Comparison of treatment regimens to sensitize in situ hybridization for low-abundance calmodulin transcripts in the white matter of the rat spinal cord (original) (raw)
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Synapse, 1990
Calmodulin is a small, acidic calcium-binding protein that regulates a number of calcium-dependent enzyme activities and is thought to be involved in neurotransmission. To begin to explore further the regulation of this important protein in the brain, we have cloned a rat calmodulin cDNA and designed an oligonucleotide probe based on this sequence. Both the cDNA and oligonucleotide probes revealed a markedly heterogeneous distribution of hybridization signal for calmodulin mRNA in the rat brain. The greatest apparent abundance of mRNA for calmodulin was seen in the hippocampus and cerebral cortex, whereas many brain regions showed relatively low hybridization signal, including the striatum and portions of the hypothalamus and brainstem.
Life Sciences, 2005
The ultrastructural distribution of the calmodulin (CaM) mRNAs transcribed from the three CaM genes was studied in the CA1 region of the adult rat hippocampus by means of electron microscopic in situ hybridization. Digoxigenin-labeled CaM gene-specific riboprobes were detected with nanogold-anti-digoxigenin antibody conjugate. The CaM mRNAs were differentially distributed in both the neuronal and glial cell compartments. The greatest difference in neuronal distribution of the CaM mRNAs was found in the dendrites, where the mRNAs transcribed from the CaM I and III genes were much more abundant than the CaM II mRNA. The neuronal perikarya were heavy labeled for all the CaM mRNAs. Interestingly, the myelinated axons and axon terminals also contained small amounts of nanogold particles for all the CaM mRNAs, which diminished with increasing distance from the soma. Most of the synaptic profiles, however, contained labeling only in the postsynaptic region. The CaM mRNAs were differentially distributed in the glial cells. While the glial cell somata were only lightly labeled, surprisingly concentrated labeling was present in the perisynaptic and perivascular astrocytic processes. In general, the CaM II mRNA was the least represented in the glial processes. Only a very low CaM gene expression was observed in the endothelial and resting microglial cells. These results provide ultrastructural evidence for differential targeting of the multiple CaM mRNA transcripts to the intracellular compartments and suggest their microdomain-specific regulation. D
Ontogeny of calmodulin gene expression in rat brain
Neuroscience, 2002
AbstractöCalmodulin (CaM), a multifunctional intracellular calcium receptor, is a key element in signaling mechanisms. It is encoded in vertebrates by multiple apparently redundant genes (CaM I, II, III). To investigate whether di¡erential expression takes place in the developing rat brain, a quantitative in situ hybridization analysis was carried out involving 15 brain areas at six ages between embryonic day 19 and postnatal day 20 (PD20) with gene-speci¢c [ 35 S]cRNA probes. A widespread, developmental stage-speci¢c and di¡erential expression of the three CaM genes was observed. The characteristic changes in the CaM mRNA levels in the examined time frame allowed the brain regions to be classi¢ed into three categories. For the majority of the areas (e.g. the piriform cortex for CaM III), the signal intensities peaked at around PD10 and the expression pro¢le was symmetric (type 1). Other regions (e.g. the cerebral cortex, layer 1 for CaM II) displayed their highest signal intensities at the earliest age measured, followed by a gradual decrease (type 2). The signal intensities in the regions in the third group (e.g. the hypothalamus for CaM III) £uctuated from age to age (type 3). Marked CaM mRNA levels were measured for each transcript corresponding to the three CaM genes in the molecular layers of the cerebral and cerebellar cortici and hippocampus, suggesting their dendritic translocation. The highest signal intensity was measured for CaM II mRNA, followed by those for CaM III and CaM I mRNAs on PD1. However, the CaM II and CaM III mRNAs subsequently decreased steeply, while the CaM I mRNAs were readily detected even on PD20. Our results suggest that during development (1) the transcription of the CaM genes is under di¡erential, area-speci¢c control, and (2) a large population of CaM mRNAs is targeted to the dendritic compartment in a gene-speci¢c manner. ß
Differential calmodulin gene expression in the rodent brain
Life Sciences, 2002
Apparently redundant members of the calmodulin (CaM) gene family encode for the same amino acid sequence. CaM, a ubiquitous cytoplasmic calcium ion receptor, regulates the function of a variety of target molecules even in a single cell. Maintenance of the fidelity of the active CaM-target interactions in different compartments of the cell requires a rather complex control of the total cellular CaM pool comprising multiple levels of regulatory circuits. Among these mechanisms, it has long been proposed that a multigene family maximizes the regulatory potentials at the level of the gene expression. CaM genes are expressed at a particularly profound level in the mammalian central nervous system (CNS), especially in the highly polarized neurons. Thus, in the search for clear evidence of the suggested differential expression of the CaM genes, much of the research has been focused on the elements of the CNS. This review aims to give a comprehensive survey on the current understanding of this field at the level of the regulation of CaM mRNA transcription and distribution in the rodent brain. The results indicate that the CaM genes are indeed expressed in a gene-specific manner in the developing and adult brain under physiological conditions. To establish local CaM pools in distant intracellular compartments (dendrites and glial processes), local protein synthesis from differentially targeted mRNAs is also employed. Moreover, the CaM genes are controlled in a unique, gene-specific fashion when responding to certain external stimuli. Additionally, putative regulatory elements have been identified on the CaM genes and mRNAs. D
Presence of Calmodulin and Calmodulin-Binding Proteins in the Nuclei of Brain Cells
Journal of Neurochemistry, 1991
The nuclear calmodulin levels have been measured in rat neurons and glial cells. The values are 1 .O and 1.1 pg/ mg of protein, respectively. These levels are about threefold higher than those in the nuclei of rat liver cells. We have also investigated the presence of several calmodulin-binding proteins in the nuclei of both brain cellular types. As similarly observed in the nuclei of liver cells, we detected the presence of a-spectrin and a 62-kDa calmodulin-binding protein in the nuclei of neurons and glial cells by immunoblotting and immunocytochemical methods. Both proteins are enriched in the purified nuclear matrix samples from both cel-lular types. In contrast to that occurring in rat hepatocytes, we have not been able to detect, by immunoblotting methods, caldesmon in the nuclear matrices of neurons and glial cells. The immunocytochemical studies suggest, however, that caldesmon can be present in the nuclei but in a fraction distinct from the nuclear matrices. Key Words: Calmodulin-Calmodulin-binding proteins-Caldesmon-a-Spectrin-Brain cortex-Neuronal and glial cell nuclei. Vendrell M. et al.
Effect of hexachlorocyclohexane isomers on calmodulin mRNA expression in the central nervous system
Molecular Brain Research, 1995
Three different calmodulin genes that encode the same protein have been found in the brain of all mammalian species so far examined. Little is known about the factors involved in regulating the expression of this gene family in the central nervous system. We have investigated the possibility of differential expression of two calmodulin genes, CaM I and CaM II, which are expressed strongly in neuronal cells in the adult rat brain, after treatment with the 3/ (lindane) and the ~ isomers of the hexachlorocyclohexane (HCH). In this study a decrease of CaM I mRNA (mainly in the 4.0 kb transcript) was found in the cortex of the rats after 24 h of isomer administration. CaM I expression seemed to be more sensitive to ~5 isomer action, whereas the 7 isomer acted mainly at CaM II level. The levels of mRNA of calmodulin CaM II gene were also found to decrease after lindane administration; ~-HCH produced an increase of this transcript. These results were obtained by Northern blot analysis and confirmed by means of in situ hybridization. Our results suggest that levels of neuronal calmodulin mRNA species are modified in response to changes in neuronal activity.
Anatomy and Embryology, 1995
A combination of either retrograde or anterograde fluorescent tracer and immunofluorescence histochemistry using the monoclonal antibody specific for the alpha isoform of calcium/calmodulin-dependent protein kinase II (CaM kinase IRz) was employed to test whether CaM kinase II(z is expressed in somata of corticospinal neurons and their axons over their whole course. After the injection of carbocyanine dye DiI into the hindlimb area of the primary motor cortex of the rat, corticospinal axons and their terminal arbors were anterogradely labeled: DiIlabeled corticospinal fibers proceeded caudally in the ipsilateral internal capsule, cerebral peduncle and medullary pyramid, crossed at the pyramidal decussation and descended in the ventralmost area of the contralateral dorsal funiculus of the spinal cord. These DiI-labeled corticospinal axons expressed strong CaM kinase IIc~ immunoreactivity along their course. However, their terminal arbors within the gray matter of the lumbar cord were very weakly immunostained. With the injection of Fast Blue into the lumbar enlargement of the rat, Somata of corticospinal neurons in layer V of the motor cortex were retrogradely labeled. The subsequent immunofluorescent histochemistry revealed that more than 80% of Fast Blue-labeled corticospinal neurons were immunostained with CaM kinase IIo~ antibody. The present immunohistochemical study demonstrated that CaM kinase IIa is strongly expressed in both somata and axons of a majority of corticospinal neurons, although we could not detect this enzyme in the corticospinal terminals in the spinal target areas.
Calmodulin Kinase II in Pure Cultured Astrocytes
Journal of Neurochemistry, 1988
Calcium-and calmodulin-dependent protein kinase activity was studied in pure neuronal and glial cultures. The addition of calcium and calmodulin stimulated '*P incorporation into several neuronal proteins including two in the 50and 60-kilodalton (kD) region which comigrated with purified forebrain calmodulin kinase I1 subunits (CaM kinase 11). In mature astrocytes, CaM kinase activity was also present, and was inhibited by trifluoroperazine and diazepam. Again in homogenates of these cells, two phosphoproteins of apparent molecular masses of 50 and 60 kD comigrated with purified CaM kinase. CaM kinase activity was absent in immature mixed glia and oligodendrocytes. The presence of CaM kinase in neurons and mature astrocytes was confirmed using monoclonal antibodies specific for the 50-kD subunit of the enzyme. No immunoreactivity was observed in oligodendrocytes. The presence of CaM kinase in astrocytes suggests a more ubiquitous role of this enzyme in regulating cellular processes than was previously recognized. Key Words: Calmodulin kinase-Astrocytes-Calcium-Cultures-Protein phosphorylation. Bronstein J. et al. Calmodulin kinase I1 in pure cultured astrocytes.