Alleviating Hypertension by Selectively Targeting Angiotensin Receptor-Expressing Vagal Sensory Neurons

Abstract

Cardiovascular homeostasis is maintained, in part, by neural signals arising from arterial baroreceptors that apprise the brain of blood volume and pressure. Here, we test whether neurons within the nodose ganglia that express angiotensin type-1a receptors (referred to as NG^AT1aR) serve as baroreceptors that differentially influence blood pressure (BP) in male and female mice. Using Agtr1a-Cre mice and Cre-dependent AAVs to direct tdTomato to NG^AT1aR, neuroanatomical studies revealed that NG^AT1aR receive input from the aortic arch, project to the caudal nucleus of the solitary tract (NTS), and synthesize mechanosensitive ion channels, Piezo1/2 To evaluate the functionality of NG^AT1aR, we directed the fluorescent calcium indicator (GCaMP6s) or the light-sensitive channelrhodopsin-2 (ChR2) to Agtr1a-containing neurons. Two-photon intravital imaging in Agtr1a-GCaMP6s mice revealed that NG^AT1aR couple their firing to elevated BP, induced by phenylephrine (i.v.). Furthermore, optical excitation of NG^AT1aR at their soma or axon terminals within the caudal NTS of Agtr1a-ChR2 mice elicited robust frequency-dependent decreases in BP and heart rate, indicating that NG^AT1aR are sufficient to elicit appropriate compensatory responses to vascular mechanosensation. Optical excitation also elicited hypotensive and bradycardic responses in ChR2-expressing mice that were subjected to deoxycorticosterone acetate (DOCA)-salt hypertension; however, the duration of these effects was altered, suggestive of hypertension-induced impairment of the baroreflex. Similarly, increased GCaMP6s fluorescence observed after administration of phenylephrine was delayed in mice subjected to DOCA-salt or chronic delivery of angiotensin II. Collectively, these results reveal the structure and function of NG^AT1aR and suggest that such neurons may be exploited to discern and relieve hypertension.

Keywords: autonomic; baroreceptor; baroreflex; blood pressure; nodose; vagus.

Figure 1.

Agtr1a mRNA(s) are localized to a subset of neurons of the NG. A–C, Representative images of an NG section showing (A) neurons labeled by NeuN (green) and (B) Agtr1a mRNA (magenta) and (C) the merged image depicting the localization of Agtr1a mRNA to NG neurons. D, Quantification of the percentage of NeuN-positive NG neurons expressing Agtr1a mRNA (n = 6; error bars = SEM). Scale bar = 25 μm.

Figure 2.

Agtr1a-tdTomato is localized to vagal afferent neurons, a subset of which expresses the mechanosensitive ion channels, Piezo1 and Piezo2. A, Illustration depicting the generation of the Agtr1a-tdTomato reporter mouse line. B, Representative image (20×) of a coronal section through the caudal NTS from an Agtr1a-tdTomato mouse. C–F, Validation of Agtr1a mRNA expression in the NG of Agtr1a-tdTomato mice. Representative images of (C) tdTomato expression in NG^AT1aR neurons (red) and (D) Agtr1a mRNA (cyan), (E) the merged image, and (E_i) the magnification of the merged image. F, Quantification of the percentage of tdTomato-expressing neurons colocalized with Agtr1a mRNA (n = 8). Representative images of (G) tdTomato expression in NG^AT1aR neurons (red) and (H) Piezo1 mRNA (cyan), (I) the merged image, and (I_i) the magnification of the merged image. J, Quantification of the percentage of tdTomato-expressing neurons colocalized with Piezo1 mRNA (n = 8). Representative images of (K) tdTomato expression in NG^AT1aR neurons (red) and (L) Piezo2 mRNA (cyan), (M) the merged image, and (M_i) the magnification of the merged image. N, Quantification of the percentage of tdTomato-expressing neurons colocalized with Piezo2 mRNA (n = 7). Error bars = SEM. Scale bars = 100 μm (B) and 25 μm (C–M).

Figure 3.

NG^AT1aR are positioned to transmit vagal sensory afferent (baroreceptor) information from the aortic arch to the NTS. A–D, Anterograde Cre-inducible AAV delivered to the NG of Agtr1a-Cre mice induces robust expression of tdTomato in Agtr1a-containing (B) cell bodies within the NG and afferent fibers (C) in the caudal NTS and (D) the aortic arch. E–H, Retrograde Cre-inducible AAV delivered to the aortic arch of Agtr1a-Cre mice also induces robust expression of tdTomato in Agtr1a-containing (F) cell bodies within the NG and afferent fibers innervating the (G) caudal NTS and (H) aortic arch. I–L, Retrograde Cre-inducible AAV delivered to the caudal NTS of Agtr1a-Cre mice induces robust expression of tdTomato in Agtr1a-containing (J) cell bodies within the NG and afferent fibers innervating the (K) caudal NTS and (L) aortic arch. Scale bar = 100 μm unless stated otherwise.

Figure 4.

Optogenetic simulation of NG^AT1aR is sufficient to reduce BP and HR, thus recapitulating baroreflex responses. A, Illustration depicting the generation of the Agtr1a-ChR2 mouse line (left) and acute recording preparation (right). While anesthetized, male and female Agtr1a-ChR2 (male, n = 8; female, n = 5–7) were catheterized to assess SBP and HR. Subsequently, the left NG was isolated in order to optogenetically stimulate NG^AT1aR neurons. B,C, Changes in SBP and HR in (B) males and (C) females during optogenetic stimulation with different photoactivation parameters (473 nm; 1, 5, or 15 Hz; 60 s). *Significantly different from baseline (time = 0 s), p < 0.05, two-way RM ANOVA or mixed-effects analysis with Dunnett's multiple-comparisons test.

Figure 5.

Optogenetic stimulation of Agtr1a-expressing input into the caudal NTS prompts glutamate release onto second-order neurons and causes robust frequency-dependent alterations in cardiovascular parameters. A, Differential interference contrast image of in vitro NTS preparation, followed by panels showing expression of ChR2-eYFP in Agtr1a-ChR2 mice and biocytin-filled cell patched in the NTS. B,C, 1 ms of stimulation with blue light produced inward currents in NTS cells voltage-clamped at −70 mV in 10/11 attempts. B, Mean normalized amplitude in 3/3 cells tested with bath application of ionotropic glutamate receptor antagonists and (C) the evoked response from a representative cell. D, Illustration of Agtr1a-ChR2 mouse and awake-behaving optical stimulation experiment setup. E–H, Changes in (E) SBP, (F) MAP, (G) DBP, and (H) HR at 30 min after optical stimulation onset with different parameters (473 nm; 1, 2, or 3 Hz). *Different from baseline (frequency = 0 Hz), p < 0.05, mixed-effects analysis followed by Dunnett's multiple-comparisons test; ⁺different from control, p < 0.05, mixed-effects analysis followed by Sidak's multiple-comparisons test; Agtr1a-ChR2, n = 7–11; Agtr1a-Cre, n = 7–9.

Figure 6.

Optical stimulation of Agtr1a-containing input into the caudal NTS prompts neural activation in key cardioregulatory areas of the brain. A, Quantification of c-fos positive nuclei in forebrain and hindbrain cardioregulatory regions. *Significant difference between Agtr1a-ChR2 and Agtr1a-Cre mice, p < 0.05, multiple unpaired t tests. p-value noted above regions in which there was not a significant difference between Agtr1a-ChR2 and Agtr1a-Cre mice. B, Representative images of c-fos expression in caudal NTS of Agtr1a-ChR2 mouse and Agtr1a-Cre mouse. (B) Scale bar = 50 μm. C,D, Representative images from Agtr1a-ChR2 and Agtr1a-Cre mice reflecting c-fos expression within the (C) forebrain and (D) hindbrain areas following optical stimulation. CeA, central nucleus of the amygdala; CVLM, caudal ventrolateral medulla; DMNV, dorsal motor nucleus of the vagus; LPBN, lateral parabrachial nucleus; LC, locus coeruleus; MnPO, median preoptic nucleus; NA, nucleus ambiguus; PVN, paraventricular nucleus of the hypothalamus; RVLM, rostral ventrolateral medulla; SON, supraoptic nucleus. All images were acquired at 20× magnification, except CeA acquired at 10× magnification. (C,D) Scale bar = 50 μm.

Figure 7.

Models of increased BP are associated with altered dynamics of calcium responses in NG^AT1aR. A, Illustration depicting the generation of Agtr1a-GCaMP6s mice. B, Experimental timeline. C, While anesthetized, male Agtr1a-GCaMP6s mice were catheterized to (D) assess SBP in response to intravenous phenylephrine (control, n = 7; DOCA, n = 5; Ang-II, n = 8) or (E) perform two-photon intravital imaging while recording changes in GCaMP6s fluorescence from NG^AT1aR in response to intravenous phenylephrine (control, n = 4; DOCA, n = 5; Ang-II, n = 3). E–G, Graphs depicting a change in SBP (ΔSBP) to intravenous phenylephrine administration (0.1 mg/kg; denoted by a dashed line at time = 60 s) in (E) control (n = 7), (F) DOCA-treated (n = 5), and (G) Ang-II–treated (n = 3) Agtr1a-GCaMP6s mice. H–J, Heat maps of the fluorescence signal (ΔF/F) from individual NG^AT1aR 60 s before, during, and after intravenous phenylephrine administration (0.1 mg/kg; denoted by red dashed lines at time = 60 s and time = 120 s) in (H) control (n = 29 neurons), (I) DOCA-treated (n = 44 neurons), and (J) Ang-II–treated mice (n = 45 neurons). Each row represents the mean activity for a single neuron. K–M, Graphs of mean fluorescence signal (ΔF/F) and AUC of mean fluorescence signal from NG^AT1aR in response to intravenous phenylephrine administration (0.1 mg/kg; denoted by a dashed line at time = 60 s) in (K) control, (L) DOCA-treated, and (M) Ang-II–treated mice. *Significantly different from baseline (time = 60 s), p < 0.05; ^∋Significantly different from control, p < 0.05; ^⊥Significantly different from DOCA, p < 0.05, two-way RM ANOVA followed by Sidak's multiple-comparisons tests.

Figure 8.

Optical stimulation of Agtr1a-containing afferents within the NTS produces robust decreases in cardiovascular parameters in normotensive and DOCA–salt-treated mice. Effects of optogenetic activation (20 ms pulses, 2 Hz, 1 min on followed by 1 min off, 60 min) of Agtr1a-expressing afferent input into the caudal NTS on cardiovascular parameters. A, Schematic depicting Agtr1a-ChR2 mouse line and fiber-optic targeting caudal NTS. B, Experimental timeline. C–H, Effects of activation under normotensive conditions and DOCA–salt conditions in (C–E) Agtr1a-Cre and (F–H) Agtr1a-ChR2 mice. The error bars indicate SEM. *Significantly different from baseline (time = 0 min), p < 0.05, two-way RM ANOVA followed by uncorrected Fisher's LSD multiple-comparisons test + significantly different from Agtr1a-Cre-light ON, p < 0.05, two-way RM ANOVA followed by uncorrected Fisher's LSD multiple-comparisons test; Agtr1a-ChR2, n = 7–11; Agtr1a-Cre, n = 8–9.