Blood pressure is controlled by endocrine, autonomic, and behavioral responses that maintain blood volume and perfusion pressure at levels optimal for survival. Although it is clear that central angiotensin type 1a receptors (AT1aR; encoded by the Agtr1a gene) influence these processes, the neuronal circuits mediating these effects are incompletely understood. The present studies characterize the structure and function of AT1aR neurons in the lamina terminalis (containing the median preoptic nucleus and organum vasculosum of the lamina terminalis), thereby evaluating their roles in blood pressure control. Using male Agtr1a-Cre mice, neuroanatomical studies reveal that AT1aR neurons in the area are largely glutamatergic and send projections to the paraventricular nucleus of the hypothalamus (PVN) that appear to synapse onto vasopressin-synthesizing neurons. To evaluate the functionality of these lamina terminalis AT1aR neurons, we virally delivered light-sensitive opsins and then optogenetically excited or inhibited the neurons while evaluating cardiovascular parameters or fluid intake. Optogenetic excitation robustly elevated blood pressure, water intake, and sodium intake, while optogenetic inhibition produced the opposite effects. Intriguingly, optogenetic excitation of these AT1aR neurons of the lamina terminalis also resulted in Fos induction in vasopressin neurons within the PVN and supraoptic nucleus. Further, within the PVN, selective optogenetic stimulation of afferents that arise from these lamina terminalis AT1aR neurons induced glutamate release onto magnocellular neurons and was sufficient to increase blood pressure. These cardiovascular effects were attenuated by systemic pretreatment with a vasopressin-1a-receptor antagonist. Collectively, these data indicate that excitation of lamina terminalis AT1aR neurons induces neuroendocrine and behavioral responses that increase blood pressure.

Aims These studies evaluate whether angiotensin type-2 receptors (AT2Rs) that are expressed on γ-aminobutyric acid (GABA) neurons in the nucleus of the solitary tract (NTS) represent a novel endogenous blood pressure lowering mechanism. Methods and Results Experiments combined advanced genetic and neuroanatomical techniques, pharmacology, electrophysiology and optogenetics in mice to define the structure and cardiovascular-related function of NTS neurons that contain AT2R. Using mice with Cre-recombinase directed to the AT2R gene, we discovered that optogenetic stimulation of AT2R-expressing neurons in the NTS increases GABA release and blood pressure. To evaluate the role of the receptor, per se, in cardiovascular regulation, we chronically delivered C21, a selective AT2R agonist, into the brains of normotensive mice and found that central AT2R activation reduces GABA-related gene expression and blunts the pressor responses induced by optogenetic excitation of NTS AT2R neurons. Next, using in situ hybridization, we found that the levels of Agtr2 mRNAs in GABAergic NTS neurons rise during experimentally-induced hypertension, and we hypothesized that this increased expression may be exploited to ameliorate the disease. Consistent with this, final experiments revealed that central administration of C21 attenuates hypertension, an effect that is abolished in mice lacking AT2R in GABAergic NTS neurons. Conclusions These studies unveil novel hindbrain circuits that maintain arterial blood pressure, and reveal a specific population of AT2R that can be engaged to alleviate hypertension. The implication is that these discrete receptors may serve as an access point for activating an endogenous depressor circuit. Translational perspective Hypertension is a widespread health problem and risk factor for cardiovascular disease and stroke. Although treatment options exist, many patients suffer from resistant hypertension, which is associated with enhanced sympathetic drive. Thus, many available therapeutics focus on dampening pressor mechanisms. The present studies take the alternative approach of treating hypertension by exploiting an endogenous depressor mechanism.

The brain maintains cardiovascular homeostasis, in part, via the arterial baroreflex which senses changes in blood pressure (BP) at the level of the aortic arch. Sensory afferents innervating the aortic arch employ baroreceptors to convert stretch exerted on the arterial wall into action potentials carried by the vagus nerve to second order neurons residing within the nucleus of the solitary tract (NTS). Although the baroreflex was described more than 80 years ago, the specific molecular, structural, and functional phenotype of the baroreceptors remain uncharacterized. This is due to the lack of tools that provide the genetic and target organ specificity that is required to selectively characterize baroreceptor afferents. Here, we use a novel approach to selectively target baroreceptors. Male mice on a C57BL/6J background were anesthetized with isoflurane, intubated, and artificially ventilated. Following sternotomy, the aortic arch was exposed, and a retrograde adeno-associated virus was applied to the aortic arch to direct the expression of channelrhoropsin-2 (ChR2) and/or tdTomato (tdTom) to sensory afferents presumably functioning as baroreceptors. Consistent with the structural characteristics of arterial baroreceptors, robust tdTom expression was observed in nerve endings surrounding the aortic arch, within the fibers of the aortic depressor and vagus nerves, cell bodies of the nodose ganglia (NDG), and neural projections to the caudal NTS (cNTS). Additionally, the tdTom labeled cell bodies within the NDG also expressed mRNAs coding for the mechanically gated ion channels, PIEZO-1 and PIEZO-2. In vitro electrophysiology revealed that pulses of blue light evoked excitatory post-synaptic currents in a subset of neurons within the cNTS, suggesting a functional connection between the labeled aortic arch sensory afferents and second order neurons. Finally, the in vivo optogenetic stimulation of the cell bodies of the baroreceptor expressing afferents in the NDG produced robust depressor responses. Together, these results establish a novel approach for selectively targeting sensory neurons innervating the aortic arch. This approach may be used to investigate arterial baroreceptors structurally and functionally, and to assess their role in the etiology or reversal of cardiovascular disease.

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 Information NIH grants HL‐125805 ...