Abstract
Relief from psychological stress confers cardio-protection by altering brain activity and lowering blood pressure; however, the neuronal circuits orchestrating these effects are unknown. Here, we used male mice to discern neuronal circuits conferring stress relief and reduced blood pressure. We found that neurons residing in the central nucleus of the amygdala (CeA) expressing angiotensin type 2 receptors (AT2R), deemed CeAAT2R, innervate brain nuclei regulating stress responding. In vivo optogenetic excitation of CeAAT2R lowered blood pressure, and this effect was abrogated by systemic hexamethonium or antagonism of GABA receptors within the CeA. Intriguingly, in vivo optogenetic excitation of CeAAT2R was also potently anxiolytic. Delivery of an AT2R agonist into the CeA recapitulated the hypotensive and anxiolytic effects, but ablating AT2R(s) from the CeA was anxiogenic. The results suggest that the excitation of CeAAT2R couples lowered blood pressure with anxiolysis. The implication is that therapeutics targeting CeAAT2R may provide stress relief and protection against cardiovascular disease.
Significance Statement
There is increasing appreciation that brain-to-body communication promotes susceptibility or resiliency to cardiovascular disease. Here, we present preclinical research that discerns a neural circuit that orchestrates brain-to-body communication and provides relief from mental stress. We discover that neurons within the central nucleus of the amygdala that express angiotensin type 2 receptors (hereafter referred to as CeAAT2R) are potent mediators of blood pressure and anxiolysis. The implication is that CeAAT2R or their angiotensin type 2 receptors can be targeted to protect against stress-induced cardiovascular disease.
Introduction
Cardiovascular disease is the leading cause of death in the United States (Curtin et al., 2023). The incidence of cardiovascular-related deaths steeply increased after the COVID-19 pandemic (Tsao et al., 2023), and this upward trajectory is projected to persist for decades (Mohebi et al., 2022). Thus, it is imperative to understand factors that drive pathogenesis or reversal of the disease. Psychological stress and hypertension are major contributors to the etiology of cardiovascular disease (Yusuf et al., 2004), and psychological stress creates changes in blood pressure and brain activity that predict pathogenesis (Ginty et al., 2017). Specifically, subjects that have increased activation of the amygdala and exaggerated elevations in blood pressure during psychological stress are more likely to experience cardiovascular disease events (Gianaros et al., 2008; Ginty et al., 2017; Tawakol et al., 2017). Interestingly, the inverse relationship is also true, and practices like meditation provide relief from psychological stress (Goyal et al., 2014), lower blood pressure (Chung et al., 2012; Palta et al., 2012), suppress activation of the amygdala (Kral et al., 2018), and protect against cardiovascular disease (Levine et al., 2017). While it is established that psychological stress impacts the amygdala to alter blood pressure, the underlying neural circuits are not well understood.
The renin-angiotensin-system (RAS) and its angiotensin type 1a (AT1aR) and type 2 (AT2R) receptors have long been implicated in blood pressure regulation and the development of cardiovascular disease. Angiotensin II (ANGII) is the effector peptide of the RAS, and it is generally accepted that overactivation of AT1aR promotes hypertension and cardiovascular disease (Chappell, 2016), but stimulation of AT2R opposes these deleterious effects (Steckelings et al., 2022). While much of the research on AT1aR and AT2R examines their effects on renal hemodynamics and/or peripheral vasoconstriction, each receptor subtype is also expressed within the brain (Sumners et al., 2020). Emerging preclinical studies using laboratory mice suggest that AT1aR and AT2R serve as phenotypic markers for neurons in the brain whose activity is coupled to emotional and cardiovascular responses to stress (Bregonzio et al., 2008; Wang et al., 2016; de Kloet et al., 2017; Elsaafien et al., 2021). For example, neurons within the central nucleus of the amygdala (CeA) express AT1aR (Mendelsohn et al., 1984; Gonzalez et al., 2012), the stimulation of which augments behavioral and cardiovascular responses to fearful stimuli (Davern et al., 2009; Yu et al., 2023). Intriguingly, AT2R(s) are also expressed in the CeA (de Kloet et al., 2016b) but their activation dampens stress responding (Yu et al., 2019). These prior reports suggest that neurons in the CeA that express angiotensin receptors can be manipulated to exacerbate or relieve psychological stress. Consequently, such neurons may comprise brain circuits that coordinate emotional and somatic responses to stress that influence the etiology of affective and cardiovascular disorders.
Here, we leverage neurons in the CeA that synthesize the AT2R (referred to as CeAAT2R) to delve deeply into brain circuits that lower blood pressure and relieve anxiety. Using mice genetically engineered to have enhanced green fluorescent protein (eGFP) or Cre-recombinase directed to cells that express AT2R, we conduct anatomical studies to reveal the neurochemistry and connectivity of CeAAT2R. Next, we use in vitro patch-clamp electrophysiology to evaluate how the selective AT2R agonist, compound 21 (C21), affects excitability of CeAAT2R. Corresponding in vivo experiments evaluate how central administration of C21 or optogenetic excitation of CeAAT2R affects blood pressure and anxiety-like behavior. Final experiments implement virally mediated gene transfer to ablate AT2R(s) within the CeA to determine whether these receptors contribute to basal levels of anxiety. Collectively, our results suggest that CeAAT2R are potent mediators of blood pressure and anxiety-like behavior. The implication is that therapeutics that activate CeAAT2R or their AT2R(s) may be ideally suited for alleviating hypertension and anxiety that manifests from heightened responses to psychological stress.
Materials and Methods
Animals
Studies were conducted in adult male mice on a C57BL/6J background. Mice were 8–10 weeks old at the initiation of the studies. All animals were maintained in temperature- and humidity-controlled rooms on 12 h light/dark cycles. In all cases, food and water were available ad libitum. All procedures were approved by the Institutional Animal Care and Use Committees at the University of Florida or Georgia State University and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
The AT2RCre knock-in mouse line was developed via the use of the CRISPR/Cas9 system by the University of Michigan. These mice were back-crossed onto a C57BL/6J background for 6+ generations upon arrival to the University of Florida (Mohammed et al., 2022). Of relevance, the Agtr2 gene is localized to the X-chromosome. Therefore, male mice were hemizygous for the AT2R-Cre gene (i.e., AT2RCre/y).
The AT2R reporter mouse line (AT2ReGFP) was initially generated by in vitro fertilization of FVB/NJ mice with Tg IP72Gsat mouse sperm (FVB/NTac × CD1(ICR) background) that was obtained from the Mutant Mouse Regional Resource Center (MMRC30278). Briefly, the construct used to produce the transgenic AT2ReGFP mouse line consists of an enhanced green fluorescent protein (eGFP) reporter gene and subsequent polyadenylation sequence that are inserted into the AT2R bacterial artificial chromosome (BAC) clone at the start codon of the first coding exon of the AT2R gene. This allows for eGFP expression that is driven by all of the regulatory sequences of the AT2R BAC gene. After successful in vitro fertilization, the AT2ReGFP reporter mouse line was back-crossed onto a C57BL/6J background for 10+ generations prior to their use in these studies. Importantly, these AT2ReGFP mice were previously validated in our laboratory by way of determining that neurons expressing eGFP are indeed neurons that synthesize Agtr2 mRNA(s) (de Kloet et al., 2016b).
Finally, the Cre/lox system was also utilized to selectively delete the Agtr2 gene from neurons within the CeA. To accomplish this, a knock-in mouse in which the first coding exon of the Agtr2 gene is surrounded by loxP sites was used (Welcome Trust Sanger Institute). These mice were provided by Drs. Ulrike Steckelings and Michael Bader and were back-crossed onto the congenic C57BL/6J strain for at least seven generations prior to their shipment (Mohammed et al., 2022). Upon arrival at the University of Florida, these mice were bred to a FLP deletor line (Jackson Laboratory Stock #011065) to remove the frt-flanked selection gene and then back-crossed onto the congenic C57BL/6J strain for an additional 6+ generations.
Viral constructs
Adeno-associated viral vectors (AAVs) that allow for the Cre-inducible expression of fluorophores (eYFPs) and/or the light-sensitive ion channel (ChR2) were obtained from the viral vector core at the University of North Carolina. For expression of eYFP and ChR2, pAAV2-EF1a-DIO-hChR2(H134R)-eYFP-WPRE (referred to as AAV-ChR2-eYFP) was used; for the expression of only eYFP, pAAV2-EF1a-DIO-eYFP (referred to as AAV-eYFP) was used. For the receptor deletion experiments, AAVs that allow for tdTomato (AAV-hSyn-dTom; referred to as tdTom) or Cre and tdTomato (AAV-hSyn-Cre-dTom; referred to as Cre-tdTom) were used.
Stereotaxic surgery
Stereotaxic surgery was performed to deliver AAVs into the central amygdala (CeA), to implant fiber optics targeting specific brain areas, and/or to implant ALZET osmotic minipumps and Brain Infusion kits (DURECT). Fiber optic implants allowed for optogenetic stimulation during cardiovascular recordings obtained under general anesthesia or during behavioral assays conducted in conscious freely moving mice, while ALZET osmotic minipumps and Brain Infusion kits (DURECT) were used to chronically deliver of C21 (7.5 ng/kg/h) or saline control into the lateral cerebral ventricle (i.c.v.) for 7 d.
In preparation for aseptic stereotaxic surgery, mice were anesthetized using isoflurane and administered analgesic (Buprenex; 0.1 mg·kg−1, s.c.). For AAV-mediated gene transfer to the CeA, Cre-inducible AAVs described above were injected bilaterally into the CeA using the following coordinates from bregma: AP: −1.4 mm, ML: ±2.75 mm, DV: −4.1 mm. For each AAV injection, the pipette was left in the region of interest for 5 min to allow for diffusion of the AAVs (100–150 nl) into the brain.
Following viral construct microinjections, a stereotactic frame was used to implant chronic dwelling fiber-optic posts terminating above the CeA using the following coordinates from bregma: AP: −1.4 mm, ML: ±2.75 mm, DV: −3.9 mm. Following that, the posts were secured in place using a skull screw and a dental acrylic as previously described (Elsaafien et al., 2021; Frazier et al., 2021).
The ALZET osmotic minipumps (model 1004; DURECT) and Brain Infusion kits (DURECT) were assembled and primed according to the manufacturer's instructions. After 48 h of priming at 37°C, the pumps were implanted subcutaneously in the intrascapular region, and the brain infusion kits were stereotactically directed to infuse C21 7.5 ng/kg/h (0.11 µl/h) or saline intracerebroventricularly using the following coordinates from the bregma: 0.2 mm posterior, 1.0 mm lateral, and 2.5 mm ventral (de Kloet et al., 2016a; Mohammed et al., 2022).
RNAscope in situ hybridization and immunohistochemistry
In order to collect tissue for neuroanatomical studies, mice were anesthetized with 0.1 ml of pentobarbital (50 mg·kg−1, i.p.) and perfused transcardially with RNase free-isotonic saline followed by 4% paraformaldehyde. Brains were then post-fixed for ∼4 h, after which they were stored in RNase-free 30% sucrose for up to 1 week before further processing. For RNAscope in situ hybridization (ISH), the CeA was sectioned at 20 µm into six serial sections using a Leica CM3050 S Cryostat (Leica). Sections were immediately mounted onto Fisherbrand Superfrost Plus Gold Microscope Slides (Thermo Fisher Scientific). After air-drying at room temperature for 20–30 min, slides were dipped in EtOH, again allowed to dry for 10–15 min, and then stored at −80°C until further processing.
RNAscope ISH was performed using the RNAscope V2 Multiplex Fluorescent Reagent Kit (Advanced Cell Diagnostics) as per the manufacturer's instructions with slight modification to the pretreatment procedure that allows for preservation of the reporter genes (i.e., eYFP), while still providing optimal mRNA signal. The probes used for these studies were as follows: Agtr2 (Mm-Agtr2; catalog #403991), vGAT (Mm-Slc32a1; catalog #319199), vGlut2 (Mm-Slc17a6-E1-E3; catalog #456759), DapB (Negative control probe-DapB; catalog #310043); and Ubc (Mm-Ubc; catalog #310771). Upon completion of the ISH, sections underwent IHC for GFP (used to amplify the eYFP signal).
Standard IHC protocols were used to amplify eYFP signal as previously described (de Kloet et al., 2017). Primary antibodies and dilutions used are as follows: GFP (Life Technologies [A10262]; 1:1,000). Secondary antibodies were purchased from Jackson ImmunoResearch, raised in donkey and used at a 1:500 dilution. Briefly, brain sections were rinsed and then incubated first in blocking solution (2% normal donkey serum and 0.2% Triton X-100 in 50 mM KPBS) for 2 h at 25°C and then in the primary antibody (diluted in blocking solution) for 18 h at 4°C. Sections were again rinsed five times for 5 min (50 mM KPBS) before incubation in the secondary antibody in blocking solution for 2 h at 25°C. After a final series of rinses, slides were allowed to air-dry and then coverslipped using ProLong Gold Antifade Mountant (Thermo Fisher Scientific).
Image capture and processing
Images were captured and processed using a laser scanning confocal microscope (Nikon Instruments). Large coronal scans captured at 10× magnification throughout the forebrain and hindbrain (coronal sections) were acquired to assess the expression of eYFP. For in situ hybridization, z-stacks of the proteins and mRNAs of interest were captured at 40× magnification. An average of 20 optical sections were collected per z-stack (0.5 µm between z-steps). Sections hybridized with the probes of interest were used to determine the exposure time and image processing required to provide optimal visualization of RNA signal. As described in detail previously (de Kloet et al., 2016b), these same parameters were then used to assess background fluorescence in sections hybridized with the negative control probe (DapB). Importantly, using these exposure times and image processing parameters, there was minimal or no fluorescence in sections hybridized with the negative control probe. All representative photomicrographs were then prepared using Adobe Photoshop 2023 to adjust brightness and contrast to provide optimal visualization. Final figures and schematics were prepared in Adobe Illustrator 2023.
Acute slice preparation
Male AT2RCre mice received stereotactic injections of the Cre-inducible AAV-ChR2-eYFP into the CeA as described above. AT2RCre or AT2ReGFP mice were anesthetized with 0.1 ml of pentobarbital (50 mg·kg−1, i.p.) and subsequently perfused through the heart with 30 ml of ice-cold aCSF sucrose solution with NaCl replaced by equal-osmol sucrose (in mM: 200 sucrose, 2.5 KCl, 1 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, 20 d-glucose, 0.4 ascorbic acid, and 2.0 CaCl2; pH 7.2, 300–305 mOsmol·L−1). The brains were rapidly dissected, and the cerebellum was sliced cleanly with a razor blade and mounted in the chamber of a vibratome (Leica VT1200s, Leica Microsystems) using superglue with the ventral surface pressed against a block of 4% agar glued to the stage. The brain was submerged in sucrose slush and bubbled constantly with 95% O2/5% CO2. Coronal slices (240 µm thickness) containing the CeA were collected and placed in a holding chamber filled with aCSF and bubbled with 95% O2/5% CO2. The aCSF is identical in composition to the sucrose solution, but with 200 mM sucrose replaced by 119 mM NaCl and 2 mM Na+-pyruvate. The slice chamber was placed in a water bath at 32°C for 20 min before placement at room temperature for a total minimum of 60 min rest before proceeding with recording.
Patch-clamp electrophysiology
Slices were placed into a chamber on the stage of a Nikon upright microscope and perfused constantly (2–3 ml/min) with aCSF bubbled continuously with 95% O2/5% CO2 and warmed to 32°C. CeAAT2R neurons containing eGFP or eYFP and ChR2 were visualized using the Dragonfly 200 laser spinning disk confocal imaging system and an iXon 888 EMCCD camera (Andor Technology). Neurons were targeted for patch clamp that were in close proximity to YFP-labeled somas and processes. Whole-cell voltage-clamp recordings were obtained using pipettes (2.5–4 MΩ) pulled from borosilicate glass (o.d. 1.5 mm, i.d. 1.17) using a P-97 Flaming/Brown horizontal micropipette puller (Sutter Instrument). To measure and amplify GABA synaptic currents, we utilized a high Cl− internal solution consisting of the following (in mM): 145 KCl, 10 HEPES, 0.9 MgCl2, 4 Mg-ATP, 0.3 Na-GTP, 6 phosphocreatine, 0.2 EGTA with pH 7.2–7.3 and 285–295 mOsmol (kg·H2O)−1. The liquid junction potential for the KCl internal was approximately +2 mV and was not corrected. Occasionally, Alexa 555 (50 μM, Thermo Fisher Scientific) was included to visualize the neuron. For voltage-clamp recordings, traces were obtained with a MultiClamp 700B amplifier (Molecular Devices) and digitized using an Axon 1440B Digitizer (Molecular Devices) at 2 kHz on a desktop computer running Clampex 10 software (Molecular Devices). Cells were clamped at −70 mV unless otherwise indicated. Data were discarded if series resistance exceeded a 25% change over the course of the recording.
Stimulating eGFP neurons pharmacologically was done through the bath application of compound 21 at 0.5 μM via a single bolus bath injection (0.5 ml) into the bath as previously described (de Kloet et al., 2016a). Mean firing activity was calculated from a 1 min period before drug application and a 1 min period around the peak effect.
ChR2 stimulation was driven by an Andor Mosaic and iQ3/Fusion integration at 488 nm using 20 ms pulses with 80 ms interpulse intervals (10 Hz). The entire field of view was stimulated. Picrotoxin (Px, 100 µM; Tocris) via a single bolus bath injection (0.5 ml) into the bath to block GABA currents.
Anesthetized assessment of blood pressure in response to optogenetic manipulations
Male AT2RCre mice received stereotaxic injections of the Cre-inducible AAV-ChR2-eYFP or AAV-eYFP into the CeA as described above. Three to four weeks later, mice were anesthetized using isoflurane and a Millar Catheter (Model SPR1000, Millar) was implanted into the aortic arch by way of the carotid artery using a similar procedure as described previously (de Kloet et al., 2017; Elsaafien et al., 2021, 2022). After catheterization, mice underwent stereotaxic surgery to lower a fiber optic into the CeA, BNST, PAG, or cNTS for optical stimulation.
To collect cardiovascular data, the Millar Catheter was connected to a PowerLab signal transduction unit (ADInstruments). Blood pressure and heart rate data were sampled/recorded at 1 kHz and analyzed using LabChart 8 software (ADInstruments). To test the effect of optical stimulation on blood pressure, mice were optically stimulated with 473 nm blue light for 60 s (10 mW output; 20 ms pulse width) at 1, 15, and 30 Hz. Mice then received injections of 0.1 ml of hexamethonium (30 mg/kg, i.p.) or saline vehicle. Once blood pressure again stabilized (∼15–20 min after the injections), mice were again subjected to optical stimulation using the same parameters. Blood pressure data were condensed into 30 s bins to perform statistical analyses and to generate the figures.
Anesthetized assessment of blood pressure in response to pharmacological injections
In a different set of experiments, AT2RCre mice received stereotaxic injections of the Cre-inducible AAV-ChR2-eYFP into the CeA, and following 3 weeks of recovery, mice were anesthetized, and a Millar Catheter (Model SPR1000, Millar) was implanted into the aortic arch. Subsequently, a dual optical injector cannula (Optical Injector, Doric Lenses) was lowered above the left CeA, allowing microinjections and optogenetic stimulation within the same site while blood pressure was simultaneously recorded, as described previously (Elsaafien et al., 2021). Either vehicle (43% ethyl alcohol, 43% dH2O, 14% trifluoroacetic acid; 1 μl) or bicuculline (10 mM; 1 μl) were microinjected and pulses of 473 nm laser light (10 mW output; 20 ms pulse width; 30 Hz; 5 min) were delivered 10 min following microinjections. Blood pressure was sampled at 30 s every 60 s for 2 and 5 min prior to and following optical stimulation, respectively.
Spectral analysis of heart rate and systolic blood pressure variability
Heart rate variability (HRV) was analyzed using a Heart Rate Variability Analysis Software provided in LabChart 8 (ADInstruments), whereas systolic blood pressure variability was analyzed using a fast Fourier transform spectral variation in systolic blood pressure using LabChart 8. Briefly, HRV and systolic blood pressure variability were sampled at 1 min bins prior to and following optogenetic stimulation, and the frequency bands were very low frequency (VLF), low frequency (LF), and high frequency (HF) at 0–0.15, 0.15–1.5, and 1.5–5 Hz, respectively. HRV was expressed as a of LF/HF ratio, which is a surrogate clinical marker of sympatho-vagal balance to the heart (Tasic et al., 2017). The low frequency domain of systolic blood pressure variability indicates the level of sympathetic vasoconstrictor activity (deBoer et al., 1987; Madwed et al., 1989).
Assessment of anxiety-like behavior
Anxiety-like behavior was assessed during the light phase (between 08:00 A.M. and 12:00 P.M.), in mice that were chronically delivered C21 or saline intracerebroventricularly, mice that were subjected to optogenetic stimulation of CeAAT2R, and mice with deletion of CeA AT2R(s), as well as their respective controls. Mice underwent stereotaxic surgical procedures and were allowed to recover for at least 7 d, as described above, prior to testing. For the optogenetics studies, mice also underwent a 2 week period of habituation to the optogenetic tethering procedure prior to initiating behavioral testing.
Elevated plus maze test
The elevated plus maze (EPM) arena has two opposing open arms and two opposing closed arms, raised 61 cm above the floor. All arms are 35 × 5 cm (l × w) with white floors, and the walls of the closed arms are made of black polycarbonate, 20 cm high. When the test was performed during optogenetic stimulation of CeAAT2R, each mouse was brought into the procedure room and tethered to the laser for 5 min within the home cage prior to the onset of the test. The mouse was then placed in the center of the EPM, facing an open arm, and optogenetic stimulation began (10 mW, 15 Hz, 20 ms pulses, 75 pulses per sequence, 5 s delay between sequences). The total duration of the test and the optogenetic stimulation was 5 min. Results, including the percentage of time spent in the open arms, the percentage of time spent in the closed arms, and the distance traveled, were recorded and analyzed by EthoVision XT 13 (Noldus Information Technology). For mice implanted with osmotic minipumps and brain infusion kits and for mice with deletion of CeA AT2R, the same procedures were performed but without tethering or optogenetic stimulation.
Open field arena test
The open field arena (OFA) test was conducted in a 45 × 45 × 30 cm (l × w × h) square arena constructed of white plastic floor and walls. When the test was performed with optogenetic stimulation, each mouse was brought into the procedure room and tethered to the laser for 5 min within the home cage prior to the onset of the test. The mouse was then placed in the center of the OFA and optogenetic stimulation began (10 mW, 15 Hz, 20 ms pulses, 75 pulses per sequence, 5 s delay between sequences), after which the mouse was untethered and returned to their home cage. Results, including total distance traveled and entries into center (located in the center of arena with 50% of total area) or edge, were recorded and analyzed using EthoVision XT 13 software (Noldus Information Technology). For mice with deletion of CeA AT2R, the same procedures were performed but without tethering or optogenetic stimulation.
Novelty-induced suppression of feeding (graham cracker test)
In order to perform the graham cracker test (GCT), mice first underwent a period of habituation, during which a small dish with ∼0.2 g graham cracker crumbs was placed in the home cage of the mouse. Mice had access to the crumbs for up to 15 min per day (1 session per day) until the latency to approach and consume the crumbs during the session was consistent (up to 10 d in a row). On the test day, the mouse was taken to the behavioral room, tethered to the laser in the home cage for 5 min, and then placed into a novel cage with access to a small dish with graham cracker crumbs. Optogenetic stimulation (10 mW, 15 Hz, 20 ms pulses, 75 pulses per sequence, 5 s delay between sequences) began when the mouse was placed into the novel cage. The latency to approach and consume the crumbs in the novel environment was then recorded as a measure of anxiety in a novel environment. The mouse was untethered and returned to its home cage following the 15 min test. For tests of mice with deletion of CeA AT2R, the same procedures were performed but without tethering or optogenetic stimulation.
Statistics
Quantification of immunohistochemical data was done in FIJI. For these data, as well as the behavioral data, unpaired Student's t tests were performed in GraphPad to determine significance. p < 0.05 (one-tailed) was considered significant.
Gap-free voltage-clamp recordings were low-pass filtered at 2 kHz. Evoked IPSCs were loaded into a custom program (https://www.researchgate.net/publication/338623194_NeuroExpress_program_for_analyzing_patch-clamp_data; NeuroExpress, Attila Szücs, Eötvös Loránd University; Hernáth et al., 2019; Rátkai et al., 2021). Noise threshold was set on a per cell basis using an average of the noise bandwidth (8–15 pA). IPSC detection threshold was set to 20 pA. Statistical analyses and graphs were generated using Prism version 9.1.0 (GraphPad Software). Example gap-free recordings generated using Igor Pro (WaveMetrics). A paired t test was used to compare ChR2-evoked IPSC amplitudes between holding potentials and before and after picrotoxin application. p < 0.05 was considered significant.
Cardiovascular parameters obtained from anesthetized mice were analyzed in LabChart 8 (ADInstruments). For all cardiovascular parameters, two-way ANOVAs with Šídák's multiple-comparisons tests were performed using GraphPad to determine significance.
All values are reported as mean ± standard error of the mean (SEM). Statistical analyses and graphs were generated using Prism version 9.1.0 (GraphPad Software).
Results
CeAAT2R connect to fore-, mid-, and hindbrain nuclei mediating autonomic outflow and stress reactivity but are primarily GABAergic
We delivered a Cre-inducible AAV synthesizing enhanced yellow fluorescent protein (eYFP) into the CeA of AT2RCre mice to visualize neurons that express AT2R(s) (Fig. 1A). Two to three weeks later, mice were perfused, brains were extracted, and sections through the CeA were processed for RNAscope in situ hybridization for Agtr2 mRNA(s) and immunohistochemistry for eYFP. Photomicrographs obtained at low power magnification found bilateral expression of eYFP within the CeA (Fig. 1B). Quantification of higher magnification images revealed that mRNA(s) encoding AT2R(s) were present within ≈90% of eYFP-labeled cells (Fig. 1C). To gain insight on how stimulation of AT2R affects the excitability of neurons, we utilized AT2ReGFP mice to obtain in vitro patch-clamp electrophysiological recordings from CeAAT2R during bath application of the specific AT2R agonist, C21 at 0.5 μM/0.5 ml (Steckelings et al., 2011). Consistent with prior reports (Zhu et al., 2001; de Kloet et al., 2016a), application of C21 significantly (p = 0.031 Wilcoxon test, n = 7) increased the firing rates in 7 of 9 neurons sampled (Fig. 1D). Collectively, these results demonstrate that Cre-recombination is directed to AT2R gene expression with the same high fidelity that we previously observed (Mohammed et al., 2022) and that stimulation of AT2R(s) elicits firing of CeAAT2R.
Genetic targeting of CeAAT2R. A, Schematics depicting the bilateral injections of a Cre- inducible AAVs into the CeA of AT2RCre mice to direct the expression of eYFP to neurons that express AT2R. B, A confocal coronal scan through the CeA demonstrating the localization of eYFP in the CeA. C, A representative image depicting the colocalization of Agtr2 mRNA to eYFP-labeled neurons in the CeA, and a bar chart of colocalization quantification. D, Top, A schematic depicting electrophysiological recordings from brain slices obtained from AT2ReGFP mice, middle, representative image of an eGFP-labeled neuron targeted for patch-clamp recordings (scale bar, 20 µm); bottom, responses to the 0.5 ml bath application of 0.5 µM C21. C21 increased action potential firing in 7/9 cells. *p < 0.05, n = 7; Wilcoxon test.
Next, we performed confocal scans of brain sections obtained from AT2RCre mice administered Cre-inducible AAV-eYFP into the CeA (Fig. 2A). These scans revealed densely labeled somas at the site of injection, as well as eYFP-labeled axons with projections that traverse the fore-, mid-, and hindbrain (Fig. 2B). Immunohistochemistry for eYFP performed on coronal brain sections confirmed the appearance of axon terminals in the bed nucleus of the stria terminalis (BNST), periaqueductal gray (PAG), and caudal nucleus of the solitary tract (cNTS; Fig. 2B, Table 1). Results from these tract-tracing experiments indicate that CeAAT2R are connected to brain nuclei implicated in autonomic outflow and stress reactivity. To ascertain the neurochemical phenotype of CeAAT2R, we performed immunohistochemistry for eYFP with RNAscope in situ hybridization for mRNA(s) encoding vesicular GABA transporter (vGAT) or vesicular glutamate transporter 2 (vGlut2), markers for GABAergic or glutamatergic neurons, respectively. As shown in Figure 2C–E, ≈90% of eYFP-labeled cells expressed vGAT mRNA but only ≈9% of such cells were found to express vGlut2 mRNA. Collectively, these results suggest that while CeAAT2R likely form local connections within the amygdala, they also send long range axonal projections to distal nuclei; however, the majority of CeAAT2R are GABAergic.
Anatomical phenotyping of CeAAT2R. A, Schematics depicting the bilateral injections of a Cre-inducible AAV synthesizing eYFP into the CeA of AT2RCre mice. B, Confocal coronal scans through the forebrain and hindbrain demonstrating somas within the CeA and fibers projecting throughout the fore-, mid-, and hindbrain. Representative image depicting the colocalization of vGAT mRNA (C) or vGlut2 (D) within eYFP-labeled neurons in the CeA. E, Bar chart of colocalization quantification.
Fiber projections of CeAAT2R throughout the brain
Excitation of CeAAT2R lowers blood pressure and this effect is mediated by the autonomic nervous system
The structural connectivity exhibited by CeAAT2R predicts that such neurons function as mediators of autonomic outflow. To begin to test this prediction, we recorded blood pressure and heart rate during acute delivery of C21 into the CeA. Mice were anesthetized and a Millar Catheter was implanted into the left common carotid artery. Mice were then placed in a stereotaxic frame, a microcraniotomy was performed, and C21 (22 ng/1 μl) was bilaterally microinjected into the CeA and cardiovascular parameters were recorded (Fig. 3A). As shown in Figure 3A, relative to controls administered saline, delivery of C21 significantly and transiently decreased blood pressure (F(26,324) = 1.2, p < 0.0001; two-way ANOVA followed by Šídák post hoc test, n = 7) but had no effect on heart rate (F(26,324) = 1.3, p = 0.1857; two-way ANOVA followed by Šídák post hoc test, n = 7).
Excitation of CeAAT2R decreases blood pressure. A, Top, Schematic depicting cardiovascular recording while bilaterally microinjecting saline or C21 (22 ng/1 µl) into the CeA of C57BL/6J mice; bottom, group data depicting systolic blood pressure (SBP) and heart rate (HR) upon the microinjection of saline or C21 into the CeA. B, Top, A schematic depicting electrophysiological recordings from brain slices obtained from AT2RCre mice injected with Cre-dependent ChR2. Middle, Image depicting a patched CeAAT2R expressing ChR2/eYFP and loaded with Alexa 555 dye (scale bar, 20 µm). Bottom, Current-clamp recordings demonstrating action potentials in response to single and repetitive stimulation with 488 nm blue light. C, Top, Schematic of the opto-cardio preparation in AT2RCre mice that received Cre-inducible AAV that directs the expression of eYFP (control; red) and/or ChR2 (experimental; blue) to AT2R-expressing cells within the CeA. Bottom: representative traces showing the blood pressure response to 473 nm blue light illumination of CeAAT2R in the control and experimental groups. Group data depicting (D) SBP and HR, (E) very low frequency (VLF-SBP) and low frequency (LF-SBP) systolic blood pressure variability, (F) SBP prior to or following the administration of 0.1 ml of hexamethonium (30 mg/kg, i.p.) with optical stimulation (473 nm, 20 ms pulse width at 10 mW for 1 min). Blue box represents period of 473 nm light illumination (15 Hz for C, E, F, 1, 15 and 30 Hz for D). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001; two-way ANOVA followed by Šídák post hoc test, †p < 0.05, ††p < 0.01, †††p < 0.001, ††††p < 0.001; repeated-measures two-way ANOVA. n = 4–10.
To gain further insight on the role that CeAAT2R play in this hypotensive response, we used optogenetics to evaluate whether excitation of CeAAT2R alters cardiovascular function. Cre-inducible AAVs, synthesizing eYFP only (control) or the light-sensitive ion channel, channelrhodopsin-2 (ChR2) and eYFP, were bilaterally administered into the CeA of AT2RCre mice (Fig. 3B). Two to three weeks later, a subset of mice was killed, and coronal sections through the CeA were used to verify ChR2 function in CeAAT2R using in vitro whole-cell voltage-clamp electrophysiological recordings. Recordings obtained from CeAAT2R found that optogenetic stimulation with varying protocols of 488 nm light pulses reliably evoked action potentials in the patched neurons, demonstrating the expression of functional opsins (Fig. 3B).
In vivo optogenetic experiments evaluated whether excitation of CeAAT2R affected blood pressure and heart rate. Mice were anesthetized and a Millar Catheter was implanted into the left common carotid artery. Mice were then placed in a stereotaxic frame, a microcraniotomy was performed, fiber optics connected to a laser light source were bilaterally inserted into the CeA, and pulses of 488 nm light were delivered to CeAAT2R while cardiovascular parameters were recorded (Fig. 3C). Relative to controls expressing only eYFP, optogenetic excitation of CeAAT2R with ChR2 significantly reduced blood pressure when pulses of 488 nm light were delivered at 15 Hz (F(14,238) = 4.05, p < 0.0001; two-way ANOVA followed by Šídák post hoc test, n = 8–10) or 30 Hz (F(16,164) = 2.4, p = 0.0043; two-way ANOVA followed by Šídák post hoc test, n = 6–7; Fig. 3C,D); however, the heart rate responses to optical stimulation were similar among the groups (15 Hz; F(14,238) = 0.6, p = 0.8674, 30 Hz; F(16,164) = 0.19, p = 0.9995; two-way ANOVA followed by Šídák post hoc test, n = 6–10; Fig. 3D). To gain insight on whether the reductions in blood pressure were associated with altered sympathetic and/or parasympathetic outflow to the vasculature, we conducted systolic blood pressure variability analyses. As shown by Figure 3E, relative to controls, optogenetic excitation of CeAAT2R significantly decreased the low frequency domain of systolic blood pressure variability (F(7,120) = 1.9, p < 0.0001; two-way ANOVA followed by Šídák post hoc test, n = 8–9). This suggests that excitation of CeAAT2R alters autonomic outflow to the vasculature by promoting sympathetic withdrawal and/or enhancing vagal tone. Finally, we systemically administered mice the nicotinic receptor antagonist, hexamethonium (0.1 ml at 30 mg/kg, i.p.), to determine whether the decrease in blood pressure that occurs with in vivo optogenetic excitation of CeAAT2R is mediated by the autonomic nervous system. As before, optogenetic excitation of CeAAT2R significantly decreased systolic blood pressure in mice expressing ChR2 relative to controls; however, this effect was completely abolished after administration of hexamethonium (F(42,371) = 2.6, p < 0.0001; two-way ANOVA followed by Tukey's post hoc test, n = 5–10; Fig. 3F). Taken together, these results suggest that excitation of CeAATR2 decreases blood pressure by suppressing sympathetic outflow and/or augmenting vagal tone.
Optogenetic excitation of distal projections originating from CeAAT2R does not lower blood pressure
Our tract-tracing and in vivo optogenetic experiments revealed that CeAAT2R send projections to brain nuclei that mediate autonomic outflow to cardiovascular tissues. Consequently, we performed follow-up experiments to test the prediction that optogenetic excitation of axonal projections originating from CeAAT2R would recapitulate the lowered blood pressure that was observed. We again bilaterally delivered Cre-inducible AAV(s) synthesizing eYFP or eYFP and ChR2 into the CeA of AT2RCre mice. Two to three weeks later, mice were anesthetized, implanted with a Millar Catheter, and placed in a stereotaxic frame, and a microcraniotomy was performed as done in the previous experiment. However, instead of delivering optical stimulation to the CeA, fiber optics now targeted axonal projections presumed to terminate in the BNST, PAG, and cNTS. Optogenetic stimulation of the BNST (15 Hz: −1.1 ± 0.5 vs −0.50 ± 0.5 mmHg; F(14,118) = 1.1, p = 0.4147, 30 Hz: −1.4 ± 1.5 vs −1.1 ± 0.67 mmHg; F(14,118) = 0.4, p = 0.9598; two-way ANOVA followed by Šídák post hoc test, n = 4–6) and PAG (15 Hz: −2.4 ± 1.5 vs −0.62 ± 1.0 mmHg; F(14,120) = 0.2, p = 0.9993, 30 Hz: −0.8 ± 0.4 vs −2.3 ± 3.3 mmHg; F(14,120) = 0.5, p = 0.9625; two-way ANOVA followed by Šídák post hoc test, n = 4–6) had no effects on blood pressure. In addition, and contrary to our prediction, activating axonal fibers in the cNTS that originated from CeAAT2R significantly elevated blood pressure (15 Hz; F(14,139) = 1.9, p = 0.0295, 30 Hz; F(14,131) = 2.0, p = 0.0203; two-way ANOVA followed by Šídák post hoc test, n = 4–8; Fig. 4). Like the prior experiment, differences in heart rate were not observed (15 Hz: −0.9 ± 0.5 vs −1.7 ± 0.6 bpm; F(14,139) = 1.1, p = 0.3671, 30 Hz: −1.9 ± 0.8 vs 0.6 ± 1.3 bpm; F(14,131) = 0.6, p = 0.8873; two-way ANOVA followed by Šídák post hoc test, n = 4–8; Fig. 4B). These results suggest that connections from CeAAT2R to distal brain nuclei may not contribute to the decreased blood pressure that was observed, but rather, this effect may be mediated by synaptic connections localized to the CeA.
Excitation of CeAAT2R fibers projecting to the NTS increase blood pressure. A, Schematic depicting the optogenetics-cardiovascular preparation in male AT2RCre mice that received Cre-inducible AAV to direct the expression of eYFP (control; red) and/or ChR2 (experimental; blue) to CeAAT2R. B, Top, Schematic depicting in vivo optogenetic stimulation of fibers originating from CeAAT2R; middle, representative traces of the blood pressure response during 473 nm blue light illumination (473 nm, 15 Hz, 20 ms pulse width at 10 mW for 5 min; 1 min ON/OFF); bottom, group data showing systolic blood pressure (SBP) and heart rate (HR) prior to and during 473 nm blue light illumination. Blue box represents period of 473 nm light illumination. *p < 0.05, ****p < 0.001; two-way ANOVA followed by Šídák post hoc test. n = 4–8.
In vitro optogenetic excitation of CeAAT2R stimulates GABA receptors on neighboring neurons, an effect that is required for the lowered blood pressure that follows in vivo optogenetic excitation of CeAAT2R
Because exciting sites distal to the CeA yielded null results or produced results contrary to those anticipated, we undertook experiments examining whether excitation of CeAAT2R engages local circuits to promote a hypotensive response. The Cre-inducible AAV synthesizing eYFP and ChR2 was again injected into the CeA of AT2RCre mice. Two to three weeks later, mice were killed, and live brain slices were used for in vitro optogenetic experiments examining local synaptic connections made by CeAAT2R. Using a combination of epifluorescence and DIC microscopy, patch-clamp electrophysiological recordings were obtained from CeA neurons that were devoid of eYFP labeling but were near eYFP/ChR2 expressing fibers originating from CeAAT2R (Fig. 5A). Delivery of 488 nm light (10 Hz for 2 s) evoked inhibitory postsynaptic currents (IPSCs) in all non-CeAAT2R patched neurons. To confirm the GABAergic nature of these synaptic events, we stimulated the ChR2 before and after the 0.5 ml bath application of the GABAA channel blocker picrotoxin (100 µM), demonstrating a significant reduction in current amplitude (p = 0.0313 Wilcoxon test, n = 6). Additionally, shifting the holding potential (Vh) from −70 to −60 mV, bringing it closer to the Cl− reversal potential, reliably reduced the amplitude of these evoked synaptic events (p = 0.125 Wilcoxon test, n = 4; Fig. 5B). The lack of a sustained inward current following the light stimulation in the patched neurons further confirmed their identity as non-CeAAT2R neurons. Collectively, these results demonstrate that CeAAT2R exert inhibition over neighboring non-CeAAT2R neurons via the local release of GABA.
Excitation of CeAAT2R evokes local GABA release that lowers blood pressure. A, Schematic depicting the electrophysiological preparation of brain slices obtained from AT2RCre mice that received Cre-inducible AAV directing the expression of eYFP and ChR2 to CeAAT2R. B, Voltage-clamp recordings of nonlabeled neurons in close proximity to ChR2-eYFP-labeled neurons. Whole field 488 nm light pulses evoked instantaneous IPSCs, indicating synaptic innervation by CeAAT2R neurons. Subsequent 0.5 ml bath application of 100 μM picrotoxin significantly reduces the amplitude of evoked IPSCs to 488 nm light pulses *p < 0.05; Wilcoxon test. n = 6. Additionally, shifting the holding potential (Vh) from −70 to −60 mV consistently reduces amplitude of evoked IPSCs to 488 nm light pulses. p = 0.13; Wilcoxon test. n = 4. C, Schematic showing optogenetic excitation with microinjection of bicuculline. D, Group data demonstrating systolic blood pressure (SBP) and heart rate (HR) upon the microinjection of vehicle (43% ethyl alcohol, 43% dH2O, 14% trifluoroacetic acid) or bicuculline (30 mM/1 μl) into the CeA. E, Group data showing SBP and HR during optical stimulation (473 nm, 30 Hz, 20 ms pulse width at 10 mW for 5 min; 1 min ON/OFF) in control and experimental mice treated with vehicle or bicuculline. Blue box represents period of 473 nm light illumination. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001; two-way ANOVA followed by Šídák post hoc test, ††p < 0.01, †††p < 0.001; repeated-measures two-way ANOVA. n = 4–7.
Next, we combined neuropharmacology with in vivo optogenetics and cardiovascular recordings to determine whether activation of GABA receptors localized to the CeA is required for the lowered blood pressure that follows excitation of CeAAT2R. Cre-inducible viruses were unilaterally delivered into the CeA of AT2RCre mice, and 2–3 weeks later, mice were prepared for cardiovascular recordings during optogenetic excitation of CeAAT2R. The methodology was like our previous experiments; however, optogenetic excitation was accomplished using a dual fiber-optic cannula that allows pulses of 488 nm light and microinjection of fluid to be delivered simultaneously to the target nuclei (Elsaafien et al., 2021; Fig. 5C). Consistent with prior reports (Ciriello and Roder, 1999; Zhang et al., 2009), microinjection of the GABAA receptor antagonist, bicuculline (10 mM/1 μl), into the CeA produced a significant but transient increase in blood pressure when compared with injection of the vehicle control (F(12,143) = 2.0, p = 0.0334; two-way ANOVA followed by Šídák post hoc test, n = 6–7; Fig. 5D). Taking this effect into account, we next tested optogenetic excitation of CeAAT2R after the pressor effects of bicuculline had subsided (e.g., ≈10 min post-bicuculline injection). Figure 5E shows the effect of optical stimulation on blood pressure in AT2RCre mice given the control AAV or an AAV expressing ChR2 after delivery of vehicle or bicuculline. As expected, optical stimulation of CeAAT2R significantly decreased blood pressure in mice expressing ChR2 when compared with controls; however, administration of bicuculline 10 min prior to optogenetic excitation of CeAAT2R completely abolished this effect (F(42,210) = 1.8, p = 0.0043; two-way ANOVA followed by Tukey's post hoc test, n = 4–5; Fig. 5E). These collective results suggest that excitation of CeAAT2R evokes the release of GABA, which acts on GABAA receptors expressed on neighboring neurons to decrease blood pressure.
Central AT2R activation or optogenetic excitation of CeAAT2R suppresses, but ablating AT2R(s) within the CeA incites anxiety-like behavior
The structure and synaptic connectivity exhibited by CeAAT2R predicts that such neurons function as mediators of stress reactivity. To begin to test this prediction, we outfitted wild-type C57BL/6J mice with mini-osmotic pumps that allow chronic infusion of the AT2R agonist, C21 (7.5 ng/kg/h), into the lateral ventricle. Subsequently, mice were tested in an EPM to determine whether activation of AT2R(s) within the brain may affect anxiety-like behavior. As shown in Figure 6, relative to saline-infused controls, chronic stimulation of brain-dwelling AT2R(s) increased the amount of time that mice spent in the open arms of the EPM (t(21) = 2.675, p = 0.0071; unpaired t test, n = 11–12) while decreasing the time spent in the closed arms (t(21) = 2.022, p = 0.028; unpaired t test, n = 11–12) and not affecting the total distance traveled (t(21) = 0.55, p = 0.294; unpaired t test, n = 11–12). These results suggest that stimulating AT2R(s) within the brain is sufficient to attenuate anxiety-like behavior.
Activation of central AT2R(s) is anxiolytic. A, Top, Schematic depicting osmotic minipumps infusing saline or C21 (7.5 ng/kg/h) into the lateral ventricles (bottom) prior to testing in the EPM. Right, Bar graphs demonstrating % of time spent in the (B) open or (C) closed arms of the maze and (D) the total distance traveled. *p < 0.05, **p < 0.01; unpaired student t test. n = 11–12.
Follow-up experiments utilized in vivo optogenetics to evaluate whether selective excitation of CeAAT2R recapitulates the anxiolytic effects of stimulating central AT2R(s). Cre-inducible AAVs synthesizing eYFP or eYFP and ChR2 were bilaterally administered into the CeA of AT2RCre mice. Following administration of AAV(s), mice were bilaterally implanted with chronic dwelling fiber optics targeting the CeA (Fig. 7A). Two to three weeks later, we initiated in vivo optogenetic experiments evaluating whether excitation of CeAAT2R affected anxiety-like behavior as assessed with the EPM, OFA, and a novelty suppressed feeding paradigm (i.e., GCT). Relative to controls, optogenetic excitation of CeAAT2R significantly increased the amount of time mice spent in the open (t(14) = 3.2, p = 0.0070, unpaired t test, n = 7–9), but decreased time spent in the closed (t(15) = 3.0, p = 0.0088, unpaired t test, n = 8–9), arms of the EPM (Fig. 7B). Consistent with these results, optogenetic excitation of CeAAT2R also significantly increased the amount of time mice spent in the center of the OFA (t(10) = 2.0, p = 0.0401, unpaired t test, n = 7–9) and decreased the latency to eat (t(7) = 2.8, p = 0.0259, unpaired t test, n = 4–5) in the GCT (Fig. 7C,D). Importantly, control mice and those expressing ChR2 traveled similar distances in the EPM (t(14) = 0.1, p = 0.8887, unpaired t test, n = 7–9) and OFA (t(14) = 0.6, p = 0.5816, unpaired t test, n = 7–9) during optical stimulation (Fig. 7B,C), indicating that the anxiolytic effects of exciting CeAAT2R are likely not attributed to alterations in locomotor activity. Taken together, these results suggest that like central AT2R stimulation, excitation of CeAAT2R is anxiolytic in male mice.
The optogenetic excitation of CeAAT2R is anxiolytic. A, Schematic depicting AT2RCre mice that received Cre-inducible AAV to direct the expression of eYFP (control; red) and/or ChR2 (experimental; blue) to CeAAT2R. Subsequently, mice were bilaterally implanted with chronic dwelling fiber optic targeting the CeA. Optogenetic stimulation of CeAAT2R during an (B) EPM, (C) OFA, and (D) GCT in control and experimental mice. Optical stimulation: 473 nm, 15 Hz, 20 ms pulse width at 10 mW for 5 min; 75 pulses per sequence; 5 s delay between sequences. *p < 0.05, **p < 0.01; unpaired student t test. n = 4–9/group.
While the results from our pharmacological and optogenetic experiments suggest that excitation of CeAAT2R is sufficient to alter anxiety-like behavior, they do not evaluate the necessity of AT2R(s), per se. To determine whether AT2R(s) localized to the amygdala contribute to basal levels of anxiety-like behavior, we bilaterally administered an AAV expressing Cre-recombinase and tdTomato into the CeA of mice with LoxP sites flanking the AT2R gene (AT2Rflox mice; Fig. 8A). This approach allows deletion of AT2R(s) from cells residing in the CeA. AT2Rflox mice delivered an AAV only expressing tdTomato served as controls. To confirm deletion of AT2R(s), a subset of mice was killed and perfused, and extracted brains were processed for RNAscope in situ hybridization for AT2R mRNA(s). Figure 8B depicts site-specific viral delivery as demonstrated by tdTomato expression localized to the CeA. Relative to controls, AT2Rflox mice given AAV expressing Cre-recombinase had significantly fewer tdTomato-labeled cells expressing AT2R mRNA(s) (tdTom: 19.5 ± 3.0 vs Cre-tdTom: 0.02 ± 0.01%, t(4) = 6.559 p = 0.0028, unpaired t test, n = 3; Fig. 8C). Importantly, control mice and those expressing Cre-recombinase had similar expression of AT2R mRNA(s) within DAPI-labeled cells in the medial amygdala (tdTom: 8.6 ± 3.1 vs Cre-tdTom: 12.9 ± 5.1%, t(4) = 0.7234, p = 0.5095, unpaired t test, n = 3), suggesting that knockdown of AT2R(s) was specific to the CeA. To ascertain the contribution of AT2R(s) to anxiety-like behavior, mice were tested in the EPM, OFA, and GCT as done previously. Contrary to exciting CeAAT2R, deleting AT2R(s) from the CeA produced effects consistent with an anxiogenic phenotype (Fig. 8D–F⇓). Specifically, AT2Rflox mice given AAV expressing Cre-recombinase spent significantly less time in the open (t(10) = 3.9, p = 0.0031, unpaired t test, n = 5–7) but more time in closed arms (t(10) = 3.7, p = 0.0043, unpaired t test, n = 5–7) of the EPM and had increased latency to eat (t(14) = 3.0, p = 0.0112, unpaired t test, n = 7–9) in the GCT; however, control mice and those with AT2R knockdown exhibited similar behaviors in the OFA. These results demonstrate that deletion of AT2R(s) from the CeA increases anxiety-like behavior and further suggest that, under basal conditions, these AT2R(s) promote anxiolysis.
The deletion of AT2R(s) within the CeA is anxiogenic. A, Schematic depicting male AT2R-flox mice that received Cre-inducible AAVs to direct the expression of tdTomato (control; white) or Cre-recombinase and tdTomato (Cre-tdTom; red) to the CeA. B, A representative coronal atlas section and corresponding photomicrograph demonstrating tdTom expression within the CeA. C, RNAscope in situ hybridization showing Agtr2 mRNA colocalization with (left) tdTomato and (right) the cell marker, DAPI, is significantly decreased in the CeA of AT2Rflox mice given AAV expressing Cre-recombinase (Cre-tdTom) relative to those given the control virus (tdTom). The performance of tdTom and Cre-tdTom mice in the (D) EPM, (E) OFA, and (F) GCT. **p < 0.01, ****p < 0.0001; unpaired student t test. n = 3–9.
Amplifying the firing of CeAAT2R silences neurons whose activation is coupled to anxiety-like behavior and elevated blood pressure. The firing of CeAAT2R is elicited by way of activating AT2R(s) using a specific agonist, C21, or by way of optogenetic stimulation of ChR2. The majority of CeAAT2R form GABAergic inhibitory synapses onto neighboring neurons that lack AT2R(s). The activity of such CeA neurons has been previously linked to anxiety-like behavior, via projections to areas like the periaqueductal gray (PAG), lateral hypothalamus (LH), and locus ceruleus (LC; Viviani et al., 2011; Paretkar and Dimitrov, 2018; Weera et al., 2021), and elevated blood pressure via projections to areas like the nucleus of the solitary tract (NTS; Saha, 2005). Here, we find that excitation of CeAAT2R leads to the release of GABA, which inhibits neighboring neurons in the CeA and quells their influence over effector nuclei that promote anxiety and sympathetically mediated vasoconstriction. While the present studies do not uncover the projection phenotypes of the non-AT2R neurons within the CeA that receive inhibitory input from CeAAT2R, they do reveal that subsets of CeAAT2R send projections to areas outside the CeA that are implicated in these processes. The present studies also reveal that the CeAAT2R that project to the NTS, in particular, are pressor neurons. Figure generated using BioRender.com.
Discussion
The current study reveals a unique population of neurons whose excitation reduces blood pressure and anxiety-like behavior (Fig. 9). These neurons synthesize AT2R(s) and reside in the CeA and are therefore identified as CeAAT2R. While some CeAAT2R synthesize glutamate and send axonal connections to distal brain nuclei, the majority of CeAAT2R are GABAergic neurons. Pharmacological stimulation of AT2R(s) in the CeA lowers blood pressure, and this effect can be recapitulated by optogenetic excitation of CeAAT2R. The lowered blood pressure that accompanies optogenetic excitation of CeAAT2R is mediated by GABAergic inhibition of neighboring neurons in the amygdala, and this reduces sympathetic drive to cardiovascular tissues. Remarkably, stimulation of AT2R(s) or optogenetic excitation of CeAAT2R also decreases anxiety-like behavior; however, ablating AT2R(s) specifically in the CeA produces the opposite effect. Collectively, these results suggest that excitation of CeAAT2R elicits hypotension and anxiolysis (Fig. 9). The implication is that CeAAT2R exerts top-down control over mind-to-body communication that promotes resiliency or susceptibility to stress-related disease.
The CeA has long been implicated in the autonomic and behavioral responses to psychological stress (Davis and Shi, 1999; Saha, 2005). The CeA is mostly composed of GABAergic neurons that form robust local connections within the extended amygdala (Cassell et al., 1999). Additional subsets of CeA neurons send projections to fore-, mid-, and hindbrain nuclei that regulate autonomic outflow to peripheral organs (Saha, 2005). We found that most CeAAT2R synthesize GABA and function as inhibitory neurons that make local connections within the CeA; however, some CeAAT2R send axonal projections to fore-, mid-, and hindbrain nuclei that regulate autonomic outflow. Thus, CeAAT2R exhibit neurochemical and structural characteristics of prototypical CeA neurons.
The connectivity of the CeA, in conjunction with an established role of the amygdala in the processing of emotional stimuli, has led to the notion that the CeA exerts descending control over somatic manifestations of fear and anxiety. In support of this notion, electrical stimulation of the CeA elevates blood pressure and heart rate (Hilton and Zbrozyna, 1963; Stock et al., 1978, 1981), and this effect is thought to be mediated by GABAergic neurons with distal projections to the hindbrain (Saha et al., 2000; Saha, 2005). Conversely, electrical stimulation of the CeA has also been found to lower blood pressure (Gelsema et al., 1987; Iwata et al., 1987), and this effect is believed to be mediated by the local release of GABA (Ciriello and Roder, 1999). These collective results indicate that excitation of the CeA may elicit pressor or depressor responses depending on which connections and neurotransmitters are engaged. Analogous to these prior studies, we found that excitation of CeAAT2R elicits pressor or depressor responses that appear to be circuit specific. That is, we found that excitation of axon terminals in the NTS originating from CeAAT2R elicited pressor, but excitation of the soma of CeAAT2R elicited depressor responses. Regarding the depressor response, neurons of the CeA with direct projections to the hindbrain are densely innervated by GABAergic synapses, suggesting that local connections within the CeA tonically inhibit sympathetic outflow to cardiovascular tissues (Sun et al., 1994). Consistent with this interpretation, our experiments found that delivery of a GABA receptor antagonist into the CeA produced a transient increase in blood pressure. Furthermore, we determined that functional GABAergic synapses were also required for the lowered blood pressure that accompanies excitation of CeAAT2R. These results suggest that CeAAT2R exert an inhibitory influence over descending pathways that evoke somatic responses to stress.
In addition to somatic responses, neurons within the CeA have long been implicated in the control of anxiety-like behavior (Walker et al., 2009), and our results indicate that CeAAT2R likely mediate these effects. We found that chronic central administration of the AT2R agonist, C21, reduces anxiety-like behavior, and our follow-up experiments determined that in vivo optogenetic excitation of CeAAT2R is potently anxiolytic. Prior research found that whole body deletion of the AT2R in mice results in an anxiogenic phenotype (Ichiki et al., 1995; Okuyama et al., 1999). Interestingly, we recapitulated this anxiogenic phenotype by selectively ablating AT2R(s) within the CeA. That is, opposite to what we observed with excitation of CeAAT2R, knockdown of AT2R(s) specifically within the CeA increases anxiety-like behavior. Taken together, these results demonstrate that agonism of the AT2R or excitation of CeAAT2R suppresses anxiety-like behavior and AT2R(s) within the CeA are necessary to maintain basal levels of anxiolysis.
Behavioral manifestations of anxiety also appear to be controlled by CeA output neurons that are under tonic inhibition. Studies by Tye et al. (2011) used in vivo optogenetics in mice to manipulate excitatory inputs to GABAergic neurons of the CeA. Driving these excitatory inputs was anxiolytic but inhibiting them increased anxiety (Tye et al., 2011). Here, our in vitro electrophysiological recordings found that optogenetic excitation of CeAAT2R directly evoked GABA-mediated iPSCs recorded from neighboring, non-CeAAT2R neurons. These responses were observed in every patched non-CeAAT2R neuron, and evoked responses were of high fidelity, even at a stimulation frequency of 10 Hz, altogether implying that, within the CeA, a local synaptic GABAergic microcircuit may mediate anxiolytic and autonomic output. Consistent with this notion, complimentary in vivo optogenetic experiments found that excitation of CeAAT2R suppressed anxiety-like behavior and lowered blood pressure, an effect that was abolished by the local blockade of GABA receptors. These results, in conjunction with prior research, suggests that excitation of CeAAT2R suppresses the activation of amygdalar outputs that heighten stress reactivity. Accordingly, we propose that AT2R(s) demarcate a subset of GABAergic neurons that potently inhibit projections from the extended amygdala that drive physiological and behavioral responses to stress. The implication is that CeAAT2R may become compromised during the onset of stress-related disease or conversely, may be recruited by therapeutics that provide stress relief. Given that CeAAT2R mediate blood pressure, as well as anxiety, future studies implementing animal models of comorbid affective and cardiovascular disorders may shed insight on the utility of targeting CeAAT2R to understand and alleviate stress-related pathologies.
Limitations and future directions
Although the current study tests hypotheses by implementing a multifaceted approach, there are limitations to consider. Many of the experiments relied on administration of viruses into a discrete region of the mouse brain (i.e., CeA). Cases deemed “hits” represent microinjections that were highly localized to the CeA; however, it is challenging to restrict microinjections exclusively within the confines of the CeA. In some instances, we did observe sparse labeling of somas in adjacent brain nuclei. To account for this limitation, we confirmed that brain nuclei targeted by axons arising from CeAAT2R (e.g., medial amygdala, subthalamic nucleus, and lateral preoptic area) were previously determined to receive projections from the CeA with methodology (e.g., virally mediated neuronal tract tracing) similar to that used in the present study (Han et al., 2017; Weera et al., 2023). Regarding in vivo optogenetic experiments, fiber optics were placed immediately dorsal to the target nucleus, which mitigates off-target stimulation that may have resulted from the spread of virus outside of the CeA. Another limitation relates to the recording of cardiovascular parameters under general anesthesia. We did not observe changes in blood pressure during optogenetic stimulation of fibers in the PAG or the BNST that originated from CeAAT2R. Despite these null findings, it is possible that projections from CeAAT2R to the PAG and/or BNST mediate cardiovascular function in conscious animals. This is especially likely under stressful conditions. The BNST and PAG are heavily implicated in cardiovascular and behavioral stress responses (Jaramillo et al., 2020; Maita et al., 2022; Matsuyama and Horiuchi, 2024). For example, the PAG, in particular, is known to mediate vasomotor responses evoked by social defeat stress (Matsuyama and Horiuchi, 2024). Finally, our studies largely interrogate the role that CeAAT2R play in mediating blood pressure and anxiety-like behavior by way of conducting sufficiency experiments (i.e., optogenetic excitation; AT2R stimulation). We did perform loss of function experiments that revealed the anxiogenic effects of knocking down AT2R(s) within the CeA. However, experiments that evaluate the necessity of the CeAAT2R in the control of blood pressure by way of optogenetically inhibiting CeAAT2R were not conducted. Consequently, the role that CeAAT2R play in the physiological regulation of blood pressure is less clear. Inhibitory optogenetic experiments are intriguing future directions, as are follow-up studies that parse the up- and downstream neural circuits mediating the anxiolytic and depressor effects associated with excitation of CeAAT2R and their AT2R(s).
Footnotes
This work was supported by the National Institutes of Health: R35HL150750 (E.G.K.), R01HL145028 (A.D.K.); AHA 23POST1020034 and K99HL175100 (K.E.).
The authors declare no competing financial interests.
- Correspondence should be addressed to Eric G. Krause at ekrause{at}gsu.edu or Annette D. de Kloet at adekloet{at}gsu.edu.
