Angiotensin AT2 receptors in the solitary tract nucleus lower blood pressure via inhibition of GABA signaling
Authors: Mohammed Mazher , Sumners Colin , Sheng Wanhui , Harden Scott W. , Frazier Charles J. , Johnson Dominique , Scott Karen A. , Krause Eric G. , de Kloet Annette D. 
Published: April 2020

Chronic CNS administration of angiotensin AT2 receptor (AT2R) agonists lowers blood pressure (BP) in normal and hypertensive rodents, but the location(s) and mechanism(s) of action are unknown. One locus may be the intermediate solitary tract nucleus (intNTS). Our previous studies demonstrated a high density of AT2R located on GABA neurons in the intNTS (de Kloet et al, Brain Struct. Func., 2016, 22:891–912), and decades of research have established increased GABA signaling in the intNTS as a pathophysiological mechanism contributing to increased BP and hypertension. These studies reveal that reductions in BP (~8 mmHg) induced by 2‐week intracerebroventricular (icv) infusion of the AT2R agonist C21 (7.5 ng/kg/h) in normotensive mice is associated with significant decreases in gene expression of GABA synthetic enzymes (GAD‐1 & GAD‐2) in the intNTS. Based on this, we developed the hypothesis that the depressor action of C21 is mediated through AT2R located on intNTS GABA neurons and a reduction of GABA signaling. To test this, we engineered a novel mouse line that has Cre‐recombinase expression directed to the AT2R gene (AT2R‐Cre), and demonstrated that Cre recombinase activity mirrors AT2R mRNA expression within the NTS. Next, we used the Cre‐LoxP system and virally (AAV)‐mediated gene transfer to direct expression of the light‐sensitive excitatory opsin channelrhodopsin‐2 (ChR2) and/or eYFP specifically to neurons in the intNTS that synthesize AT2R. Using these mice, we performed electrophysiological‐ or cardiovascular recordings during optogenetic manipulations (blue light) that selectively excite AT2R‐expressing neurons in the intNTS. For the electrophysiological studies, optical stimulation produced a strong inward current in voltage clamp (611.80 ± 78.55 pA; n=9), and robust frequency dependent firing of action potentials in current‐clamp, validating functional opsin expression within AT2R‐expressing neurons. Optical activation of the Chr2/eYFP‐containing AT2R‐expressing neurons in brain slices resulted in activation of adjacent (non‐eYFP‐labeled) neurons, as indicated by light‐evoked currents (n=31), an effect blocked by GABA receptor antagonists (PTX and CGP). Cardiovascular studies revealed that (i) Optogenetic stimulation of the AT2R intNTS neurons expressing ChR2 in isoflurane anesthetized mice elicited significant elevations in BP compared with control mice that express only eYFP; (ii) The AT2R‐containing neurons that were activated by light stimulation are GABAergic, as indicated by increased expression and co‐localization of c‐Fos mRNA with GAD‐1 mRNA and eYFP; (iii) Chronic icv infusion of C21 as above significantly blunted the light‐induced increases in BP, an effect that persisted across all patterns of optical stimulation. Collectively, these data indicate that AT2R‐mediated decreases in BP involve a blunting of GABA signaling in the intNTS, and suggest that neurons expressing AT2R in the intNTS may be an access point for cardiovascular control.Support or Funding InformationNIH grants HL‐125805 (AdK), HL‐145028 (AdK), HL‐136595 (EGK/CS)