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Identification of Cell-Specific Messenger Ribonucleic Acids in Oxytocinergic and Vasopressinergic Magnocellular Neurons in Rat Supraoptic Nucleus by Single-Cell Differential Hybridization

Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Harold Gainer, Ph.D., Chief, Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 36, Room 4D20, Bethesda, Maryland 20892. E-mail: gainerh{at}ninds.nih.gov.

Magnocellular neurons (MCNs) in the hypothalamo-neurohypophysial system synthesize high levels of the peptides oxytocin (OT) and vasopressin (VP) in separate cells. We used RT-PCR amplification of the RNA from single-cells dissected from supraoptic nuclei of lactating rats to produce cDNAs from identified OT or VP MCNs, which were used to construct OT- and VP-MCN-specific cDNA libraries. These cDNA libraries were then screened using labeled probes from the OT- and VP-cells’ amplified cDNAs. Differentially hybridized colonies were isolated and characterized by slot blot hybridization, Southern blot hybridization, DNA sequencing, and in situ hybridization histochemistry. Using this approach, several novel cell-specific mRNAs were identified in the MCNs. One cell-specific clone, phosphofructokinase-C, was isolated from the OT-cell library, and five cell-specific clones were isolated from the VP-cell library. These were identified as paternally expressed gene (Peg)5/neuronatin, metallothionein III, Peg3, synaptotagmin V, and a 3'-phosphoadenosine 5'-phosphosulfate synthase 2-related mRNA. None of these genes would have been predicted to be differentially expressed in OT and VP MCNs, based on our current knowledge; and hence, this single cell differential gene expression approach has begun to further define the MCN phenotypes by identifying selectively expressed molecules in them.

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