Arcuate Angiotensin II Increases Arterial Pressure via Coordinated Increases in Sympathetic Nerve Activity and Vasopressin Secretion

Visual Abstract


Introduction
The hypothalamic arcuate nucleus (ArcN) is a well-established integrative hub for the regulation of energy balance and reproduction. The ArcN has also been identified as a site important in autonomic control of the cardiovascular system (Sapru, 2013). For example, the metabolic hormones, insulin and leptin, each act in the ArcN to increase arterial pressure (AP) and sympathetic nerve activity (SNA) to several organs, including skeletal muscle, the splanchnic circulation, and the kidneys (Cassaglia et al., 2011;Harlan and Rahmouni, 2013). Like insulin and leptin, ArcN Angiotensin II (AngII) increases AP (Arakawa et al., 2011). ArcN AngII also influences reproduction and energy balance (Mehay et al., 2021), implicating ArcN AngII as a candidate integrative neuropeptide. However, the mechanisms by which AngII increases AP, the cellular mechanisms of integration, and downstream neurocircuitry are unknown.
Two major ArcN cell types that influence AP and SNA are inhibitory neuropeptide Y (NPY) neurons and excitatory proopiomelanocortin (POMC) neurons, which release a-melanocyte stimulating hormone (a-MSH). Indeed, both leptin (Shi et al., 2015b) and insulin (Ward et al., 2011;Cassaglia et al., 2016) increase SNA via suppression of tonically sympathoinhibitory NPY neurons and activation of sympathoexcitatory POMC neurons. At least in mice, AngII type 1 receptors (AT1aRs) are highly expressed in ArcN AgRP neurons (Claflin et al., 2017), almost all of which also express NPY (Broberger et al., 1998). AgRP/NPY neurons inhibit SNA via release of NPY in two hypothalamic sites: the paraventricular nucleus (PVN) and dorsal medial hypothalamus (DMH; Shi et al., 2017). Therefore, we first tested whether ArcN AngII increases SNA in part by inhibiting NPY neurons that project to the PVN or the DMH. Second, because suppression of tonic PVN NPY sympathoinhibition can unveil the sympathoexcitatory effects of a-MSH at melanocortin type 3 or 4 receptors (MC3/4R; Cassaglia et al., 2014;Shi et al., 2015b), we also determined whether a-MSH contributes to the sympathoexcitatory effects of ArcN AngII.
Cardiovascular diseases that are sexually dimorphic, like hypertension, exhibit stark sex differences in a dependence on the renin-angiotensin system (RAS; for review, see Xue et al., 2013;Brooks et al., 2015;Ramirez and Sullivan, 2018). AT1aR are expressed more highly in the ArcN in females than in males, with the greatest levels observed during estrus/diestrus compared with proestrus (Seltzer et al., 1993;Jöhren et al., 1997). However, whether ArcN AngII also elicits cardiovascular and autonomic effects in females has not been previously investigated. Therefore, we next tested whether ArcN AngII increases SNA and AP in female rats and whether the response varies during the reproductive cycle. Second, as in males, we tested whether the sympathoexcitatory response relies on inverse changes in the activity of ArcN NPY and POMC neurons that project to the PVN.
Pregnancy increases SNA, likely due in part to the central actions of AngII (Brooks et al., 2020); however, the brain sites are unknown. The ArcN supports increased SNA during pregnancy (Shi et al., 2015a), but neither leptin nor insulin are involved (Shi et al., 2019b). Thus, the hormonal mediator has not been identified. Therefore, to begin to test the hypothesis that AngII acts in the ArcN to increase SNA during normal pregnancy, we determined whether ArcN AngII is sympathoexcitatory in late pregnant rats and whether this is associated with increased ArcN AT1AR expression. Aberrant activity of the RAS contributes to the often-fatal hypertensive disorder, preeclampsia, which increases SNA even more (Brooks et al., 2020). Thus, this information from normal pregnancy will also provide a basis for studies to test whether central actions of the RAS contribute to the excessive sympathoexcitation observed in females with pregnancy-induced hypertensive disorders.
Lastly, while the AT1aR was frequently found in NPY neurons in mice (Claflin et al., 2017), whether the same is true in rats is unknown. Therefore, we systematically explored the expression pattern and cellular phenotypes of the AT1aR in the ArcN of male rats and of female rats in various reproductive stages using fluorescent in situ hybridization (FISH).

Materials and Methods
Experiments were performed using male and female Sprague Dawley rats (13-17 weeks, Charles River Laboratories, Inc). All the rats were acclimated for more than or equal to one week before experimentation in a room with a 12/12 h light/dark cycle, with food (LabDiet 5001) and water provided ad libitum. Rats were generally housed in pairs. Vaginal epithelial cytology was examined daily to establish the 4-to 5-d estrous cycle. Rats were usually impregnated by housing with a male, and the presence of vaginal sperm was designated pregnancy day 0 (P0). Alternatively, timed pregnant rats were obtained from Charles River Laboratories, Inc. Pregnant rats were housed singly, until the experiment on pregnancy day 20 (P20). All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional (Oregon Health & Science University or University of Virginia) Animal Care and Use Committee.

Surgical preparation
Anesthesia was induced and maintained with 2-5% isoflurane in 100% oxygen. Body temperature was maintained at 37 6 1°C using a rectal thermistor and heating pad. A tracheal tube, a femoral arterial catheter, and two venous catheters were placed for artificial ventilation, the measurement of mean AP (MAP), and drug infusions, respectively. The lumbar sympathetic nerve was located after a midline abdominal incision, and the splanchnic nerve was exposed after a flank incision. Bipolar stainless-steel electrodes were positioned and secured around the nerves using lightweight silicone material (Kwik-Sil, WPI, Inc). The rat was then placed in a stereotaxic instrument (David Kopf Instruments), and, following a midline incision on the top of the skull, a hole was burred near the midline to allow for ArcN, PVN, or DMH nanoinjections. After completion of surgery, isoflurane anesthesia was slowly withdrawn over 30 min, and a continuous intravenous (iv) infusion of a-chloralose was begun and continued for the duration of the experiment (50 mg/kg loading dose over 30 min; 25 mg/kg/h maintenance dose; Sigma-Aldrich). Pregnant rats received an a-chloralose dose equivalent to a virgin rat at a similar age. Throughout the experiment, the rats were continuously artificially ventilated with 100% oxygen, and respiratory rate and tidal volume were adjusted to maintain expired CO 2 at 3.5-4.5%. Anesthetic depth was regularly confirmed by the lack of a pressor response to a foot or tail pinch; if necessary, additional a-chloralose was administered iv. After completion of surgery and the a-chloralose loading dose, rats were allowed to stabilize for !60 min before experimentation.
Experimental protocols were then performed to answer the following questions. (1) Does ArcN AngII increase SNA in males and females, and does the response vary during the estrous cycle or with pregnancy? After collecting baseline data, 30 nl of AngII (1 mM/l, Tocris) or aCSF was injected bilaterally into the ArcN and recordings continued for 90 min. (2) Is the SNA response mediated by ArcN AT1aR? A total of 60 nl of candesartan (0.5 mmol/l, Tocris Bioscience) or aCSF was injected bilaterally into the ArcN, and 10-15 min later, 30 nl of AngII [1 mmol/l (Arakawa et al., 2011), Tocris] or aCSF was injected  (Ward et al., 2011;Shi et al., 2015b;Cassaglia et al., 2016). Therefore, prior blockade of NPY Y1R would not be At the end of each experiment, ;60 nl of fluorescent polystyrene microbeads (FluoSpheres, F8803, 1:200; Invitrogen) were administered using the same pipette and coordinates to verify the injection sites using a standard anatomic atlas (Paxinos and Watson, 2007). Rats were then euthanized via iv administration of a barbiturate (Euthasol; Virbac AH).

Data analysis
Throughout the experiment, pulsatile AP, MAP, and heart rate (HR) were continuously collected using a Biopac MP100 data acquisition and analysis system (Biopac Systems), sampling at 2000 Hz. SNA was bandpass filtered (100-3000 Hz) and amplified (Â10,000). After data collection, postmortem SNA was quantified and subtracted from values of SNA recorded during the experiment. The SNA signal was then rectified, integrated in 1-s bins, and for the figures was normalized to basal values (% of control). Response values of LSNA, SSNA, MAP, and HR were the difference between the averages of All data are presented as means 6 SEM. Between group differences were assessed using 2-way repeated measures ANOVA and the post hoc Newman-Keuls test (GB-Stat v10, Dynamic Microsystems); p , 0.05 was considered statistically significant.

RNAscope fluorescent FISH Brain sectioning and FISH protocol
Rats were deeply anesthetized with pentobarbital and perfused transcardially with 400-to 500-ml ice-cold isotonic saline, followed by 4% paraformaldehyde (pH 7.4, 100 ml). The brains were removed and postfixed for 6-18 h at 4°C. Brains were sectioned (15-30 mm) and either mounted directly onto Superfrost Plus slides (Fisher Scientific) and stored at À80°C, or placed in cryoprotectant (30% ethylene glycol, 20% glycerol, and 50 mM sodium phosphate buffer, pH 7.4) at À20°C until further processing. Sections stored in cryoprotectant were briefly washed in sterile PBS before mounting on charged slides, and dried overnight. All sections for an experimental "run" were mounted and reacted on the same slide and thus experienced the same experimental conditions and solutions. Sections mounted were selected every 90, 120, 180, or 360 mm (depending on experiment) throughout the ArcN (from À1.92 to À3.60 mm from bregma). After two rinses in sterile water, sections were incubated with protease IV from the RNAscope Multiplex Fluorescent Assay kit [Advanced Cell Diagnostics (ACD); RRID:SCR_012481] for 30 min at 40°C. Sections were rinsed twice in sterile water and incubated in RNAscope catalog oligonucleotide probes (described in Table 1) for 2 h at 40°C. The rest of the FISH was done per manufacturer's instructions. When more than one probe was incubated simultaneously, different probes were in unique channels and tagged with unique fluorophores.

Mapping and imaging
Sections were imaged at 20Â on a Zeiss ApoTome2 on AxioImager with a 20 Â 0.8 PlanApo objective or 63Â, 1.4 (oil) Plan Apo objective. Filter settings for Alexa Fluor 488, Atto 550, and Atto 647 fluorophores were as follows: Alexa Fluor 488, excitation of 500 nm, emission of 535 nm; Atto 550, excitation of 545, emission of 605 nm; Atto 647, excitation of 640 nm, emission of 690 nm. Neurons were plotted with the Neurolucida software (Micro Brightfield; RRID: SCR_001775). Only cell profiles that included a nucleus and more than or equal to three fluorescent grains were counted and/or mapped. Sections were matched as closely as possible to brain levels with reference to bregma using the atlas of Paxinos and Watson (2007) or Paxinos and Watson (2013). Cells were counted and mapped unilaterally. To determine whether AT1aR neurons were close by neurons with other phenotypes, we used the Colocalization Representative experiments (left) and grouped data (right; n = 5) showing that PVN SHU9119 transiently decreases LSNA and SSNA after ArcN AngII. Decreases in MAP and HR were also observed, but these responses did not achieve statistical significance (data not shown); *p , 0.05 compared with baseline (time 0); †p , 0.05 compared with values just before PVN injections of SHU9119. Error bars represent SEM. Two camera systems were used to image the sections. In one, photographs were taken with a Hamamatsu C11440 Orca-Flash 4.0LT digital camera (resolution 2048 Â 2048 pixels) and the resulting TIFF files were first exported into Fiji (RRID:SCR_002285) and the unsharp mask filter and/or brightness/contrast were adjusted for clarity and to reflect true rendering as much as possible. Images were not otherwise altered. TIFF images were imported into Canvas v10 (ACD; RRID:SCR_014312) for labeling and final presentation. In the second system, photographs were taken with a Zeiss AxioCam 506 mono camera (2752 Â 2208 pixels) using a Apotome.2 grid illumination device (five phase translations per image). Raw image data files were processed with default settings in Zeiss ZEN 2.3 software and the resulting .czi files were adjusted for brightness/contrast for clarity and to reflect true rendering as much as possible. Images were not otherwise altered. Images were analyzed by using the positive and negative controls to set imaging processing parameters and background, respectively.

Sympathoexcitatory effects of ArcN AngII in male rats.
There were no differences in the baseline values of MAP and HR between groups (Table 2).

ArcN AngII nanoinjections increase SNA by activating AT1aR
In initial experiments, unilateral ArcN injections of AngII (1 mmol/l) only transiently increased SNA in male rats; therefore, the remaining experiments used bilateral    injections. When administered bilaterally, AngII instead produced a slowly developing and sustained increase in LSNA, SSNA, MAP, and HR ( Fig. 1). While bilateral ArcN candesartan injections had no effects, candesartan pretreatment completely prevented the responses to injections of AngII into the ArcN 10-15 min later (Fig. 1). Nevertheless, 90 min after ArcN AngII, ArcN candesartan administration failed to significantly reverse AngII-induced sympathoexcitation (n = 3; data not shown). Therefore, ArcN AngII activates AT1aR to increase LSNA and SSNA via a poorly reversible mechanism.
Role of NPY projections to the PVN and DMH in ArcN AngII-induced sympathoexcitation As expected (Cassaglia et al., 2014(Cassaglia et al., , 2016Shi et al., 2015b), after ArcN aCSF, bilateral PVN injections of the high affinity NPY Y1R antagonist, BIBO3304, produced small but significant increases in LSNA, SSNA, MAP, and HR (Fig. 2), indicating that NPY tonically suppresses SNA via PVN Y1R. However, after ArcN AngII, the increases in these variables were the same following PVN BIBO3304 as following PVN aCSF (Fig. 2), suggesting that ArcN AngII suppresses tonic PVN NPY inhibition. As previously reported in mice (Shi et al., 2017), bilateral injections of BIBO3304 into the DMH also increased LSNA, SSNA, MAP, and HR (Fig. 3). In contrast to the PVN, DMH injections of BIBO3304 60 min after ArcN AngII elicited even further increases in these variables (relative to DMH aCSF). Collectively, these data indicate that ArcN AngII increases LSNA and SSNA, in part, by inhibition of ArcN NPY neurons that tonically suppress the activity of PVN presympathetic neurons via Y1R. On the other hand, these results do not support the hypothesis that ArcN AngII similarly suppresses tonic NPY sympathoinhibition via DMH Y1R, although these data alone do not eliminate a possible action of DMH NPY at other receptor subtypes.

Role of PVN MC3/4R
In initial experiments, we tested whether PVN administration of the MC3/4R antagonist SHU9119 reversed the increases in LSNA/SSNA after ArcN AngII nanoinjections. PVN SHU9119 did decrease SNA, albeit only partially and transiently (Fig. 4), as previously noted after insulin or in pregnant rats (Ward et al., 2011;Shi et al., 2015a). The failure of PVN SHU9119 to completely reverse the effects of ArcN AngII may be due in part to the induction of MC3/ 4R (or ArcN AT1aR) signaling that is not rapidly reversed, in parallel to the inability of candesartan to reverse the effects of ArcN AngII. Therefore, we next tested SHU9119 pretreatment. Bilateral injections of SHU9119 into the PVN of rats that subsequently received ArcN aCSF did not alter SNA, MAP, or HR (Fig. 5). However, PVN SHU9119 pretreatment completely prevented the effects of subsequent injection of AngII into the ArcN (Fig. 5). Collectively, these data indicate that ArcN AngII sympathoexcitation relies on POMC projections to the PVN. The ability of PVN SHU9119 to completely block the effects of ArcN AngII, coupled with the failure of ArcN AngII to lessen tonic NPY-Y1R inhibition of the DMH, suggests that ArcN-to-DMH projections of NPY neurons, or parallel excitatory neurons, do not directly participate in the sympathoexcitatory effects of AngII. Figure 6 illustrates the injection sites for physiological experiments in male rats.

Sympathoexcitatory effects of ArcN AngII in female rats
Basal values (Table 3) Body weight was similar in rats throughout the estrous cycle. As expected, uterine weight was elevated in rats in proestrus, compared with estrous or diestrous rats. Changes in MAP, HR, LSNA, and SSNA were not detectable during the estrous cycle. However, pregnancy markedly decreased MAP and increased body weight, HR, LSNA, and SSNA.

Increases in SNA and MAP in response to ArcN AngII vary during the estrous cycle
As shown in representative tracings (Fig. 7) and the grouped data (Fig. 8), bilateral nanoinjections of AngII rapidly (within 10 min) increased MAP in all groups. The pressor response was sustained in estrous rats, but recovered toward baseline in proestrous and diestrous rats. This rapid response is somewhat distinct from that in male rats: while in some males ArcN did elicit an initial pressor response (e.g., representative experiments in Figs. 1-3), overall, the change in MAP within the first 10min was variable (4.2 6 2.9 mmHg; n = 15; no difference from baseline or females). In females as in males, ArcN AngII also elicited a slowly developing increase in LSNA, SSNA, and HR during estrus and diestrus, but not during proestrus. On the other hand, these variables did not change in cycling rats that received bilateral nanoinjections of aCSF (Fig. 8).
In separate experiments in rats in estrus (Fig. 10), blockade of PVN MC3/4R with SHU9119, 2 h after ArcN nanoinjections of AngII, decreased LSNA, HR, and MAP. Because PVN SHU9119 does not alter these variables in otherwise untreated virgin female (Shi et al., 2015a, b) and male rats (Fig. 5), we conclude that, as in males, ArcN AngII increases LSNA at least in part by activating ArcN POMC neurons that release a-MSH in the PVN.

ArcN AngII increases MAP and SNA in late pregnant rats
As in cycling rats, ArcN AngII immediately increased MAP in late pregnant rats (Fig. 11). Like rats in estrus, but not in diestrus or proestrus, the pressor response was sustained for 90 min (Fig. 11). During pregnancy, ArcN AngII also slowly increased LSNA, SSNA, and HR (Fig. 11). Figure 11B compares these responses to those from cycling female rats. The initial pressor response was similar between groups, but at the end of the 90 min observation period, the increase in MAP was greater in pregnant rats compared with diestrous or proestrous rats. The increase in LSNA (% of control) was smaller in pregnant compared with estrous rats; however, the absolute LSNA baseline was higher during pregnancy (Table 3), and LSNA increased to similar absolute levels in pregnant and estrous rats (estrus, 2.6 6 0.6 mV; P20, 4.7 6 0.3 mV; NS (not significant)). Therefore, the % change may have been smaller, because LSNA in pregnant rats started from a higher baseline and the AngII-induced increases reached similar maximal absolute levels in both groups. On the other hand, the increase in LSNA induced by ArcN AngII in pregnant rats, was greater than proestrous rats, but similar to diestrous rats (Fig. 11B). ArcN AngII increased SSNA (% control) and HR similarly during pregnancy and estrus, but more than during proestrus and diestrus (Fig. 11B).

Vasopressin contributes to the pressor response to ArcN AngII in estrous and pregnant rats
ArcN AngII produced a rapid increase in MAP in all groups of females, before significant increases in SNA or HR, suggesting a hormonal mediator may be involved. Recently, ArcN neurokinin B neurons were shown to project to and regulate vasopressin neurons in the supraoptic nucleus (Pineda et al., 2016). Therefore, we tested the role of vasopressin in the initial pressor response. In estrous rats, the AVP V1aR antagonist given iv had no effects on baseline MAP, LSNA, SSNA, and HR, and these variables Figure 11. ArcN AngII increases LSNA, SSNA, MAP, and HR in late pregnant rats. A, Grouped time course data showing that bilateral nanoinjections of AngII, but not aCSF (at arrow), increase MAP, LSNA, SSNA, and HR in late pregnant rats (P20; n = 6). B, Statistical comparison of data from late pregnant rats (n = 6) to cycling rats (data and n same as Fig. 8) obtained at baseline (time 0) as well as the maximum changes between 5 and 10 min (time 10 min), and the maximum changes between 88 and 90 min (time 90 min) after injecting AngII or aCSF; *compared with time 0; †compared with proestrus at the same time; ‡compared with diestrus at the same time; #compared with estrus at the same time. Error bars represent SEM. remained stable in rats given ArcN aCSF (data not shown). However, AVPV1x pretreatment abolished the early, but not the late increase in MAP induced by ArcN AngII (Fig. 12). After intravenous V1ax, the initial increase in SSNA was greater; however, neither the early nor late changes in LSNA or HR were significantly altered by systemic blockade of AVP V1aR (Fig. 12).
During pregnancy, intravenous V1ax significantly decreased MAP, which slowly returned to baseline over the 90 min protocol (Fig. 13). This pretreatment totally abolished the ArcN AngII pressor response; after intravenous AVP1x, the change in MAP in rats that received ArcN nanoinjections of AngII was the same as in rats that received ArcN aCSF. An initial AngII-induced decrease in SSNA was transformed into a significant increase following V1ax; however, the increase in HR was unaltered. The increase in LSNA following ArcN AngII was largely unchanged by blocking systemic AVP V1aR, although ultimately a lower level was achieved in this group. These data indicate that during pregnancy, AngII-induced vasopressin release completely mediates the pressor response induced by ArcN AngII, without a significant contribution from the parallel increases in SNA, likely because of reduced vascular responsiveness to norepinephrine. Figure 14 summarizes the ArcN and PVN nanoinjections sites for the experiments in female rats.

ArcN AT1aR expression profiles in male and female rats
Our data show that stimulation of ArcN AngII AT1aR increases SSNA, LSNA, MAP, and HR in male rats and in female rats that are pregnant or in estrus/diestrus; in proestrous rats, AngII only increases MAP. The autonomic responses depend on POMC/a-MSH activation and simultaneous suppression of tonic NPY-mediated inhibition, of preautonomic neurons in the PVN. In females, we further show that the initial pressor response is mediated by increased vasopressin secretion. To begin to understand the cellular mechanisms, we next performed a comprehensive survey of ArcN AT1aR expression in male and female rats.
Previous studies in male rats that employed autoradiography or ISH were unable to detect AT1aR expression in the ArcN (Jöhren et al., 1997;Lenkei et al., 1997). Therefore, we used RNAscope, which amplifies expression, to examine AT1aR throughout the ArcN in males. As expected, the signal was weak, compared with neighboring hypothalamic nuclei, like the DMH or VMH (Fig. 15), but clearly evident. AT1aR-positive cells were observed in all levels of the ArcN, but were particularly prominent in the mid-to-caudal segments (Fig. 15). Similarly to mice (Claflin et al., 2017), AT1aRs were expressed in NPY neurons, albeit at a lower level (9%; Fig. 15). AT1aR expression was also detectable in POMC neurons, but rarely (Fig. 15). Thus, in male rats, most AT1aR-expressing cells were neither NPY nor POMC neurons.
In females, we confirm (Jöhren et al., 1997) that ArcN AT1aR expression varies throughout the reproductive cycle, with the highest levels observed in estrus compared with diestrus; proestrus AT1aR expression was nearly undetectable, even using RNAscope (Fig. 16). As in males, AT1aR expression was observed throughout the rostral to caudal ArcN (Fig. 16). A major novel finding, however, was that ArcN AT1aR expression increased dramatically during pregnancy [estrus (n = 3) with 73 6 11 AT1aR-positive cells counted in five sections per animal; pregnancy (n = 3) with 121 6 7 AT1aR-positive cells counted in five equivalent sections; p , 0.05; Fig. 16], with the greatest increases observed in the more caudal levels (data not shown). As a result, much of the rest of our analysis was conducted in pregnant rats.

Discussion
While previous work indicated that systemic AngII activates the ArcN (Davern and Head, 2007), Sapru and colleagues were the first to demonstrate that ArcN AngII increases AP (Arakawa et al., 2011). Here, we show that the pressor response exhibits two phases, both of which are mediated by activation of AT1aR: an initial rapid phase, particularly prominent in females, is mediated by vasopressin-induced vasoconstriction, and the second phase evident in both sexes is associated with slowly developing increases in LSNA, SSNA, and HR. In females, we further show that the effects of ArcN AngII vary during the estrous cycle, with significant increases in LSNA, SSNA, HR, and MAP occurring during diestrus and estrus, but only a pressor response during proestrus, and that pregnancy markedly increases the expression of AT1aR in the ArcN with parallel substantial AngII-induced increases in SNA and MAP. In both sexes, the sympathoexcitation relied on suppression of tonic sympathoinhibitory NPY inputs, and activation of POMC/ a-MSH projections, to the PVN; DMH Y1R were not involved. Our finding that few or no NPY or POMC neurons express the AT1aR suggests that AngII elicits these effects at least in part indirectly via local interneurons. However, the lack of co-expression with kisspeptin eliminated this neuronal type as a candidate. Instead, AT1aRs were found in TH (presumed dopaminergic) neurons that are largely GABAergic. Collectively, these data suggest that ArcN AngII increases SNA and AP at least in part via TH interneurons, resulting in suppression of tonic NPY sympathoinhibitory, and stimulation of POMC sympathoexcitatory, projections to the PVN.
While a role for the ArcN in cardiovascular control is well accepted, the present results are the first to show that the ArcN-AngII-induced pressor response is mediated in part by sympathoexcitation; more specifically, bilateral (but not unilateral) ArcN AngII activation of AT1aR produced a slowly developing and sustained increase in the activity of sympathetic nerves innervating the hindlimb and the splanchnic circulation, which implicates engagement of ArcN cellular signaling mechanisms. These responses were observed in both males and females, although estrous and pregnant females exhibited the greatest increases in AP and SNA, in parallel with increased ArcN AT1aR expression. In both sexes, prior injections of AngII into the ArcN prevented the sympathoexcitatory response normally induced by blockade of PVN NPY Y1R. The failure of PVN BIBO3304 to increase SNA after ArcN AngII injections is not because of a ceiling effect, since DMH BIBO3304 triggered a further normal increase in SNA after ArcN AngII, and because other agonists that increase SNA via NPY/POMC projections to the PVN, like insulin (Cassaglia et al., 2011(Cassaglia et al., , 2016Ward et al., 2011), can produce even greater increases in SNA. Therefore, we conclude that tonically inhibitory NPY inputs to the PVN were silenced by ArcN AngII. This conclusion is consistent with a previous study in mice showing that genetic deletion of AT1aR from ArcN AgRP neurons increased NPY expression within the ArcN (Morselli et al., 2018). ArcN AngII also recruits sympathoexcitatory POMC inputs into the PVN, since SHU9119 decreased SNA after ArcN AngII in both sexes. More importantly, prior PVN SHU9119 pretreatment completely prevented the sympathoexcitatory effects of ArcN AngII in males, indicating that ArcN POMC neurons that project to the PVN are a major component of the sympathoexcitatory response to ArcN AngII. The synergism between the decreases in NPY and increases in POMC inputs into the PVN is consistent with prior studies showing that all PVN presympathetic neurons that are inhibited by NPY are activated by a-MSH (Cassaglia et al., 2014), that stimulation of PVN presympathetic neurons by a-MSH requires simultaneous withdrawal of tonic NPY inhibition (Shi et al., 2015b), that blockade of PVN MC3/4R with SHU9119 prevents the increase in SNA induced by PVN BIBO3304 (Cassaglia et al., 2014), and that experimental or physiological states that increase SNA via the ArcN, like leptin (Shi et al., 2015b) or insulin (Ward et al., 2011;Cassaglia et al., 2016) administration, pregnancy (Shi et al., 2015a), or obesity (Shi et al., 2019a(Shi et al., , 2020b, are all mediated by decreased PVN NPY Y1R and increased PVN MC3/4R activity. On the other hand, while ArcN NPY neurons via Y1R (Shi et al., 2017; and likely also POMC neurons) are capable of influencing SNA via an action in the DMH, our data do not support a role for this linkage in the sympathoexcitatory effects of ArcN AngII.
Using RNAscope, AT1aRs were found throughout the ArcN, although in males and diestrus or proestrus females, at much lower levels than in nearby hypothalamic nuclei, like the DMH or VMH. However, only a small Figure 16. AT1aR are highly expressed in the ArcN during pregnancy. A-F, Sections from a representative pregnant rat various levels throughout the ArcN (mm behind bregma shown in lower left corner) showing high expression levels of the AT1aR (red puncta) compared with rats in diestrus (G; low signal, similar to males), proestrus (H; almost undetectable as in Jöhren et al. (1997), or estrus (I; higher than diestrus/proestrus, but not as high as pregnancy). The images in the second panel are enlarged from the boxed areas directly above (A-D). Insets in G and H are enlargements from the boxed areas in each image. As in males, the limited colocalization of AT1aR (red) with NPY (white; A-H) appears in the mid-ArcN levels. AT1aR mRNA (Agtr1, red) colocalizes more often with VGAT mRNA (Slc32a1, blue) than with Vglut2 mRNA (Slc17a6, green) in both estrus (I) and pregnant rats (J). Thus, most AT1aR in the rat ArcN is expressed in VGAT, non-NPY non-POMC, neurons. Scale bars: 50 mm (A-H) and 20 mm (I, J). fraction (;10%) of AT1aRs were expressed in NPY neurons in both males and females, and a scattered few (males) or no (females) AT1aRs were found in POMC neurons. These findings raise several important questions. First, how can ArcN AngII increase SNA in male and diestrous rats, if receptor expression is low? Based on the slowly-developing nature of the response, and the failure of candesartan to reverse the response, signaling mechanisms may be engaged that amplify the initial signal, as is typical of G-protein coupling. In addition, ArcN AngII may increase the expression of its own receptor, as in other hypothalamic areas (Xue et al., 2012). In this context, it is notable that leptin can also induce the expression of its own receptor (Shi et al., 2020a) and that obesity and leptin (Hilzendeger et al., 2012), and in females progesterone (Jöhren et al., 1997;Donadio et al., 2006), can increase AT1aR expression.
The present results suggest two mechanisms by which AngII could inhibit NPY neurons. First, since AT1aR do colocalize with some NPY neurons, AngII may directly hyperpolarize or inhibit this cohort, although to our knowledge AT1aR-mediated neuronal inhibition has not been reported previously. Second, as the majority of AT1aR neurons also express VGat, and are often nearby NPY neurons, AngII-AT1aR-mediated stimulation of GABAergic neurons could locally inhibit nearby NPY neurons. On the other hand, the paucity of AT1aR expression in POMC neurons raises a second important question: how are POMC neurons activated to drive the sympathoexcitatory response? Multiple mechanisms could be involved. First, AngII-induced loss of tonic NPY sympathoinhibition within the PVN could unveil unfettered tonic POMC sympathoexcitation. Second, ArcN POMC presympathetic neurons, which are likely a small component of the entire POMC population, may be among the few POMC neurons that express AT1aR (only in males). In support, in obese males (not females), POMC presympathetic neurons become sensitized to the sympathoexcitatory effects of leptin and insulin (Shi et al., 2019a(Shi et al., , 2020b; however, simultaneously, most ArcN POMC neurons of obese males are resistant to the anorectic effects of leptin and insulin (Prior et al., 2010;Mark, 2013). Third, AT1aR-induced (direct or indirect) hyperpolarization of NPY neurons might release neighboring POMC neurons from tonic NPY inhibition (Roseberry et al., 2004;Atasoy et al., 2012), thereby increasing their activity. Finally, AngII could excite ArcN AT1aR interneurons, which in turn activate POMC neurons. Kisspeptin neurons were considered a strong candidate, since kisspeptin neurons can stimulate POMC neurons via release of glutamate (Qiu et al., 2018). However, glutamatergic AT1aR neurons were relatively few and rarely nearby POMC neurons. More importantly, coexpression of kisspeptin and AT1aR was never observed.
As previously noted in sex-steroid-treated female rats (Jöhren et al., 1997), we found instead that a significant fraction of AT1aR-positve cells also expressed TH. Most TH and AT1aR-expressing cells were localized within the dorsomedial (dm) ArcN, and previous studies in rats (Zoli et al., 1993) and mice (Zhang and van den Pol, 2015) revealed that TH neurons in the dm ArcN express and release dopamine, rather than norepinephrine or epinephrine, in addition to GABA (confirmed here). ArcN TH neurons send axons locally van den Pol, 2015, 2016) and inhibit a large fraction of nearby neurons (both TH and non-TH) via GABA release (Zhang and van den Pol, 2015). Yet, in 11 cases in mice, no electrophysiologically apparent synaptic connection between ArcN TH neurons and identified NPY neurons was observed (Zhang and van den Pol, 2016). Therefore, if AT1aR-TH neurons that release GABA inhibit NPY neurons, this must occur via a subset of TH-AT1aR neurons with direct connections to NPY neurons or via bulk diffusion of GABA to extrasynaptic sites (Belelli et al., 2009;Lee and Maguire, 2014). Collectively, current information suggests that ArcN AngII increases SNA via release of a-MSH in the PVN from ArcN POMC neurons, by disinhibition of NPY neurons both in the PVN and likely also the ArcN.
Pregnancy slowly increases basal SNA, to reach very high levels just before delivery (Brooks et al., 2020). However, the mechanism is unknown. One candidate is central AngII actions. Indirect support includes the findings that pregnancy increases plasma AngII levels in parallel with the increases in SNA, that the increase in muscle SNA in women correlates with the increase in renin, that the pressor response to intracerebroventricular AngII is larger in pregnant compared with nonpregnant rats due in part to greater activation of the sympathetic nervous system, and that intracerebroventricular administration of losartan, an AT1aR antagonist, decreases RSNA (relative to MAP) in late pregnant conscious rabbits (for review, see Brooks et al., 2020). However, the central sites at which AngII binds to AT1aR to support elevated SNA have not been identified. The present results suggest that the ArcN may be one candidate, since pregnancy markedly increased ArcN AT1aR expression. In addition, pregnancy enhanced the sympathoexcitatory and pressor responses to ArcN AngII, at least compared with proestrus, another reproductive state with high gonadal hormone levels. However, proof of this hypothesis requires evidence that blockade of ArcN AT1aR decreases SNA in late pregnant individuals.
AngII was originally shown to stimulate vasopressin secretion 50 years ago (Bonjour and Malvin, 1970) by acting centrally (Mouw et al., 1971). Since these initial observations, a large body of work indicates that AngII binds to AT1R in circumventricular organs (Brooks and Malvin, 1993;McKinley et al., 2004) as well as hypothalamic sites behind the blood-brain barrier, such as the PVN and supraoptic nucleus (Prager-Khoutorsky and Bourque, 2010) to enhance vasopressin release. The present results reveal a new site of action for AngII to stimulate vasopressin, the ArcN, since blockade of systemic vasopressin type 1 receptors prevented the initial pressor response to ArcN AngII nanoinjections in nonpregnant rats and completely prevented the AP rise during pregnancy. Our data do not explain the mechanisms by which ArcN AngII stimulates vasopressin secretion, but there are many possibilities. First, AT1aR-expressing neurons in the PVN project to the inner zone of the median eminence (ME; de Kloet et al., 2017; where vasopressin magnocellular neurons travel to the posterior pituitary and can be activated; Holmes et al., 1986); therefore, in parallel, ArcN-AT1aR activation may stimulate vasopressin magnocellular vasopressin neurons in passage in the ME. Indeed, it is well established that ArcN DA neurons project to the ME to inhibit prolactin secretion and that that the majority of ArcN TH (DA) neurons express the AT1aR (Jöhren et al., 1997;Fig. Figure 19. Hypothetical model summarizing the results and conclusions. AngII binds to AT1aR and stimulates TH GABAergic interneurons, which suppress NPY neuronal activity. Alternatively, AngII binding to AT1aR may directly inhibit NPY neurons. NPY neurons tonically inhibit PVN preautonomic neurons. Release of this NPY inhibition allows ArcN POMC neuronal activity to activate PVN MC4R on presympathetic neurons, by both disinhibition of POMC neurons in the ArcN and also by unfettered activation of PVN presympathetic neurons by a-MSH. As a result, SNA increases. Simultaneously, AT1aR activation of ArcN TH neurons that project to the PVN, ME, or pituitary may release DA to stimulate vasopressin secretion via D1 receptors. Solid arrows indicate known functional connectivity. Dotted arrows require further experimentation to establish. 18). Moreover, the ME and posterior pituitary express excitatory D1 receptors and are innervated by DA neurons (Björklund et al., 1973;Huang et al., 1992), and ArcN DA stimulates vasopressin secretion (Gerstberger et al., 1987;Rossi, 1998;Gálfi et al., 2001). Thus, it is tempting to speculate that ArcN AngII stimulates posterior pituitary vasopressin secretion via DA-D1 receptor stimulation in the ME and/or posterior pituitary. Alternatively, the rapidity of the response implicates the actions of a fast neurotransmitter, like glutamate, dopamine, or GABA, possibly via ArcN projections to magnocellular neurons in the PVN or SON. Prior studies revealed that ArcN neurons that express neurokinin B/kisspeptin, and are largely glutamatergic, project to and activate vasopressin neurons in the SON and PVN (Pineda et al., 2016;Stincic et al., 2021). However, here we show that, at least in the rat, kisspeptin neurons do not express the AT1aR. On the other hand, ArcN TH neurons project to the PVN (Zhang and van den Pol, 2016), and PVN magnocellular neurons express excitatory D1 receptors (Ran et al., 2019). Thus, AngII-induced excitation of ArcN TH neurons could stimulate vasopressin secretion via activation of D1 receptors in the PVN. Future research is required to test these and other possible hypotheses to identify the mechanisms by which ArcN AngII stimulates vasopressin release.
Pregnancy increases vasopressin secretion, such that the relationship between plasma vasopressin levels and osmolality is left-shifted, producing frank hyponatremia/ decreased plasma osmolality. Indeed, in the present study, iv injection of the vasopressin antagonist lowered AP in anesthetized, acutely prepared pregnant, but not virgin, rats, indirectly suggesting relatively elevated vasopressin levels during pregnancy. Current evidence suggests that the relative increase in vasopressin is mediated by relaxin, which synergizes with AngII, in the lamina terminalis (for review, see McKinley et al., 2004;Brunton et al., 2008;Brooks et al., 2020). Our finding that ArcN AngII likely elicits enhanced vasopressin secretion during pregnancy identifies the ArcN as a potentially additional site at which AngII stimulates vasopressin secretion in pregnant animals.
Collectively, these data suggest the following functional model by which ArcN AngII increases SNA and BP (Fig.  19): AngII binding to AT1aR directly inhibits ArcN NPY neurons and/or stimulates TH GABAergic interneurons, which suppress NPY neuronal activity. ArcN NPY neurons tonically inhibit PVN preautonomic neurons. Release of this NPY inhibition allows ArcN POMC neuronal activity to activate PVN MC4R on presympathetic neurons, by both disinhibition of POMC neurons in the ArcN and also by unfettered activation of PVN presympathetic neurons by a-MSH. We hypothesize that simultaneously, particularly in females, AT1aR activation of ArcN TH neurons that project to the ME, pituitary, or PVN release DA to stimulate vasopressin secretion.
The ArcN is a key integrative site in the control of reproduction and energy balance, which in turn are influenced by ArcN AngII-AT1aR (Steele, 1992;Donadio et al., 2006;Deng and Grobe, 2019). The present results further demonstrate that ArcN AngII actions at AT1aR increases AP through stimulation of SNA via projections to the PVN and also via vasopressin secretion. Since the original discovery by Vander and colleagues that psychosocial stress stimulates renin secretion (Clamage et al., 1976), it has become increasingly clear that central activation of AT1aR contributes to a multitude of both physical and psychological stress responses, including increases in vasopressin secretion and activation of the sympathetic nervous system (for review, see Saavedra et al., 2005Saavedra et al., , 2011Mayorov, 2011). Thus, ArcN AT1aR are well-poised to facilitate integration of these modalities with stress. Indeed, ArcN AT1aR are required for stress to inhibit prolactin secretion (Donadio et al., 2004). Moreover, because brain TH neurons, through the release of norepinephrine and DA, also mediate multiple stress responses (Anisman and Zacharko, 1986), the association of AT1aR with TH in neurons that project within and outside the ArcN further points to a local/regional integrative role with stress. This local role could be similar to that recently described for AT1aR-CRF crosstalk within the PVN to control both the HPA axis and autonomic control of AP in the context of stress (de Kloet et al., 2017;Elsaafien et al., 2021).
The initial cardiovascular event during pregnancy is profound vasodilation, which tends to lower AP and activate the RAS (Brooks et al., 2020), and as such presents a physical stress. Intriguingly, in females, ArcN AT1aR expression is dramatically increased by high progesterone in association with estrogen, such as shown here during pregnancy, as well as during estrus (Jöhren et al., 1997). Thus, as described above, increased actions of ArcN AT1aR (because of both increased AT1aR and AngII) may contribute to ArcN support of elevated SNA and BP during pregnancy (Shi et al., 2015a). The elevated AT1aR may also suppress prolactin secretion (Steele, 1992), until just before delivery when progesterone levels plunge and prolactin levels rise in preparation for delivery and lactation, when brain AT1aR are low (Speth et al., 1999). Clearly, a direct testing of such an ArcN AT1aR-TH integrative role with stress and/or pregnancy awaits further research.