Anatomical Characterization Of Gαi2 Expressing Hypothalamic Paraventricular Nucleus Neurons

Abstract

BackgroundHypertension is a global health burden, and excess dietary salt is an established cause of hypertension. The hypothalamic paraventricular nucleus (PVN) is an essential component of the neuronal network that regulates sodium homeostasis and blood pressure. Previously, we have shown PVN specific GPCR‐coupled Gαi2 subunit proteins are essential to counter the development of salt‐sensitive hypertension by mediating the sympathoinhibitory and natriuretic responses to increased dietary sodium intake to maintain sodium homeostasis and normotension. However, the cellular localization and identity of PVN Gαi2 expressing neurons is currently unknown.ObjectiveTo determine the anatomical characterization of Gαi2 expressing PVN neurons.MethodologyThree‐month‐old male (n=3) and female (n=3) Sprague Dawley rats were subjected to transcardial perfusion, and brains were post fixed in 4% paraformaldehyde and 30% sucrose. 20μm frozen brain slices were collected. Using in‐situ hybridization (ACD RNAscope kits) neuronal expression of Gαi2, vGLUT2, GAD67, corticotropin‐releasing hormone (CRH), tyrosine hydroxylase (TH), oxytocin, and arginine vasopressin (AVP) in hypothalamic PVN neurons was determined. The visualization and evaluation of co‐localization of Gαi2 expressing PVN parvocellular neurons with vGLUT2, GAD67, CRH, TH, oxytocin and AVP was done using a Keyence Bz‐9000e microscope and Image J respectively.ResultsWe observed no differences among male and female rats, therefore, all data is presented as n=6. Our data show that neurons containing Gαi2 mRNA are localized in parvocellular but not magnocellular region of hypothalamic PVN. In the parvocellular region Gαi2 mRNA co‐localized with 87.5 ± 3.0% of GABA expressing neurons and 25.1 ± 2.7 % of vGLUT2 neurons, signifying that the Gαi2 mRNA is highly co‐localized with GABAergic neurons. Additionally, Gαi2 co‐localized with 76.3 ± 5.2% of CRH neurons and 33.5 ± 1.8% of TH expressing parvocellular neurons. We observed no localization of Gαi2 neurons with oxytocin or AVP expressing magnocellular neurons.ConclusionGiven 1) our prior data demonstrate PVN Gαi2 proteins play a critical role in mediating sympathoinhibition and, 2) that the GABAB receptor signals predominantly through a Gαi2 pathway we speculate, given the high co‐localization of Gαi2 mRNA with GABAergic neurons, that Gαi2 proteins modulate GABAergic signaling to impact sympathetic outflow and blood pressure.Support or Funding InformationT32 HL007224 to PCR01 HL139867 to RDWR01 HL141406 to RDWR56 AG057687 to RDW