Summary
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1.
The use of radioactive and biotinylated oligonucleotide probes has been optimized to detect and analyze byin situ hybridization, neurons expressing neuropeptide genes (vasopressin, oxytocin, somatostatin).
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2.
In situ hybridization was performed on cryostat-cut sections obtained from tissues perfused with 1% formaldehyde. Radioactive probes were labeled by tailing with35S-dATP and revealed with autoradiography. Biotinylated probes were obtained either by the incorporation of 11-biotin dUTP or by the addition of biotinylated nucleotides to the oligonucleotide during its synthesis. Biotin was revealed with streptavidin alkaline phosphatase and the appropriate substrate.
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In the adult rat brain, radioactive and biotinylated probes revealed peptidergic neurons. The biotinylated probes provided an optimal cellular and subcellular resolution with a sensitivity similar to that observed with radioactive probes. Staining was selectively restricted to the cytoplasm and to the proximal part of processes.
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Biotinylated vasopressin probes with 10 biotins added demonstrated magnocellular neurons and parvocellular neurons in the suprachiasmatic nucleus and the bed nucleus stria terminalis.
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Vasopressin gene expression was studied during ontogeny in the rat fetus and neonate. Vasopressin mRNA was first detectable at gestational day 16 in the supraoptic nucleus in neurons of neuroblastic appearance. An aspect similar to the one present in adult was found at gestational day 19 in magnocellular neurons and at day 3 postnatal in parvocellular neurons.
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6.
The results confirm that radioactive oligonucleotide probes are efficient tools to investigate neuropeptide gene expression byin situ hybridization and demonstrate that biotinylated oligonucleotides are very efficient and provide a much higher resolution than radioactive probes with a reasonable sensitivity.
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Bloch, B., Guitteny, A.F., Chouham, S. et al. Topography and ontogeny of the neurons expressing vasopressin, oxytocin, and somatostatin genes in the rat brain: An analysis using radioactive and biotinylated oligonucleotides. Cell Mol Neurobiol 10, 99–112 (1990). https://doi.org/10.1007/BF00733638
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DOI: https://doi.org/10.1007/BF00733638