Laser Capture Microscopy (LCM) and real time RT‐PCR: Gene expression in PVN of transgenic vasopressin‐enhanced green fluorescent protein (VP‐eGFP) rats

Water deprivation decreases plasma volume and increases plasma osmolality and vasopressin (VP) release. Immunohistochemistry and in situ hybridization indicate that osmotic stimuli deplete magnocellular neurons of VP content, while increasing VP gene expression. The current experiments tested feasibility of using LCM and real time RT‐PCR to evaluate mRNA expression in hypothalamic paraventricular nucleus (PVN) VP cells. Transgenic VP‐eGFP Wistar rats were water deprived for 48 hrs. VP‐eGFP expressing cells in the magnocellular PVN from euhydrated (E, n= 3) and dehydrated (D, n= 3) rats were identified in frozen hypothalamic sections (5 μm) with fluorescent microscopy and laser dissected (spot size~7.5 μm; power = 75mW) onto LCM caps (Arcturus). RNA was extracted, cDNA synthesized and real time RT‐PCR was performed for mRNA’s of the ribosomal protein L‐32 (housekeeper), and VP. Relative expression of VP was increased 3.5 X in the PVN of D compared to E rats. In contrast, expression of GAPDH (another housekeeping gene) relative to L‐32 in PVN VP neurons was not different between D and E rats. Thus, the use of brain tissue from VP‐eGFP rats circumvents potential RNA degradation during standard post‐sectioning immunohistochemistry and allows for evaluation of mRNA expression of target genes in identified VP neurons. (NIH R01 HL36245, CMH and R01 HL68725, JES)