which may be important in fine tuning the output from individual glomeruli.

Animals↗
PubMed1999

Calbindin-D(28k) controls [Ca(2+)](i) and insulin release. Evidence obtained from calbindin-d(28k) knockout mice and beta cell lines.

The role of the calcium-binding protein, calbindin-D(28k) in potassium/depolarization-stimulated increases in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and insulin release was investigated in pancreatic islets from calbindin-D(28k) nullmutant mice (knockouts; KO) or wild type mice and beta cell lines stably transfected and overexpressing calbindin. Using single islets from KO mice and stimulation with 45 mM KCl, the peak of [Ca(2+)](i) was 3.5-fold greater in islets from KO mice compared with wild type islets (p < 0.01) and [Ca(2+)](i) remained higher during the plateau phase. In addition to the increase in [Ca(2+)](i) in response to KCl there was also a significant increase in insulin release in islets isolated from KO mice. Evidence for modulation by calbindin of [Ca(2+)](i) and insulin release was also noted using beta cell lines. Rat calbindin was stably expressed in betaTC-3 and betaHC-13 cells. In response to depolarizing concentrations of K(+), insulin release was decreased by 45-47% in calbindin expressing betaTC cells and was decreased by 70-80% in calbindin expressing betaHC cells compared with insulin release from vector transfected betaTC or betaHC cells (p < 0.01). In addition, the K(+)-stimulated intracellular calcium peak was markedly inhibited in calbindin expressing betaHC cells compared with vector transfected cells (225 nM versus 1,100 nM, respectively). Buffering of the depolarization-induced rise in [Ca(2+)](i) was also observed in calbindin expressing betaTC cells. In summary, our findings, using both isolated islets from calbindin-D(28k) KO mice and beta cell lines, establish a role for calbindin in the modulation of depolarization-stimulated insulin release and suggest that calbindin can control the rate of insulin release via regulation of [Ca(2+)](i).

1-Methyl-3-isobutylxanthine↗
PubMed1994

Reductive methylation and pKa determination of the lysine side chains in calbindin D9k.

The Lys residues in the 75-residue Ca(2+)-binding protein calbindin D9k were reductively methylated with 13C-enriched formaldehyde. The possible structural effects resulting from the chemical modification were critically investigated by comparing two-dimensional NMR spectra and the exchange rates of some of the amide protons of the native and the modified protein. Our results show that the protein retains its structure even though 10 Lys out of a total of 75 amino acid residues were modified. In the Ca(2+)- and apo-forms of the protein, the 13C-methylated Lys residues can be detected with high sensitivity and resolution using two-dimensional (1H, 13C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. The pKa values of the individual Lys residues in Ca(2+)-calbindin D9k and apo-calbindin D9k were obtained by combining pH titration experiments and (1H, 13C)-HMQC NMR spectroscopy. Each Lys residue in the Ca(2+)- and apo-forms of calbindin D9k has a unique pKa value. The Lys pKa values in the calcium protein range from 9.3 to 10.9, while those in the apo-protein vary between 9.7 and 10.7. Although apo-calbindin D9k has a very similar structure compared to Ca(2+)-calbindin D9k, the removal of two Ca2+ ions from the protein leads to an increase of the pKa values of the Lys residues.

Calbindins↗
PubMed2005

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.

Animals↗
PubMed2002

AP2-like cis element is required for calretinin gene promoter activity in cells of neuronal phenotype differentiated from multipotent human cell line DEV.

Calretinin (CR) is an EF-hand calcium binding protein expressed at high level in neurons. To identify regulatory elements in CR gene promoter, cultured rat cortical cells were transfected with constructs containing its 5'-end deletion mutants and the luciferase reporter gene. A fragment ending at -115 bp upstream of the transcription start site had high promoter activity and was able to induce expression of luciferase specifically in neuronal cells of cortical cultures. The wild type sequence of -115/+54 CR promoter fragment preferentially drove the expression of green fluorescent protein analog in cells of neuronal phenotype differentiated from multipotent human cell line DEV. Electrophoretic mobility shift assays (EMSA) revealed that the -115/-71 CR gene promoter region contains a binding site for a factor present in brain nuclear extract. Among oligonucleotides containing consensus binding sites for transcription factors within this region, the one representing AP2 binding site was able to compete formation of a protein complex. Mutations of this site prevented the binding between brain protein(s) and the -115/+54 CR gene promoter region and abolished the preferential expression of reporter gene in neuronal cells of DEV line. Thus, the AP2-like element seems to be essential for the neuron-specific activity of the CR gene promoter.

Adaptor Protein Complex 2↗
PubMed2006

Developmental changes in Ca2+-regulated functions of early postnatal Purkinje neurons.

Ca(2+) influx through L-type Ca(2+) channels regulates several different cellular processes in developing Purkinje neurons, including activation of transcription factors and expression of cellular proteins. In the current studies, we examined the age dependence of these actions of Ca(2+) during the early developmental period. Purkinje neurons acutely isolated from postnatal day 4-8 rat pups were studied. We also examined the sensitivity of the Ca(2+)-regulated processes to a toxic environmental factor (ethanol) known to show age-dependent actions on developing Purkinje neurons. Results show that Ca(2+) activation of the transcription factor cAMP-responsive element binding protein (CREB) and Ca(2+)-induced alterations in the level of the apoptotic enzyme caspase 3 show both dose and age dependence in the early-developing Purkinje neurons. Interestingly, the age dependence was opposite for the two proteins. Ca(2+) regulation of calbindin, a Ca(2+) binding protein, was dose dependent but showed little age dependence. Exposure to ethanol altered Ca(2+) activation of pCREB in an age-dependent manner but did not alter Ca(2+) regulation of caspase 3 or calbindin levels. Taken together, these results show that the downstream effects of Ca(2+) signaling have age-dependent components during early Purkinje neuron development. This age dependence may play an important role in the normal developmental program and could contribute to the critical window of sensitivity observed for certain toxic agents during early development.

Age Factors↗
PubMed1996

Calbindin D28K forms a Ca(2+)-dissociable complex with mellitin in vitro.

Calbindin D28K (CB), a cytosolic calcium binding protein (CBP), forms a macromolecular complex with the polypeptide mellitin (ME) the absence of calcium, which can be reversibly dissociated by the addition of Ca2+. The molar ratio of CB:ME constituted in this complex is 1:4, suggesting that CB interacts with the tetrameric form of ME. Like free tetrameric ME, the CB:ME complex does not migrate into 15% non-denaturing polyacrylamide electrophoretic gels, although both constituents migrate normally after irreversible complex denaturation by heating in sodium dodecyl sulphate (SDS). The interaction of these two proteins can be distinguished from the association of calmodulin (CM) with ME, which forms a reversibly dissociable, equimolar complex in the presence of Ca2+ and a stable non-migrating complex (molar ratio = 1:12) in its absence. Thus, CB and CM appear to bind ME under different Ca2+ regulatory control, suggesting possible roles for CB as a Ca(2+)-dependent regulatory binding protein.

Animals↗
PubMed2006

Typical and atypical antipsychotic drugs target dopamine and cyclic AMP-regulated phosphoprotein, 32 kDa and neurotensin-containing neurons, but not GABAergic interneurons in the shell of nucleus accumbens of ventral striatum.

Specific neurons in the brain are the primary targets of the action of antipsychotic drugs. Identification and characterization of the nature of these neurons are important for understanding how antipsychotic drugs produce their effects. In previous studies GABAergic/dynorphinergic neurons were identified as a principal cell target of antipsychotic drugs in the shell of nucleus accumbens. In the present study, we further characterized which subpopulations of GABAergic neurons in this area respond after acute administration of antipsychotic drugs. Rats were treated with the typical antipsychotic haloperidol, or the prototype atypical antipsychotic clozapine and killed two hours after treatment. In appropriate sections of brain, double immunofluorescence labeling was performed with antibodies directed against markers specific to candidate cell types and Fos-like proteins (a marker to identify drug-induced cell activation). We reported here that haloperidol- and clozapine-activated neurons showed the following features: 1) approximately 54-57% of them express dopamine and cyclic AMP-regulated phosphoprotein, 32 kDa (a marker for GABAergic medium spiny projection neurons), 2) they appear rarely to be GABAergic interneurons, marked by the calcium binding proteins, parvalbumin, calretinin or calbindin-D28K, 3) about 84-86% of them express the neuropeptide neurotensin (a neurotransmitter most often associated with projection neurons in the site tested). The results suggest that most of the antipsychotic drug-activated neurons in the shell of nucleus accumbens are likely to be neurotensin containing projection neurons.

Animals↗
PubMed2005

Expression of PrP(C) in the rat brain and characterization of a subset of cortical neurons.

The cellular prion protein (PrP(C)) is a membrane-bound glycoprotein mainly present in the CNS. The scrapie prion protein (PrP(Sc)) is an isoform of PrP(C), and it is responsible for transmissible spongiform encephalopathies (TSEs), a group of neurodegenerative diseases affecting both humans and animals. The presence of the cellular form is necessary for the establishment and further evolution of prion diseases. Here, we map the regional distribution of PrP(C) in the rat brain and study the chemical nature of these immunopositive neurons. Our observations are congruent with retrograde transport of prions, as shown by the ubiquitous distribution of PrP(C) throughout the rat brain, but especially in the damaged areas that send projections to primarily affected nuclei in fatal familial insomnia. On the other hand, the presence of the cellular isoform in a subset of GABAergic neurons containing calcium-binding proteins suggests that PrP(C) plays a role in the metabolism of calcium. The lack of immunostaining in neurons ensheathed by perineuronal nets indicates that prions do not directly interact with components of these nets. The destruction of these nets is more likely to be the consequence of a factor needed for prions during the early stages of TSEs. This would cause destruction of these nets and death of the surrounded neurons. Our results support the view that destruction of this extracellular matrix is caused by the pathogenic effect of prions and not a primary event in TSEs.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed2002

Effect of Brn-3a deficiency on parvalbumin-, calbindin D-28k-, calretinin- and calcitonin gene-related peptide-immunoreactive primary sensory neurons in the trigeminal ganglion.

Immunohistochemistry for parvalbumin, calbindin D-28k, calretinin and calcitonin gene-related peptide (CGRP) was performed on the trigeminal ganglion and oro-facial tissues in Brn-3a wildtype and knockout mice at embryonic day 18.5 and postnatal day 0. In wildtype mice, the trigeminal ganglion contained abundant parvalbumin-, calbindin D-28k- and CGRP-immunoreactive neurons while the ganglion was almost devoid of calretinin-immunoreactive neurons. In Brn-3a knockout mice, a 63% decrease of parvalbumin-immunoreactive neurons was detected. In contrast, the absence of Brn-3a dramatically increased the number of calbindin D-28k-immunoreactive (3.5-fold increase) and calretinin-immunoreactive neurons (91-fold increase). The number of CGRP-immunoreactive neurons, however, was not altered by the Brn-3a deficiency. Cell size analysis indicated that loss of Brn-3a increased the proportions of small (<100 microm (2)) parvalbumin-, calbindin D-28k- and CGRP-immunoreactive neurons while it decreased those of large (>200 microm(2)) immunoreactive cells. Calretinin-immunoreactive neurons were either small or medium (100-200 microm (2)) in mutant mice. The oro-facial tissues contained parvalbumin-, calbindin D-28k- and CGRP-immunoreactive fibers, but not calretinin-immunoreactive ones in wildtype mice. In Brn-3a knockout mice, the number of parvalbumin-immunoreactive fibers markedly decreased in the infraorbital nerve and parvalbumin-immunoreactive endings disappeared in the vibrissa. In contrast, the number of calbindin D-28k-immunoreactive fibers increased significantly in the infraorbital and mental nerves. In addition, calbindin D-28k-immunoreactive endings appeared in the vibrissa. As well, some fibers showed calretinin-immunoreactivity in the infraorbital nerve of the mutant. However, no obvious change of CGRP-immunoreactive fibers was observed in the oro-facial region of knockout mice. Taken together, our data suggest that Brn-3a deficiency has effects on the expression of neurochemical substances in the trigeminal ganglion.

Animals↗
PubMed2005

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

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

Animals↗
PubMed1994

Cytosolic Ca2+ binding proteins during rat brain ageing: loss of calbindin and calretinin in the hippocampus, with no change in the cerebellum.

The expression of two cytosolic, high affinity Ca(2+)-binding proteins, calbindin-28 and calretinin, has been investigated in the cerebellum and hippocampus of young and old rats (from 12 days to 30 months) by combining immunofluorescence and Western blotting. Three markers, calreticulin (the major Ca2+ binding protein within the lumen of the endoplasmic reticulum), MAP-2 (a microtubule binding protein concentrated in neuronal dendrites) and synaptophysin (an integral protein of synaptic vesicles), were studied in parallel. In the cerebellar cortex a rise from 12 to 60 days was observed with calbindin-28 and, especially, calretinin, concentrated in the Purkinje and granule neurons, respectively. The level of expression of the two proteins subsequently remained high and the distribution was unchanged, even in the cerebellum of old animals. A completely different pattern was observed in the hippocampus. Here calretinin, present especially in fibres and interneurons, was abundant in the young, decreased in the adult and reached low values in the old rats. Calbindin-28 accumulated during growth, especially in a subpopulation of CA1 pyramidal cells and in the mossy fibres of CA3, then declined, although irregularly, during ageing. These changes of the two proteins were more marked in the dorsal and central parts than in the ventral part of the hippocampus. In the same brain areas the levels of expression of the three additional markers and their distribution within neurons and synapses were unchanged by ageing.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed2002

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↗
PubMed2004

Molecular and functional analysis of hyperpolarisation-activated nucleotide-gated (HCN) channels in the enteric nervous system.

Hyperpolarisation-activated non-specific cation currents (Ih currents) are important for the regulation of cell excitability. These currents are carried by channels of the hyperpolarisation-activated nucleotide-gated (HCN) family, of which there are four known subtypes. In the enteric nervous system (ENS), the Ih current is prominent in AH neurons. We investigated the expression and localization of HCN isoforms in the ENS of mice, rats and guinea-pigs. HCN1, HCN2 and HCN4 were expressed in enteric neurons. Immunoreactivity for HCN1 was observed on neuronal cell membranes of Dogiel type II neurons in rat and mouse. HCN2 channel immunoreactivity occurred in the majority of enteric neurons in the guinea-pig, rat and mouse. Immunoreactivity for HCN4 protein was revealed on the cell membranes of many neurons, including Dogiel type II neurons, in the guinea-pig. HCN4 was expressed by glial cells in guinea-pig. There was no evidence of HCN3 channel protein in any species with either immunohistochemistry or Western analysis. RT-PCR (polymerase chain reaction) using mouse HCN primers revealed mRNA for all four channels in the longitudinal muscle plus myenteric plexus of mouse distal colon. Sequencing confirmed the identity of the mRNA. Quantitative PCR demonstrated that HCN2 was the most highly expressed HCN channel subtype in the myenteric plexus of mouse distal colon. HCN1 and HCN4 were expressed at lower levels. HCN3 subtype mRNA was 0.2% of HCN2. We used intracellular recording to identify neurons having Ih currents and intracellular dye filling to locate the neurons for the immunohistochemical determination of channel expression. AH neurons with Ih currents were HCN2 and HCN4 channel positive. There was no correlation between the magnitude of the Ih and intensity of channel immunoreactivity. Our results indicate that HCN1, 2 and 4 genes and protein are expressed in the ENS. AH/Dogiel type II neurons, which have a prominent Ih, express HCN2 and 4 in guinea-pig and HCN1 and 2 in mouse and rat.

Animals↗