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Research Paper

Angiotensin type 1A receptor expression in C1 neurons of the rostral ventrolateral medulla contributes to the development of angiotensin-dependent hypertension Nikola Jancovski1 , David A. Carter1 , Angela A. Connelly1 , Elyse Stevens1 , Jaspreet K. Bassi1 , Clement Menuet1 and Andrew M. Allen1,2 1

Experimental Physiology

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Department of Physiology, University of Melbourne, Melbourne, Victoria 3010, Australia Florey Institute of Neuroscience and Mental Health, University of Melbourne, Melbourne, Victoria 3010, Australia

New Findings r What is the central question of this study? This study addresses the mechanism by which deletion of angiotensin II type 1A receptors from catecholaminergic neurons reduces angiotensin-dependent hypertension, as well as the identity of the cells involved. r What is the main finding and its importance? Deletion of angiotensin II type 1A receptors from catecholaminergic neurons results in reduced sympathetic nerve activation and fluid and electrolyte retention during angiotensin infusion. The C1 neurons of the rostral ventrolateral medulla are involved in the later phase of the hypertension. We demonstrate that at least two different populations of catecholaminergic neurons are involved in the sympathetic nerve activation required for the full development of angiotensin-dependent hypertension.

Chronic low-dose systemic infusion of angiotensin II induces hypertension via activation of the angiotensin II type 1A receptor (AT1A R). Previously, we have demonstrated that expression of the AT1A R on catecholaminergic neurons is necessary for the full development of angiotensin-dependent hypertension. In the present study, we examined the mechanism by which selective deletion of the AT1A R from these cells affects the development of hypertension. We also tested the hypothesis that AT1A Rs expressed by catecholaminergic C1 neurons in the rostral ventrolateral medulla play an important role in angiotensin-induced hypertension. A Cre-lox approach was used to delete the AT1A R from all catecholaminergic cells or from C1 neurons selectively. Subcutaneous administration of angiotensin II induced hypertension in all mice, with delayed onset and reduced maximal response in the global AT1A R catecholaminergic knockout mice. The AT1A R catecholaminergic knockout mice had decreased renal fluid and electrolyte retention and urinary noradrenaline excretion. The blood pressure response was reduced only during the second week of angiotensin II infusion in the mice with selective C1 AT1A R deletion, demonstrating that AT1A R expression by C1 neurons plays a moderate role in angiotensin-induced hypertension. The difference in the time course of development of hypertension between the mice with global AT1A R knockout from catecholaminergic cells and the mice with C1 AT1A R deletion suggests that other catecholaminergic neurons are important. (Received 16 July 2014; accepted after revision 15 September 2014; first published online 18 September 2014) Corresponding author A. M. Allen: Department of Physiology, University of Melbourne, Victoria 3010, Australia. Email: [email protected]

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DOI: 10.1113/expphysiol.2014.082073

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Introduction Essential hypertension is a multifactorial disorder that is a major risk factor for the development of multiple cardiovascular diseases, thus leading to increased morbidity and mortality (Roger et al. 2012). The homeostatic regulation of blood pressure (BP) involves complex interactions among multiple neuronal, hormonal and renal regulatory systems (Guyton, 1991), and despite the extensive research efforts, the exact mechanisms responsible for generation and maintenance of high BP remain unclear. The renin–angiotensin system (RAS) plays a pivotal role in the regulation of BP, and it is well established that angiotensin II (Ang II), which is the major effector hormone of the RAS, acts through several targets, including the brain, kidney, vasculature, adrenal glands and heart (Bader et al. 2001; Bader, 2010). As it is well established that RAS overactivity occurs in some forms of hypertension, chronic infusion of Ang II has become a commonly used model of experimental hypertension. Hypertension induced by long-term infusion of Ang II involves the brain (Marvar et al. 2010). Acting through the blood–brain barrier (BBB)-deficient circumventricular organs, circulating Ang II promotes thirst, salt appetite, secretion of vasopressin and corticotrophin-releasing hormone and modulation of autonomic nervous system activity (Allen et al. 2009; Cuadra et al. 2010). In addition to the blood-borne Ang II, the existence of a brain RAS and centrally-generated Ang II that affects cardiovascular homeostasis is well established (O’Callaghan et al. 2013). Studies in humans and experimental animals have postulated a link between elevated sympathetic nervous system activity (SNA) and hypertension (Schlaich et al. 2004; Malpas, 2010), and there is evidence suggesting that the hypertensive response induced by Ang II infusion is, at least in part, sympathetically mediated (Luft et al. 1989; Jancovski et al. 2013). The involvement of SNA in the pathogenesis of hypertension is highlighted by the efficacy of catheter-based renal sympathetic denervation as a treatment strategy for patients with drug-resistant hypertension (Schlaich et al. 2009; Mahfoud et al. 2013). Recently, we reported that in a mouse with deletion of the Ang II type 1A receptor (AT1A R) from all catecholaminergic cells (CAT-KO), baseline physiological characteristics are normal, but there is a delay in the onset of angiotensin-dependent hypertension and reduced maximal increase in BP (Jancovski et al. 2013). In addition, the BP response was associated with decreased SNA and reduced production of reactive oxygen species in the rostral ventrolateral medulla (RVLM), a nucleus that contains sympathoexcitatory, catecholaminergic C1 neurons (Abbott et al. 2009; Chen et al. 2010). The AT1A R is expressed on C1 neurons (Chen et al. 2012), and exogenous microinjection of Ang II in the RVLM

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produces a dose-dependent sympathetically mediated increase in BP (Allen et al. 1988), while blockade of these receptors decreases SNA and BP in animal models of hypertension (Allen, 2001; Allen et al. 2009). The pressor actions of exogenous Ang II depend on AT1A R expression on C1 neurons (Chen et al. 2012; Jancovski et al. 2013). However, several other catecholaminergic cell groups also express the AT1A R, including the caudal ventrolateral medulla, nucleus of the solitary tract, locus coeruleus and the sympathetic ganglia. Hence, in the present study we sought to gain further knowledge regarding the role of AT1A Rs on catecholaminergic cells and sympathetic activation during Ang II-induced hypertension. Specifically, we tested the hypothesis that deletion of the AT1A R from C1 neurons of the RVLM would reduce angiotensin-dependent hypertension to the same extent as that observed in CAT-KO mice. In addition, we employed metabolic cages and urine analysis to test whether deletion of the AT1A R from all catecholaminergic cells affects urinary catecholamine concentrations as well as fluid and electrolyte excretion during Ang II-dependent hypertension. The hypotheses were tested in transgenic mice using the Cre recombinase (Cre)-loxP approach (Gaveriaux-Ruff & Kieffer, 2007; Bouabe & Okkenhaug, 2013) either to delete AT1A R expression from tyrosine hydroxylase (TH)-expressing cells (CAT-KO; Jancovski et al. 2013) or to delete AT1A R expression only from C1 neurons in the RVLM by microinjection of a lentivirus expressing Cre under the control of a phox2 binding site promoter (Chen et al. 2012). Methods Ethical approval

The experimental procedures were approved by a University of Melbourne Animal Ethics and Experimentation Committee, and mice were handled in accordance with the guidelines of National Health and Medical Research Council of Australia ‘Australian code for the care and use of animals for scientific purposes (8th edition)’. Animals

Experiments were performed on adult (3- to 6-month-old) male mice that were housed in standard cages on a 12 h–12 h light–dark cycle, with lights on between 06.00 and 18.00 h. Animals had free access to standard chow and water at all times, unless otherwise stated. The AT1A Rfl/fl mice were originally obtained from Drs S. Gurley and T. Coffman at Duke University (Gurley et al. 2011). These were used as control animals and also for the lentiviral delivery of Cre (n = 24).  C 2014 The Authors. Experimental Physiology  C 2014 The Physiological Society

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Angiotensin II type 1A receptors on C1 neurons

The TH-IRES-Cre+/− mice were obtained from Dr T. Ebendal’s laboratory via the European mutant mouse consortium (Lindeberg et al. 2004). Both strains were on a C57Bl/6 background and were bred in the Biological Resources Facility of the University of Melbourne, Australia. Crossing AT1A Rfl/fl ;TH-IRES-Cre+/− fl/fl and AT1A R ;TH-IRES-Cre−/− mice produced fl/fl AT1A R ;TH-IRES-Cre+/− (CAT-KO; n = 22) and AT1A Rfl/fl ;TH-IRES-Cre−/− (WT; n = 25) littermates that were used for experiments. Prior to experimentation, all mice were genotyped using standard PCR protocols (Chen et al. 2012; Jancovski et al. 2013). Cre reporter mice (ROSA26-eYFP; n = 3) were obtained from Professor F. Costantini, Columbia University, New York, NY, USA(Srinivas et al. 2001) and maintained at the Florey Neurosciences Institute, University of Melbourne. Measurement of blood pressure and heart rate

Systolic BP (sBP) was measured in awake mice by a non-invasive computerized tail-cuff system (SC1000 Blood Pressure Analysis System; Hatteras Instruments, Cary, NC, USA). Mice were placed in a metal animal holder on a heated specimen platform (35–37 °C), and the BP was detected by a sensor system that tracks changes in the tail blood flow. Four preliminary and eight experimental measurements were taken per mouse at each time point, and the values for sBP and heart rate (HR) were obtained by averaging the eight experimental measurements. In order to minimize stress-related variations in BP, mice were trained for 3 weeks prior to the beginning of the experiment. Baseline sBP measurements were then made on five consecutive days in CAT-KO (n = 8), WT (n = 8) and AT1A Rfl/fl mice (n = 8) or on 4 days in Lv-PRSx8-Cre (n = 8) and Lv-PRSx8-GFP mice (n = 8) before the implantation of osmotic minipumps. The cardiovascular parameters were recorded every day after the implantation until day 16 in CAT-KO, WT and AT1A Rfl/fl mice or until day 15 in Lv-PRSx8-Cre and Lv-PRSx8-GFP mice. Osmotic minipump implantation

Osmotic minipumps (model 1004, ALZET, Cupertino, CA, USA) prepared to deliver Ang II (500 ng kg−1 min−1 ) were implanted subcutaneously in isoflurane-anaesthetized mice (2.0–2.5% via a nose cone as required for loss of pedal withdrawal reflexes) as previously reported (Jancovski et al. 2013). Measurement of fluid and electrolyte homeostasis

Mice (CAT-KO, n = 9; WT, n = 11) were housed individually in metabolic cages (Techniplast, Varese, Lombardy, Italy) with free access to a gel diet (Nutra-gel; Bio Serv, Frenchtown, NJ, USA; Gurley et al. 2011). The gel diet was prepared by mixing 10 g of powder and

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3 ml of hot water. The amount of water in the diet was taken into account for determination of the total amount of water intake. Mice were allowed to acclimate for 24 h prior to the commencement of any measurements. The 24 h water intake and urine excretion were then recorded at baseline and at days 6–7 and 12–13 after commencement of Ang II infusion. The urine volume was measured and urine stored at −80 °C for future analysis. Urinary sodium and potassium were measured by flame photometry (Model 410c; Sherwood Scientific Ltd, Cambridge, UK) and osmolality with a vapour pressure osmometer (Vapro 5220; Wescor, Logan, UT, USA). Urine catecholamine levels

The concentrations of adrenaline and noradrenaline in the urine were measured using a commercially available enzyme-linked immunosorbent assay [2-CAT (A-N) Research ELISA; LDN, Nordhorn, Germany]. Sodium metabisulfite (final concentration 4 mmol l−1 ) and EDTA (final concentration 1 mmol l−1 ) were added to the urine collector to prevent catecholamine degradation. The assay was performed in accordance with the instructions provided by the manufacturer. The daily excretion of adrenaline and noradrenaline was calculated from the urine concentration and 24 h urinary volume measurements. Microinjection of lentiviruses into the RVLM

In a separate cohort of AT1A Rfl/fl mice, lentiviral vectors were microinjected into the RVLM to induce cell and regional selective deletion of the AT1A R in adult animals. Mice were initially anaesthetized by inhalation of isoflurane (4% in a small closed container) and then injected with ketamine (80 mg kg−1 ) and xylazine (10 mg kg−1 ) I.P. If any further anaesthetic was required, ketamine only was injected. After determination of a surgical plane of anaesthesia, as evidenced by loss of pedal withdrawal to a noxious stimulus, they were placed in a stereotaxic apparatus (Benchmark Stereotaxic Instruments; myNeuroLab, St Louis, MO, USA), with the skull surface horizontal between lambda and bregma. Before the surgery, mice were injected with an analgesic [carprofen, 0.5 mg (100 g)−1 I.P.; Norbrook, Tullamarine, Vic., Australia]. The occipital bone overlying the cerebellum was partly removed, and bilateral microinjections of a lentivirus expressing either Cre-recombinase (Lv-PRSx8-Cre, n = 8) or green fluorescent protein (GFP; Lv-PRSx8-GFP, n = 8) were made into the RVLM (Chen et al. 2012). Lentivirus was microinjected (100 nl) using pressurized nitrogen gas delivered through a pneumatic pressure device (model SYS-PV820;WPI Inc., Sarasota, FL, USA) using the following co-ordinates: 1.7 and 1.9 mm caudal from

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lambda; 1.2 mm lateral to the mid-line; and 5.3 mm below the brain surface. Following surgery, the incision was sutured and the mice were placed on a heat blanket. They were allowed 4 weeks for recovery and viral transgene expression, during which time no differences in food and water intake or behaviour were observed compared with unoperated mice. Blood pressure was then measured using the tail-cuff procedure described above (see ‘Measurement of blood pressure and heart rate’) and osmotic minipumps were implanted. Immunohistochemistry

At the completion of the BP recording protocol in Lv-PRSx8-Cre or Lv-PRSx8-GFPmice, general anaesthesia was induced by inhalation of isoflurane and then injection of ketamine/xylazine as described in the previous subsection. Mice were perfused through the heart with saline (0.9% NaCl) followed by a solution of 4% formaldehyde in 0.1 mol l−1 sodium phosphate buffer. The brains were removed, post-fixed in formaldehyde for 1–2 h, then cryoprotected in 20% sucrose overnight at 4 °C. Brainstem sections were cut on a cryostat (Microm International, Walldorf, Germany) and serial, coronal 40 μm sections were collected in four series and stored in cryoprotectant [30% (w/v) sucrose, 30% (v/v) ethylene glycol and 1% (w/v) polyvinyl-pyrrolidone in 50 mM phosphate buffer, pH 7.2] until immunohistochemical staining (Llewellyn-Smith et al. 2003; Chen et al. 2010). Brains were processed for immunohistochemical double staining of TH and GFP, or TH and the macrophage marker CD68, which is a reliable marker for the injection site (Card et al. 2006). The following primary antibodies were used: rat anti-mouse CD68(1:1000 dilution; AbD Serotec, Raleigh, NC, USA), rabbit anti-TH(1:1000 dilution; Chemicon International, Temecula, CA, USA) and chicken anti-GFP(1:1000 dilution; Chemicon International). The secondary antibodies were Alexa Fluor 488-conjugated goat anti-chicken (1:200 dilution), Cy3-conjugated donkey anti-rabbit (1:200 dilution), Dylight 549 goat anti-rat (1:200 dilution; AbD Serotec) or Alexa Fluor 488 goat anti-rabbit (1:200 dilution; Vector Laboratories, Burlingame, CA, USA) as required. Immunofluorescence was imaged using an Axio Imager D.1 microscope connected to an AxioCam MRc5 digital camera (both from Zeiss, Australia) and mapping performed with reference to a mouse brain atlas (Paxinos & Franklin, 2004). Verification of lentivirus-induced Cre expression

Bilateral microinjections of Lv-PRSx8-Cre were made into the RVLM of ROSA26-eYFP (n = 3) mice using the approach described above (see ‘Microinjection of lentiviruses into the RVLM’). Approximately 4–5 weeks after the viral injection, mice were deeply anaesthetized,

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perfused with 4% paraformaldehyde and brains prepared for immunohistochemical localization of TH and GFP as described in the previous subsection. Statistical analysis

The grouped data are presented as means±SEM. The only exception is Fig. 1A, where the group data are presented as means ± SD. All longitudinal measurements were analysed by two-way repeated-measures ANOVA comparing genotype, time and their interaction, using SigmaPlot version 11.2 (Systat Software Inc., San Jose, CA, USA). Post hoc analysis employed the Holm–Sidak or Bonferroni method. Values were considered significant at P < 0.05. As all animals in both experimental groups were examined by one operator during the same period, using the same BP measuring equipment and protocol, we further combined all groups into one statistical analysis using two-way repeated-measures ANOVA as described above. To normalize for the small difference in basal sBP measures, data were analysed as the change in sBP. These data are included in Fig. 6 and enable comparison between the different control groups and between the CAT-KO and the Lv-PRSx8-Cre groups. Results Basal cardiovascular, fluid and electrolyte parameters

Basal sBP and HR were measured by a non-invasive tail-cuff method. As expected, initial recordings show high variability between consecutive measurements in the same animal within the same recording session (Fig. 1A). Overall, all mice had elevated sBP values during these early recordings. Over the period of 3 weeks of daily training, by the same operator, this variability and elevated sBP declined in all strains such that average sBP values were not different from those obtained with telemetry (compare Fig. 1 and reference Jancovski et al. 2013). During the subsequent period of baseline recording, we observed no difference in sBP between CAT-KO, WT and AT1A Rfl/fl mice (Fig. 1B). During the baseline period, 24 h measures of food and water intake, urinary volume and electrolyte concentration, and catecholamine levels were not different between CAT-KO and WT mice (Fig. 2A–F). Likewise, there were no differences in body weight between WT and CAT-KO mice throughout development (Fig. 2G). Angiotensin-dependent hypertension

Subcutaneous infusion of Ang II (500 ng kg−1 min−1 ) gradually increased sBP, with a significant elevation above baseline at day 4 of infusion in WT, day 5 in AT1A Rfl/fl  C 2014 The Authors. Experimental Physiology  C 2014 The Physiological Society

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and day 6 in CAT-KO mice (Fig. 1A). The sBP was significantly elevated in WT and AT1A Rfl/fl compared with CAT-KO mice from day 4 or 6, respectively, until the end of the experimental period. The CAT-KO mice showed a significantly lower maximal increase in sBP (CAT-KO 129 ± 5.6 mmHg; P < 0.001) compared with the other control strains (WT 141 ± 5.2 mmHg; AT1A Rfl/fl 143 ± 4.2 mmHg; Fig. 1A and C). These results are very similar to those reported previously in telemetered CAT-KO and WT mice (Jancovski et al. 2013).

of the Ang II infusion. Whilst there was a significant increase in food intake at day 12 compared with baseline in both groups, no difference in food intake was observed at any time point between the WT and CAT-KO mice (Fig. 3A). Both WT and CAT-KO mice showed a significant increase in water intake during the Ang II infusion, with no difference observed between groups (Fig. 3B). Urine excretion significantly increased during the Ang II infusion in both groups, with the CAT-KO showing a significant increase in urine excretion at day 12 when compared with the WT mice (Fig. 3C). Sodium excretion also increased in both groups during the Ang II infusion, with a significant increase observed at day 12 in the CAT-KO compared with the WT mice (Fig. 3D). Urinary K+ excretion increased during the infusion but was not different between the WT

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Figure 1. Attenuation of angiotensin II (Ang II)-dependent hypertension following angiotensin II type 1A (AT1A R) deletion from catecholaminergic neurons A, grouped data showing the systolic blood pressure (sBP) before (training and baseline) and after [days (D)1–16] subcutaneous infusion of Ang II (500 ng kg−1 min−1 ) in wild-type (WT; n = 8), AT1AR deletion from catecholamine knockout cells (CAT-KO; n = 8) and AT1A Rfl/fl mice (n = 8). Significant differences between WT/AT1A Rfl/fl and CAT-KO are indicated as follows: ∗ P < 0.05, † P < 0.01 or ‡ P < 0.001; # indicates the first time point of increase above baseline (P < 0.05) and, with the exception of day 7 of CAT-KO, a significant increase at all subsequent points. Data are presented as means ± SD. B and C, bar graphs comparing basal systolic blood pressure and the maximal change in sBP in response to chronic Ang II infusion in WT, AT1A Rfl/fl and CAT-KO mice; ∗∗ P < 0.01. Data are shown as means + SEM.

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compared with CAT-KO mice [WT 9.3 ± 3.4 μg (24 h)−1 ; CAT-KO 1.7 ± 0.4 μg (24 h)−1 ].

and CAT-KO mice at any time point (Fig. 3E). Whilst there was a trend towards an increase in the WT mice, urine osmolality increased during the Ang II infusion only in the CAT-KO mice, and at day 12 a significant difference was observed between the groups (Fig. 3F).

Lentivirus-induced Cre expression into the RVLM of ROSA26-eYFP mice

Lv-PRSx8-Cre was microinjected into the RVLM of ROSA26-eYFP mice to verify the selectivity of the PRSx8 promoter and the level of Cre recombinase expression. Microinjections of a lentivirus expressing Cre recombinase under the control of a phox2 binding site promoter in the RVLM induced Cre expression primarily in C1 catecholaminergic neurons as evidenced by robust eYFP fluorescence that was colocalized with immunoreactivity for tyrosine hydroxylase (Fig. 5A and B).

Urinary catecholamine excretion

In a previous report, we used spectral analysis of the BP and HR signals derived from telemetric recordings to infer that there was a large increase in sympathetic activity in WT mice during Ang II infusion, whilst CAT-KO mice showed no change (Jancovski et al. 2013). In the present cohort, we measured urinary catecholamine concentrations. Adrenaline excretion showed a small increase with Ang II infusion, but was not different at any time point between mouse strains (Fig. 4A). Urinary excretion of noradrenaline progressively increased in WT mice during the Ang II infusion, whilst there was no change in levels measured in the CAT-KO mice (Fig. 4B). The urinary noradrenaline excretion in WT mice was significantly increased at day 12–13 post-Ang II infusion

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the BP response to subcutaneous Ang II infusion (500 ng kg−1 min−1 ) was measured. There was no difference in basal sBP between the groups, and the data are presented as change in sBP (Fig. 6A). The initial sBP increase observed during days 1–6 of infusion was similar in the Lv-PRSx8-Cre and Lv-PRSx8-GFP mice (Fig. 6A). A divergence in the sBP response was observed from day 8 onwards (Fig. 6A). The maximal increase in sBP was significantly less in the Crecompared with GFP-injected mice (sBP: Lv-PRSx8-Cre 23 ± 2.1 mmHg; Lv-PRSx8-GFP 31 ± 1.7 mmHg). No consistent changes were observed in the HR response after Ang II infusion. Postmortem analysis demonstrated that the lentiviral injections were within the region of the C1 neurons of the RVLM (Fig. 6B and C and 7A), and 75–80% of C1 neurons located within 300 μm of the caudal pole of the facial nucleus were transduced (Fig. 7B). More caudal TH-positive neurons were not targeted by the injections and were not transduced (Fig. 7B). There was no difference in basal BP between any experimental group and its control(s), and data were normalized. Statistical comparison was made with data from all five groups; however, for clarity these data are presented in two separate graphs (Fig. 6D and

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E). With the exception of an early spike in sBP at day 9 in Lv-PRSx8-GFP mice, all three control groups showed a similar timecourse of BP increase, although the maximal increase was greater in the WT and AT1A Rfl/fl groups compared with the Lv-PRSx8-GFP at days 13 and 15 (sBP: Lv-PRSx8-GFP 31 ± 1.7 mmHg; WT 40 ± 1.4 mmHg; AT1A Rfl/fl 39 ± 2.1 mmHg). Sustained increases in sBP above baseline were observed in all control groups and Lv-PRSx8-Cre starting at day 5. The sBP increase in the Lv-PRSx8-Cre group diverged from the control groups at day 11 and then remained significantly reduced for the remainder of the recording period. In the CAT-KO mice, the initial increase in sBP above baseline occurred first at day 6 but did not remain consistently above baseline until day 10 (Fig. 6E). This increase in sBP in the CAT-KO mice was significantly reduced compared with all control groups from day 6 onwards and never reached the same maximal level.On days 9, 10 and 13, the sBP of the CAT-KO mice was significantly lower than that of the Lv-PRSx8-Cre mice (Fig. 6E). The CAT-KO mice did show a similar maximal BP to the Lv-PRSx8-Cre mice (sBP: CAT-KO 19 ± 3 mmHg; Lv-PRSx8-Cre 22 ± 2 mmHg) occurring at day 14.

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Figure 3. Altered fluid and electrolyte homeostasis in response to chronic Ang II infusion following AT1A R deletion from catecholaminergic cells Bar graphs showing 24 h measurement of food intake (A), water intake (B), urine output (C), urinary sodium excretion (D), urinary potassium excretion (E) and urine osmolality (F) at baseline and 6 and 12 days after commencing systemic Ang II infusion in WT (n = 11) and CAT-KO mice (n = 9). ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001. Data are presented as means ± SEM.

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Discussion Continuous infusion of a low dose of Ang II results in increased BP that develops over several days and is associated with increased SNA and fluid and electrolyte retention by the kidney. In this study, we demonstrate that the onset of hypertension in response to Ang II infusion is delayed and the maximal increase reduced in CAT-KO mice. This receptor deletion dramatically reduces the Ang II-induced increase in SNA and ameliorates the fluid and electrolyte retention. Part of the reduced response to Ang II infusion involves AT1A R expression by the catecholaminergic C1 neurons of the RVLM in the brainstem. However, these neurons appear to play a role only in the later phase of the Ang II-induced hypertension. Using two different approaches, frequency analysis of the BP and HR signal from telemetered mice (Jancovski et al. 2013) and measurement of urinary noradrenaline excretion, we have now demonstrated activation of SNA during the course of Ang II-induced hypertension in WT mice and an attenuated response in CAT-KO mice. These data are consistent with the view that Ang II activates SNA, with subsequent modification of renal function (Grisk & Rettig, 2004). Reduced sodium and fluid retention by the kidney in the CAT-KO mice suggest altered neural modulation of renal function that most probably contributes to the reduced BP response in these animals. Long-term regulation of BP involves multiple mechanisms that maintain a balance between BP and the urinary concentration of salt and water, and this balance can be affected by several factors, including renal SNA (Dampney et al. 2005). Increased renal SNA is associated with kidney RAS overactivity, altered fluid and electrolyte handling, as well as changes in renal blood flow (DiBona, 2000). Interestingly, elevated sympathetic tone to other organs may also be important in Ang II-induced hypertension

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(DiBona, 2002). In a rat model of Ang II–salt hypertension, organ-specific changes of SNA maintain the hypertensive response that is determined by elevations in splanchnic SNA, decreased renal SNA or no changes in the skeletal muscle SNA (Osborn & Fink, 2010). The same group has shown that chronic infusion of a low dose of Ang II in combination with a high-salt diet markedly increases splanchnic SNA, which in turn increases the venomotor tone and contributes to hypertension (King et al. 2007). Unlike rabbits (Guild et al. 2012) and rats (Yoshimoto et al. 2010), in conscious mice, long-term direct recordings of SNA are still challenging owing to technical issues and problems associated with the maintenance of a stable signal over a long time. Thus, information regarding altered SNA to specific tissues and organs, as well as its contribution to hypertension, is not presently available. Experimental work in rabbits has shown that Ang II-induced hypertension is characterized by an initial baroreflex-mediated decrease in the renal SNA (Barrett et al. 2005). However, chronic low-dose infusion of Ang II, either alone or in combination with a high-salt diet, subsequently produces chronic elevations of the renal SNA (Guild et al. 2012; Moretti et al. 2012). We observed decreased sBP in the CAT-KO mice throughout the period of Ang II infusion, supporting the observation of Northcott et al. (2010) that neuronal effects of Ang II even contribute to the early phase of the hypertension. When the AT1A R was deleted only from C1 neurons, a decrease in Ang II-induced hypertension occurred only in the later phase of the hypertension. We suggest that the early interaction may involve catecholaminergic neurons of the peripheral nervous system, whilst the central effects of Ang II occur later. We clearly demonstrate that part, albeit a relatively moderate contribution, of the later phase of the BP response to Ang II infusion involves the catecholaminergic C1 neurons of the RVLM. The RVLM C1 neurons

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Figure 4. Urinary catecholamine excretion before and during Ang II infusion in WT and CAT-KO mice A and B, grouped data showing 24 h urinary excretion of adrenaline and noradrenaline at baseline and 6 and 12 days after commencing Ang II infusion in WT (n = 5) and CAT-KO mice (n = 5). ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001. Data are presented as means ± SEM.

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Angiotensin II type 1A receptors on C1 neurons

have monosynaptic connections with sympathetic preganglionic neurons in the intermediolateral cell column of the spinal cord and provide the essential excitatory drive for the generation of sympathetic vasomotor tone (Allen et al. 2009). We have previously reported that the pressor actions of exogenous Ang II depend on AT1A R expression on C1 neurons (Chen et al. 2012). Expression of the AT1A R in the RVLM is significantly increased in response to chronic systemic infusion of Ang II (Braga, 2011), and the increased production of reactive oxygen species induced in the RVLM by Ang II infusion is reduced in the CAT-KO mice. Thus, we conclude that RVLM C1 neurons do play a role in the pathogenesis of Ang II-dependent hypertension. It is generally accepted that hypertension induced by systemic infusion of Ang II involves a central component, but the pathways and mechanisms involved remain unclear. Angiotensin II is a lipophobic octapeptide that is unable to cross the BBB under normal circumstances. The most prevalent hypothesis is that systemic Ang II will act on the BBB-deficient circumventricular organs to stimulate polysynaptic pathways involved in the generation of SNA (O’Callaghan et al. 2013). Most circumventricular organs express AT1A Rs (Mendelsohn et al. 1984; Allen et al. 1998), and it has been demonstrated that the lamina terminalis is essential for the hypertensive response to Ang II infusion (Vieira et al. 2010; Young et al. 2012). It is thought that central Ang II, acting via AT1A Rs, can modulate the gain of this pathway at multiple sites. We have confirmed that Ang II-dependent hypertension elicits production of reactive oxygen species via the subfornical organ–paraventricular nucleus–RVLM pathway and that formation of reactive oxygen species at the RVLM is reduced in CAT-KO mice; the RVLM being the first point in this pathway where the AT1A R is expressed on catecholaminergic neurons (Jancovski et al. 2013). Our studies do not shed light on the source of Ang II that acts on these central synapses. A recent study has reported that increased circulating levels of Ang II, during pathophysiological conditions such as hypertension, can compromise the permeability of the BBB in the brainstem and hypothalamic nuclei (Biancardi et al. 2014). Consequently, disruptionof the BBB allows the circulating Ang II to gain access to its receptors in cardiovascular nuclei located behind the BBB, including the RVLM, and contribute to the developmentof hypertension. A major challenge has been to delineate the individual contribution of AT1A R activation in different cell types to the development and maintenance of high BP. Experimental studies have demonstrated an important role of kidney AT1A R in the pathogenesis of hypertension (Crowley et al. 2006) and that cell-selective deletion of AT1A Rs from the proximal tubules in the kidney decreases basal BP and reduces Ang II-induced hypertension (Gurley

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et al. 2011; Li et al. 2011). Cre-lox approaches to delete AT1A Rs selectively either from vascular smooth muscle cells in large vessels (Sparks et al. 2011) or from T lymphocytes (Zhang et al. 2012) do not influence baseline BP or Ang II-induced hypertension. Interestingly, adoptive transfer of T cells from AT1A R knockout mice (AT1A R-KO) into B- and T-lymphocyte-deficient mice (RAG1−/− ) does implicate AT1A R expression by T lymphocytes in the development of hypertension, as the hypertensive response to Ang II is blunted (Guzik et al. 2007). We now clearly demonstrate that AT1A Rs expressed by catecholaminergic cells also participate in the development of Ang II-dependent hypertension (Jancovski et al. 2013). Does the difference in the magnitude of Ang II-induced hypertension in the CAT-KO mice and in mice with conditional deletion of AT1A R from C1 neurons in the RVLM reflect an involvement of AT1A Rs expressed by non-C1 catecholaminergic cells or the fact that transduction of C1 neurons was not complete? We used replication-deficient lentiviral vectors under the control of an artificial phox2 binding site promoter, PRSx8, to induce transgene expression in C1 neurons in the RVLM

A RVLM

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Figure 5. Microinjection of Lv-PRSx8-Cre into the rostral ventrolateral medulla (RVLM) of an ROSA26-eYFP mouse A, a schematic coronal section depicting the RVLM (modified from the atlas of Paxinos & Franklin, 2004), showing the region of the RVLM that is photographed in higher power in B. Distance from bregma, −6.72 mm. B, representative photomicrographs of fluorescence immunohistochemistry from anROSA26-eYFP mouse (n = 3) injected with Lv-PRSx8-Cre showing tyrosine hydroxylase (TH; red), green fluorescent protein (GFP; green) and the merged images. The outlined areas are shown at higher power in the images below. The scale bar represents 200 μm.

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and reliably achieved transduction of 80% of neurons in the rostral part of the RVLM. Consistent with the results from this study, previous work has also reported that despite its high selectivity, the PRSx8 promoter does not target all of the C1 neurons in the RVLM and might induce some transgene expression in a small number of non-C1 cells (Abbott et al. 2009, 2012; Chen et al. 2010, 2012). It is apparent that 100% transduction of C1 neurons is unlikely using this approach. It is therefore conceivable that the small portion of neurons still expressing AT1A Rs could affect the response to angiotensin

infusion. However, this is not likely, because employing the same approach, we have previously demonstrated that cell-selective deletion of AT1A Rs from C1 neurons of the RVLM dramatically attenuated the response to exogenous Ang II in anaesthetized mice (Chen et al. 2012). It is also apparent that the C1 neurons are affecting the later stage of the angiotensin-dependent hypertension, whilst in the CAT-KO mice divergence in the BP occurs early in the infusion. Potentially, the effect exerted through the AT1A R on C1 neurons is only a transient effect, although it is unclear from these results whether the lack of difference

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Figure 6. Region- and cell-type-selective deletion of AT1A Rs from catecholaminergic C1 neurons in the RVLM of adult animals reduces the blood pressure response to systemic Ang II infusion A, change in systolic BP (sBP), recorded using a non-invasive tail-cuff system, following systemic infusion of Ang II (500 ng kg−1 min−1 ) in AT1A Rfl/fl mice that had previously been injected in the RVLM with a lentivirus expressing GFP (Lv-PRSx8-GFP; n = 8) or Cre-recombinase (Lv-PRSx8-Cre; n = 8) under the control of a phox2 promoter. ∗ P < 0.05; # indicates the first time point of increase above baseline (P < 0.05) and a significant increase at all subsequent points. B, photomicrographs of coronal sections of medulla at the level of the RVLM from an Lv-PRSx8-GFP-injected mouse, showing fluorescence immunohistochemistry for tyrosine hydroxylase (TH), green fluorescent protein (GFP) and the merged images. C, photomicrographs of coronal sections of medulla at the level of the RVLM from an Lv-PRSx8-Cre-injected mouse, showing fluorescence immunohistochemistry for TH, the macrophage marker (CD68) and the merged images. The scale bar represents 200 μm. D, line graph showing the change in sBP following Ang II infusion in the three different control groups; # indicates the first time point of increase above baseline (P < 0.05) and a significant increase at all subsequent points, and † P < 0.05 comparing Lv-PRSx8-GFP with either WT or AT Rfl/fl . E, line graph showing the change in sBP 1A following Ang II infusion in the two experimental groups; # indicates the first time point of increase above baseline (P < 0.05) and, with the exception of day 7 of CAT-KO, a significant increase at all subsequent points, and ∗ P < 0.05 indicates differences between the groups. Data are means ± SEM.

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(Allen et al. 1998). Moreover, AT1A Rs are expressed by catecholaminergic sympathetic postganglionic neurons and adrenal medullary chromaffin cells (Castren et al. 1987; Marley et al. 1989).Our data suggest little role for the adrenal medulla, because whilst urinary adrenaline excretion showed a small, but significant, change over the course of Ang II infusion, it was not different between groups. It is well established that Ang II activates sympathetic postganglionic neurons directly (Lewis & Reit, 1965), and Ma and co-workers have reported that in mice Ang II increases renal SNA by direct stimulation of neurons in the sympathetic ganglia (Ma et al. 2004). We suggest that AT1A Rs on postganglionic neurons might be an important target for Ang II-induced hypertension either via a direct effect on the cell excitability or via presynaptic modulation of neurotransmitter release (Story & Ziogas, 1987).

between the mice with selective deletion of the AT1A R from C1 neurons and control animals after day 13 is due to physiological compensation or to technical issues associated with the infusion of Ang II. Our conclusion is that another group of catecholaminergic neurons is affected early in the angiotensin infusion and that the angiotensinergic stimulation of C1 neurons contributes only to the later phase. The question remains regarding which other AT1A R-expressing catecholaminergic neurons are responsible for the majority of the reduced response to Ang II observed in the CAT-KO mice. There is a strong overlap between the distribution of neurons expressing the AT1A R and those expressing TH in the CNS. These regions include the RLVM, caudal ventrolateral medulla, nucleus of the solitary tract, area postrema, locus coeruleus and some hypothalamic nuclei

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Figure 7. Distribution of GFP-expressing neurons in the rostral ventrolateral medulla A, schematic coronal sections of the mouse medulla oblongata (modified from the atlas of Paxinos & Franklin, 2004) showing the centres of the microinjections of Lv-PRSx8-GFP in all AT1A Rfl/fl mice (n = 8). B, histograms showing grouped data depicting the number of neurons expressing TH, the number of these that express TH and GFP, or that express GFP but not TH at different levels of the RVLM. Data are shown as means ± SEM. Abbreviations: 7 N, facial motor nucleus; Amb, nucleus ambiguus; and RVLM, rostral ventrolateral medulla.

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In summary, our findings highlight the importance of AT1A R expression by catecholaminergic cells, including the C1 neurons in the RVLM, in the development and maintenance of Ang II-dependent hypertension. Whilst transgenic mice lacking AT1A Rs, either on all catecholaminergic cells or exclusively on C1 neurons in the RVLM, have normal baseline BP, they show an altered BP response to Ang II infusion. Although WT mice show increased urine noradrenaline concentrations and fluid and electrolyte retention, these are attenuated in the CAT-KO mice. The evidence suggests that Ang II acting via AT1A Rs on catecholaminergic cells to increase renal sympathetic nerve activity is an important mechanism in Ang II-induced hypertension.

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Mendelsohn FA, Quirion R, Saavedra JM, Aguilera G & Catt KJ (1984). Autoradiographic localization of angiotensin II receptors in rat brain. Proc Natl Acad Sci U S A 81, 1575–1579. Moretti JL, Burke SL, Davern PJ, Evans RG, Lambert GW & Head GA (2012). Renal sympathetic activation from long-term low-dose angiotensin II infusion in rabbits. J Hypertens 30, 551–560. Northcott CA, Watts S, Chen Y, Morris M, Chen A & Haywood JR (2010). Adenoviral inhibition of AT1A receptors in the paraventricular nucleus inhibits acute increases in mean arterial blood pressure in the rat. Am J Physiol Regul Integr Comp Physiol 299, R1202–R1211. O’Callaghan EL, Choong YT, Jancovski N & Allen AM (2013). Central angiotensinergic mechanisms associated with hypertension. Auton Neurosci 175, 85–92. Osborn JW & Fink GD (2010). Region-specific changes in sympathetic nerve activity in angiotensin II–salt hypertension in the rat. Exp Physiol 95, 61–68. Paxinos G & Franklin KBJ (2004). The Mouse Brain in Stereotaxic Coordinates. Elsevier Academic Press, San Diego, CA, USA. Roger VL, Go AS, Lloyd-Jones DM, Benjamin EJ, Berry JD, Borden WB, Bravata DM, Dai S, Ford ES, Fox CS, Fullerton HJ, Gillespie C, Hailpern SM, Heit JA, Howard VJ, Kissela BM, Kittner SJ, Lackland DT, Lichtman JH, Lisabeth LD, Makuc DM, Marcus GM, Marelli A, Matchar DB, Moy CS, Mozaffarian D, Mussolino ME, Nichol G, Paynter NP, Soliman EZ, Sorlie PD, Sotoodehnia N, Turan TN, Virani SS, Wong ND, Woo D & Turner MB (2012). Executive summary: heart disease and stroke statistics—2012 update: a report from the American Heart Association. Circulation 125, 188–197. Schlaich MP, Lambert E, Kaye DM, Krozowski Z, Campbell DJ, Lambert G, Hastings J, Aggarwal A & Esler MD (2004). Sympathetic augmentation in hypertension: role of nerve firing, norepinephrine reuptake, and angiotensin neuromodulation. Hypertension 43, 169–175. Schlaich MP, Sobotka PA, Krum H, Lambert E & Esler MD (2009). Renal sympathetic-nerve ablation for uncontrolled hypertension. New Engl J Med 361, 932–934. Sparks MA, Parsons KK, Stegbauer J, Gurley SB, Vivekanandan-Giri A, Fortner CN, Snouwaert J, Raasch EW, Griffiths RC, Haystead TA, Le TH, Pennathur S, Koller B & Coffman TM (2011). Angiotensin II type 1A receptors in vascular smooth muscle cells do not influence aortic remodeling in hypertension. Hypertension 57, 577–585. Srinivas S, Watanabe T, Lin CS, William CM, Tanabe Y, Jessell TM & Costantini F (2001). Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol 1, 4. Story DF & Ziogas J (1987). Interaction of angiotensin with noradrenergic neuroeffector transmission. Trends Pharmacol Sci 8, 269–271. Vieira AA, Nahey DB & Collister JP (2010). Role of the organum vasculosum of the lamina terminalis for the chronic cardiovascular effects produced by endogenous and exogenous ANG II in conscious rats. Am J Physiol Regul Integr Comp Physiol 299, R1564–R1571.

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Additional information Competing interests None declared.

Exp Physiol 99.12 (2014) pp 1597–1610

Author contributions N.J. and A.M.A. conceived and designed the research. N.J., J.K.B., D.A.C., A.A.C. and E.S. collected the data. N.J., C.M. and A.M.A. interpreted the data. N.J., C.M. and A.M.A. prepared the manuscript. All authors approved the final version for publication. Funding This work was supported by the Australian National Health and Medical Research Council (#1029396, #1007451) and the Australian Research Council (DP1094301). Acknowledgements The authors gratefully acknowledge the assistance of John Brandi and Alan Lambell with the urine analysis.

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Angiotensin type 1A receptor expression in C1 neurons of the rostral ventrolateral medulla contributes to the development of angiotensin-dependent hypertension.

Chronic low-dose systemic infusion of angiotensin II induces hypertension via activation of the angiotensin II type 1A receptor (AT1AR). Previously, w...
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The rostral ventrolateral medulla (RVLM) plays a key role in cardiovascular regulation. It has been reported that tonically active glutamatergic input to the RVLM is increased in hypertensive rats, whereas angiotensin-converting enzyme 2 (ACE2) in th

ANG II modulates both slow and rapid baroreflex responses of barosensitive bulbospinal neurons in the rabbit rostral ventrolateral medulla.
This study investigated the effects of ANG II on slow and rapid baroreflex responses of barosensitive bulbospinal neurons in the rostral ventrolateral medulla (RVLM) in urethane-anesthetized rabbits to determine whether the sympathetic baroreflex mod

Immunohistochemical evidence of tissue hypoxia and astrogliosis in the rostral ventrolateral medulla of spontaneously hypertensive rats.
Increased activity of the sympathetic nervous system has been highlighted as a key factor that contributes to the development and maintenance of arterial hypertension. However, the factors that precipitate sustained increases in sympathetic activity