AT1a receptors in the subfornical organ increase splenic nerve activity and blood pressure in response to chronic Angiotensin II

Abstract

Circumventricular organs (CVOs) are important control centers of blood pressure (BP) homeostasis, able to respond to circulating Angiotensin II (AngII) and consequently modulating the activity of peripheral autonomic nervous system. The aim of this study was to investigate whether AngII Type 1a Receptors (AT1aR) in CVOs neurons are directly involved in modulating the splenic sympathetic nerve activity (SSNA) and BP during chronic AngII. First, by injecting fluorescent AngII into femoral vein of C57Bl/6J mice, we observed positivity in the CVOs, specifically in the subfornical organ (SFO) and brainstem, demonstrating that AngII crosses the blood-brain barrier. To investigate whether circulating AngII directly activates neurons in CVOs and whether these neurons are involved in the modulation of BP levels during hypertension development, we took advance of a mouse model expressing an iCreERT2 under the control of an activity-dependent Fos promoter and crossed with a fluorescent reporter line to generate a FosTRAP2;TdTomato mice. FosTRAP2;TdTomato mice were infused with AngII or vehicle as control by osmotic minipumps. In our previous studies we demonstrated that AngII is able to induce the onset of hypertension and at the same time to evoke an increase in SSNA after 3 days from starting the administration. To investigate neurons activated at this time point, we treated FosTRAP2;TdTomato with tamoxifen (TAM) i.p. after 3 days after AngII or Veh. Then, mice were monitored for 28 days administration, when BP levels were chronically increased. Brains were collected and stained with cFos primary antibody to identify neurons activated at the end of protocol. We found that 3 days of AngII increased the activity of neurons in the SFO and brainstem as compared to control mice. Additionally, PVN and thalamus had moderate activation of neurons at 3 days from AngII and a higher expression of neurons at 28 days as compared to Veh mice. These findings demonstrated that SFO neusons were activated early during hypertension development. Additionally, by combining RNA scope (AT1R+) with immunofluorescence (cFos+), we further demonstrated that AT1aR-expressing SFO neurons are activated during chronic exposure to AngII. To evaluate the role of AT1aRs in SFO, we generated a mouse model carrying a deletion in the AT1aR gene selectively in the SFO, by stereotaxis injecting directly into the SFO of AT1aRflox mice an adenoviral vector (AdCre-GFP) expressing both Cre-recombinase and a fluorescent protein (GFP). As a control, mice of the same AT1aRflox genotype were used, but they were injected with an adenoviral vector expressing only GFP (AdGFP). AT1aRflox mice treated with AdCreGFP, compared to AT1aRflox mice treated with AdGFP alone, showed a significant reduction in SSNA and a protection from AngII-induced BP increase. We demonstrated that AT1aR-expressing neurons in SFO play a pivotal role in modulating SSNA and BP in AngII-induced hypertension.