BRIEF REPORT |
Correspondence to: Karoly Gulya, Dept. of Zoology and Cell Biology, University of Szeged, 2 Egyetem St., POB 659, Szeged H-6722, Hungary. E-mail: gulyak@bio.u-szeged.hu
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Summary |
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We investigated the intracellular distribution of the mRNAs corresponding to the three non-allelic CaM genes in cultured hippocampal cells by in situ hybridization with digoxigenin-labeled gene-specific riboprobes. In neurons the perikaryon was heavily stained and strong dendritic mRNA targeting was detected for all three CaM genes. The color labeling exhibited a punctate distribution, suggesting that CaM mRNAs are transported in RNA granules. Immunocytochemistry for S100 demonstrated that glial cells express CaM mRNAs at a very low level. A minority of the cultured cells were negative for either labeling. (J Histochem Cytochem 51:541544, 2003)
Key Words: calmodulin, mRNA localization, hippocampal cell culture, S100 immunocytochemistry, dendritic, intracellular
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Introduction |
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CALMODULIN (CaM) is a multifunctional, highly conserved Ca2+-binding protein particularly abundant in neural tissue. It is involved in a wealth of cell functions by interacting with various target proteins, which sometimes even possess opposing activities. In the central nervous system (CNS), a vast majority of the CaM is synthesized by neurons, where it resides in several different intracellular pools, whereas the glial CaM expression is much weaker (
In rodent brain, the CaM mRNAs are heavily distributed in the neuronal cell somata (
In the present study, we carried out CaM gene-specific, non-radioactive in situ hybridization (ISH) cytochemistry on rat primary hippocampal cultures. This system was selected for the following reasons: (a) unlike many immortalized cell lines, it may represent the nearest approximation to in vivo CNS neurons; (b) it mimics several in vivo characteristics of these cells (e.g., dendrites and axons are present, synapse formation takes place); (c) its monolayer structure allows resolution of the subcellular compartments; and (d) notably, in vivo hippocampal pyramidal cells have directly been demonstrated to target CaM I mRNAs into dendrites (e.g.,
On embryonic day 18, rat hippocampi were trypsinized, cells were dissociated by trituration, and plated onto poly-L-lysine-coated (Sigma; St Louis, MO) coverslips at a density of 105 cells/coverslip (
A majority of the cultured cells exhibited neuronal morphology (
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To identify the cells with nondetectable CaM expression, glia-specific S100 immunocytochemistry was carried out after the ISH protocol. Because the S100 protein is mainly confined to the nucleus, whereas the CaM mRNAs are localized in the cytoplasm, the parallel use of the two detection systems would feasibly allow discrimination among CaM-positive/S100-negative or CaM-negative/S100-positive cells. The vast majority of the CaM-negative cells proved to be S100-positive, whereas the neurons expressing the CaM genes did not express the S100 marker (Fig 1B). Moreover, a relatively small number of cells were negative for both the CaM mRNAs and the S100 protein. It should be noted, however, that "negative" can simply mean a very low but still appropriate expression level for the cell. Nevertheless, the identity of the "double-negative" cells remains unknown.
Here, by utilizing nonradioactive ISH cytochemistry, we demonstrate directly that mRNAs transcribed from each CaM gene are subjected to dendritic translocation in the hippocampal neurons in primary culture. In addition, a punctate localization of the CaM mRNAs was revealed, which is consistent with previous findings demonstrating that mRNAs translocate in granular structures. Direct in vivo evidence of the dendritic CaM mRNA pool has been obtained only for the CaM I mRNAs, and exclusively in pyramidal and Purkinje neurons of the developing rat brain (
This study demonstrates that, at least in primary hippocampal neurons, (a) the capacity for dendritic translocation is not restricted to CaM I transcripts but is a more general characteristic of the CaM transcripts, (b) CaM mRNA transport most probably occurs in granular units, and (c) S100-positive (glial) cells do not express CaM in a detectable amount. Further studies are needed to clarify the significance of these findings under in vivo conditions.
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Acknowledgments |
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Supported by grants from the National Scientific Research Fund, Hungary (OTKA T034621) and the Ministry of Health, Hungary (57/2000) to KG.
The skillful technical assistance of Ms Zsuzsa Ambrus is highly appreciated.
Received for publication June 17, 2002; accepted December 18, 2002.
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