Abstract P621: Cellular Localization of Angiotensin Type 1a Receptor mRNA in the Paraventricular Nucleus of the Hypothalamus

The chronic neurogenic hypertension that involves increased effects of angiotensin II (Ang II) via its type 1 receptor (AT1R) within brain cardiovascular control centers is associated with induction of microglial activation and neuroinflammation in the paraventricular nucleus of the hypothalamus (PVN). However, whether Ang II exerts direct effects via AT1R located on microglia is not established. Therefore, the objective of this study was to determine the cellular localization of AT1R in the PVN. Naive twelve-week old normotensive (Sprague Dawley, SD; Wistar Kyoto, WKY) rats and spontaneously hypertensive rats (SHR) were euthanized and perfused with 4% paraformaldehyde. Brains were removed and sectioned coronally at the level of the PVN. Sections throughout the entire PVN underwent RNAscope fluorescence in situ hybridization to determine AT1aR mRNA expression, combined with immunohistochemistry using microglia (Ionized calcium binding adaptor molecule 1; Iba1)-, neuron (HuC/D)-, or astrocyte (Glial fibrillary acidic protein; GFAP)-specific markers. The results, obtained from at least 3 SD and 3 WKY rats indicate strong co-localization of AT1aR transcripts with neurons in the neuroendocrine and parvocellular PVN regions, as expected. By contrast, there was no detectable co-localization of AT1aR mRNA with either microglia or astrocytes throughout the PVN of these rats. Further, qRT-PCR revealed that while both AT1aR- and AT1bR mRNAs were detectable in SD rat hypothalamus (1.00±0.14; 0.40±0.12; n=7), neither transcript was detectable in microglia cultured from the hypothalamus of SD rats (n=6). The pattern of AT1aR mRNA expression in the PVN of SHR, a hypertensive model that exhibits over activity of Ang II/AT1R actions at the PVN, was similar to that observed in the SD and WKY rats, i.e. strong co-localization with HuC/D-positive cells, and no detectable co-localization with either Iba1 or GFAP-positive cells. Collectively, these findings indicate that AT1R are localized to neurons, not glia, in the PVN of normotensive rats or SHR in situ . Further, they suggest that it is unlikely that Ang II exerts direct effects at microglia in the PVN, and that induction of microglial activation at this site following Ang II infusion is likely an indirect action.