Characterization of Perineuronal Nets (PNNs) in the Paraventricular Nucleus of the Hypothalamus (PVN) and their alteration in neurogenic hypertension

The paraventricular nucleus of the hypothalamus (PVN) is critical to integrate peripheral signals to modulate neurohumoral and sympathetic output. During hypertension, overactivation of the PVN contributes to neurohumoral dysregulation and increased sympathetic tone. Perineuronal nets (PNNs) are lattice-like extracellular structures that surround the soma and proximal dendrites of neurons and modulate neuronal firing and circuit plasticity. These structures are expressed throughout the brain, including the hypothalamus and PVN, and are of particular interest because changes in their deposition have been associated with multiple neurological disorders including stress, PTSD, and depression. Here, we explore changes in PNNs in the PVN during DOCA-salt hypertension, a model of neurogenic hypertension, specifically testing the hypothesis that PNNs deposition would be decreased in the PVN. 5-month old male C57BL6 mice were implanted with a s.c. deoxycorticosterone acetate (DOCA) pellet and received 0.9% NaCl in the drinking water. Blood pressure was monitored twice weekly by tail-cuff plethysmography. After 21-days of DOCA-salt (n=3) and sham control (n=4), mice were perfused with 2% paraformaldehyde to quantify PNNs using the marker N-acetylgalactosamine-binding Wisteria floribunda agglutinin (WFA). We found a trend towards less WFA+ neurons in the PVN in DOCA-salt vs control (42.29 ± 7.93 neurons/mm 2 in DOCA vs 68.97 ± 10.80 in control; p=0.1231), but observed no difference in the overall PVN area that was positive for WFA+ staining (26.53 ±0.80 % in DOCA vs 29.75 ± 2.02 % in control). Next, we quantified WFA intensity (PMID 28713865) and observed a decrease in individual PNN intensity in the PVN of DOCA-salt mice (40.08 ± 7.46 vs 67.39 ± 7.42 a.u. in control), as well as a decrease in total WFA+ intensity in the PVN (26.04 ± 3.48 vs 33.80 ± 0.29 a.u. in control; p=0.0458). We conclude that PNN deposition is decreased in the PVN during DOCA-salt hypertension, a potentially important mechanism which has been previously unexplored. PNN intensity has been previously associated with neuronal firing; therefore, ongoing studies are investigating the contribution of PNNs in the PVN to sympathetic tone and blood pressure regulation during DOCA-salt hypertension.

The paraventricular nucleus of the hypothalamus (PVN) and the perifornical area of the anterior hypothalamus (PeFAH) are crucial for the regulation of blood pressure. Hypertension is associated with increased sympathetic tone, partly resulting from over-activation of the PVN. The PeFAH, directly adjacent to the PVN, also contributes to an overall increase in sympathetic tone, though whether and how it contributes to the overall overexcitation of the PVN is not well understood. Several molecular mechanisms modulate neuronal activity including the deposition of perineuronal net (PNNs), lattice-like dynamic extracellular structures that surround the soma and proximal dendrites of neurons regulating their firing and contributing to circuit plasticity. These structures are of particular interest because changes in their deposition have been associated with multiple neurological disorders including Alzheimer’s disease for which hypertension is the highest modifiable vascular risk factor. However, whether PNNs are modulated during hypertension and whether they contribute to blood pressure regulation is not known. Therefore, this study aims to understand how hypertension modulates PNNs and thereby neuronal activity within the PVN and PeFAH. First, we created a comprehensive map of neuronal cell types surrounded by PNNs within the PVN from 3–5-month-old naïve female and male C57BL6 mice. We co-localized known neuronal populations within the PVN, including oxytocin, vasopressin, and neuronal nitric oxide synthase. (nNOS) and quantified the percentage of those neurons surrounded by PNNs. Then, we employed the DOCA-salt hypertension model to test the hypothesis that PNNs deposition is altered in both the PVN and the PeFAH during hypertension. To this end, 3-5-month old male C57BL6 mice were implanted with a subcutaneous (s.c.) deoxycorticosterone acetate (DOCA) pellet and received 0.9% NaCl in the drinking water. After 21-days of DOCA-salt, mice exhibited sustained increased blood pressure as compared to sham control and we found an increase in the number of PNNs within the PVN of DOCA-salt (n=8) vs control (n=9) (44.59 ± 5.37 PNNs/millimeter (mm) 2 in DOCA vs 23.81 ± 6.00 in control; p=0.0443). However, we observed no difference in the overall PVN area that was positive for PNNs (1.03 ± 0.11 % in DOCA vs 1.00 ± 0.03% in control, p = 0.5097). We found a significant decrease in the area that was positive for PNNs within the PeFAH (0.75 ± 0.03 % in DOCA vs 0.98 ± 0.005% in control, p = 0.0052). Taken together, we conclude that PNN number is increased in the PVN during DOCA-salt hypertension potentially activating previously ‘silenced’ neurons which could be a contributing mechanism to the overactivation of the PVN. Additionally, a decrease in the PNN coverage in the PeFAH, a highly inhibitory nucleus, may further contribute to the overactivation of the PVN by decreasing the firing of inhibitory neurons thereby disinhibiting the PVN. Ongoing studies are investigating which neuronal population has increased PNN deposition within the PVN and how PNNs contribute to sympathetic tone and blood pressure regulation during DOCA-salt hypertension. NINDS, D-SPAN/F99/K00 Award, K00 phase (K00NS130872), PI: Ismary Blanco NINDS/NIA Career Transition Award (K22 NS123507), PI: Monica M Santisteban This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

During hibernation, mammals like the 13-lined ground squirrel cycle between physiological extremes. Most of the hibernation season is spent in bouts of torpor, where body temperature, heart rate, and cerebral blood flow are all very low. However, the ground squirrels periodically enter into interbout arousals (IBAs), where physiological parameters return to non-hibernating levels. During torpor, neurons in many brain regions shrink and become electrically quiescent, but reconnect and regain activity during IBA. Previous work showed evidence of extracellular matrix (ECM) changes occurring in the hypothalamus during hibernation that could be associated with this plasticity. Here, we examined expression of a specialized ECM structure, the perineuronal net (PNN), in the forebrain of ground squirrels in torpor, IBA, and summer (non-hibernating). PNNs are known to restrict plasticity, and could be important for retaining essential connections in the brain during hibernation. We found PNNs in three regions of the hypothalamus: ventrolateral hypothalamus, paraventricular nucleus (PVN), and anterior hypothalamic area. We also found PNNs throughout the cerebral cortex, amygdala, and lateral septum. The total area covered by PNNs within the PVN was significantly higher during IBA compared to non-hibernating and torpor (P < 0.01). Additionally, the amount of PNN coverage area per Nissl-stained neuron in the PVN was significantly higher in hibernation compared to non-hibernating (P < 0.05). No other significant differences were found across seasons. The PVN is involved in food intake and homeostasis, and PNNs found here could be essential for retaining vital life functions during hibernation.

Increased glutamatergic input, particularly N-methyl-D-aspartate receptor (NMDAR) activity, in the paraventricular nucleus (PVN) of the hypothalamus is closely associated with high sympathetic outflow in essential hypertension. The molecular mechanisms underlying augmented NMDAR activity in hypertension are unclear. GluN2 subunit composition at the synaptic site critically determines NMDAR functional properties. Here, we found that evoked NMDAR-excitatory postsynaptic currents (EPSCs) of retrogradely labeled spinally projecting PVN neurons displayed a larger amplitude and shorter decay time in spontaneously hypertensive rats (SHRs) than in Wistar-Kyoto (WKY) rats. Blocking GluN2B caused a smaller decrease in NMDAR-EPSCs of PVN neurons in SHRs than in WKY rats. In contrast, GluN2A blockade resulted in a larger reduction in evoked NMDAR-EPSCs and puff NMDA-elicited currents of PVN neurons in SHRs than in WKY rats. Blocking presynaptic GluN2A, but not GluN2B, significantly reduced the frequency of miniature EPSCs and the firing activity of PVN neurons in SHRs. The mRNA and total protein levels of GluN2A and GluN2B in the PVN were greater in SHRs than in WKY rats. Furthermore, the GluN2B Ser(1480) phosphorylation level and the synaptosomal GluN2A protein level in the PVN were significantly higher in SHRs than in WKY rats. Inhibition of protein kinase CK2 normalized the GluN2B Ser(1480) phosphorylation level and the contribution of GluN2A to NMDAR-EPSCs and miniature EPSCs of PVN neurons in SHRs. Collectively, our findings suggest that CK2-mediated GluN2B phosphorylation contributes to increased synaptic GluN2A, which potentiates pre- and postsynaptic NMDAR activity and the excitability of PVN presympathetic neurons in hypertension.

Overactivity of the renin-angiotensin system, oxidative stress, and cyclooxygenases (COX) in the brain are implicated in the pathogenesis of hypertension. We previously reported that angiotensin-converting enzyme 2 (ACE2) overexpression in the brain attenuates the development of deoxycorticosterone acetate-salt hypertension, a neurogenic hypertension model with enhanced brain renin-angiotensin system and sympathetic activity. To elucidate the mechanisms involved, we investigated whether oxidative stress, mitogen-activated protein kinase signaling and cyclooxygenase (COX) activation in the brain are modulated by ACE2 in neurogenic hypertension. Deoxycorticosterone acetate-salt hypertension significantly increased expression of Nox-2 (+61±5%), Nox-4 (+50±13%), and nitrotyrosine (+89±32%) and reduced activity of the antioxidant enzymes, catalase (-29±4%) and superoxide dismutase (-31±7%), indicating increased oxidative stress in the brain of nontransgenic mice. This increased oxidative stress was attenuated in transgenic mice overexpressing ACE2 in the brain. Deoxycorticosterone acetate-salt-induced reduction of neuronal nitric oxide synthase expression (-26±7%) and phosphorylated endothelial nitric oxide synthase/total endothelial nitric oxide synthase (-30±3%), and enhanced phosphorylation of protein kinase B and extracellular signal-regulated kinase 1/2 in the paraventricular nucleus, were reversed by ACE2 overexpression. In addition, ACE2 overexpression blunted the hypertension-mediated increase in gene and protein expression of COX-1 and COX-2 in the paraventricular nucleus. Furthermore, gene silencing of either COX-1 or COX-2 in the brain, reduced microglial activation and accompanied neuroinflammation, ultimately attenuating Deoxycorticosterone acetate-salt hypertension. Together, these data provide evidence that brain ACE2 overexpression reduces oxidative stress and COX-mediated neuroinflammation, improves antioxidant and nitric oxide signaling, and thereby attenuates the development of neurogenic hypertension.

PROVIDING SUFFICIENT MILK to nursing young is necessary for infant survival and depends on periodic bolus secretion of oxytocin (OT) from the neurohypophysis during suckling. This pulsatile release maximizes myoepithelial cell contractions in the mammary gland by avoiding OT receptor desensitization (37, 44). Underlying the periodicity is the brief (4 – 6 s), synchronous, and explosive bursting of OT neurons in the supraoptic (SON) and paraventricular nuclei (PVN), the axons of which terminate at the neurohypophyseal neurohemal contact zone. These bursts (and the resultant OT release) appear with remarkably long intervals (5–20 min) despite the continual nipple stimulation provided by pups (30, 32, 38, 39), and are seldom observed during other periods of enhanced OT release. The bursting pattern maximizes frequency-dependent facilitation of OT release at neurohypophyseal terminals, and minimizes release fatigue (3–5). Understanding this periodicity remains one of the greatest challenges for OT neurobiologists. This system undergoes astonishing physiological plastic changes during pregnancy and lactation that should provide instructive clues to the process. It is now appreciated that OT

1. Arginine vasopressin (AVP) is synthesized in specific brain regions including the magnocellular and parvocellular divisions of the paraventricular nucleus (PVN). Whereas magnocellular AVP responds to osmotic stimuli and functions mainly--although not exclusively--as an antidiuretic hormone, that produced in the parvocellular region controls the hypothalamus-pituitary-adrenal (HPA) axis, in conjunction with CRF. 2. In view of the reported sex differences in control of the HPA axis, we studied if these also pertain to AVP mRNA in the PVN of ovariectomized-estrogenized female rats and male rats determined by in situ hybridization. AVP mRNA was measured in intact rats, adrenalectomized (ADX) rats and ADX receiving dexamethasone (DEX) of both sexes. 3. Computerized autoradiography showed that in both sexes, AVP mRNA levels in the parvocellular division of the PVN increased after adrenalectomy and decreased following DEX. However, the reduction by DEX was more pronounced in female rats. No changes were found for the magnocellular region. Grain counting analysis of the medial-medial (MMP) and medial-lateral (MLP) subdivisions of the parvocellular region showed that the average number of grains per cell area in the MMP region of adrenally intact female rats was higher than that in males. However, in females there was no clear-cut effect of adrenalectomy on AVP mRNA levels, although the reduction after DEX treatment was again greater than that in male rats. Frequency histograms constructed by plotting the number of cells vs the number of grains per area substantiated the enhanced glucocorticoid negative control of AVP mRNA in the MMP and MLP of female rats. 4. The results indicated a sexual dimorphism in the glucocorticoid-dependent plasticity of AVP mRNA levels in the PVN. Because AVP mRNA expression differs between sexes under basal levels, after adrenalectomy, and after DEX treatment, these plastic changes may differentially condition the response to stress. Taking into consideration that stress and AVP may play a role in neurogenic hypertension, the possibility of sexual dimorphisms in AVP control may be important to assess the role of sex hormones in stress and steroid-derived hypertension.

The hypothalamic paraventricular nucleus (PVN) integrates neuroendocrine and autonomic signals that regulate blood pressure and metabolism. Although the renin-angiotensin system (RAS) is implicated in neurogenic hypertension and obesity, cell-type-specific expression and regulation of its components within the PVN remain poorly understood. Here, we employed single-nucleus RNA sequencing (snRNA-seq) to profile the transcriptomic landscape of the PVN in male mice under baseline conditions and in models of DOCA-salt–induced hypertension and high-fat diet (HFD)-induced obesity.We identified major PVN cell types, including neurons, astrocytes, precursor oligodendrocytes, oligodendrocytes, microglia and endothelial cells, and further resolved eight transcriptionally distinct neuronal subtypes. Expression of RAS-related genes was highly cell-type specific: Agt (angiotensinogen) was enriched in astrocytes, whereas Ace (angiotensin-converting enzyme), Atp6ap2 (also known as the (pro)renin receptor [PRR]), Agtr1a (angiotensin II type 1a receptor, aka AT1aR), Lnpep (leucyl/cystinyl aminopeptidase, aka angiotensin 4 receptor [AT4R]), and the Mas1 proto-oncogene were predominantly expressed in neurons. DOCA-salt treatment increased the proportion of GABAergic and vasopressin neurons and enhanced neuronal Agt and Atp6ap2 expression, while reducing astrocytic Agt, suggesting activation of a vasoconstrictive RAS axis. HFD exposure increased excitatory and stress-responsive neuronal subtypes (glutamatergic, vasopressin, corticotropin-releasing hormone) and upregulated Atp6ap2, Agtr1b, Lnpep, and Mas1 in vasopressin neurons, while downregulating multiple RAS genes in GABAergic neurons.These findings reveal dynamic, cell-type–specific remodeling of RAS signaling in the PVN in response to hypertensive and metabolic stress, providing a transcriptomic atlas of RAS expression in the PVN and identifying potential cellular targets for therapeutic strategies addressing cardiometabolic disorders.

Microelectrophoresis of cholinergic and aminergic drugs on paraventricular neurons

See related article, pp 319–325 Chronic sympathetic hyperactivity, characteristic of the majority of patients with hypertension or heart failure, can contribute to cardiovascular morbidity and mortality via a number of actions. Pharmacological strategies to prevent these adverse effects have had variable success. β-Blockers clearly benefit patients with heart failure but have less definitive effects in patients with hypertension, whereas α1-blockers or centrally acting agents have shown mixed results, and all of these classes can cause bothersome adverse effects. Device-based approaches, such as baroreflex activation therapy and renal denervation, have been shown to lower sympathetic activity in patients with hypertension, but beneficial actions on cardiovascular outcomes have yet to be demonstrated. So, where do the central nervous system (CNS) actions of angiotensin II (Ang II), aldosterone, and, hence, Ang II type 1 (AT1) receptor and mineralocorticoid receptor (MR) blockers fit in? Experimental studies have demonstrated that both circulating Ang II and aldosterone act within the CNS to cause sympatho-excitation and raise blood pressure.1,2 Ang II stimulates AT1 receptors in nuclei of the lamina terminalis and thereby activates mainly angiotensinergic pathways to the paraventricular nucleus (PVN) and rostral ventrolateral medulla. Circulating Ang II, in addition, activates an MR-endogenous ouabain pathway. This slowly acting, neuromodulatory pathway appears responsible for most of the persistent neuronal activation in, for example, the PVN, and the progressive hypertension induced by circulating Ang II. Studies using central infusions of an aldosterone synthase inhibitor suggest that the CNS MR activation by Ang II largely depends on locally produced aldosterone rather than circulation-derived aldosterone.1 However, the progressive hypertension caused by a chronic increase in circulating aldosterone can also be prevented by specific CNS blockade of either MR or AT1 receptors,2 suggesting that both circulating aldosterone and Ang II may activate …

See related article, pages 263–270 The brain renin–angiotensin system (RAS) plays a critical role in maintaining blood pressure regulation and volume homeostasis. Components of the RAS, including angiotensinogen, renin, angiotensin (Ang)-converting enzymes, and Ang receptors, are expressed in various nuclei located between the anteroventral region of the third ventricle (AV3V) and the brain stem.1,2 Accordingly, formation of the various Ang peptides, notably Ang II and Ang(1-7) can take place in the brain, independently of the endocrine RAS, and participate in the regulation of water intake, salt appetite, cardiac baroreflex and autonomic functions. Upregulation of Ang II type 1 (AT1) receptors in these nuclei has been shown to reduce baroreflex sensitivity and increase sympathetic tone, thus contributing to the development and maintenance of hypertension and heart failure, ultimately leading to end-organ damage.3 On the other hand, treatment with ACE inhibitors and Ang receptor blockers can prevent RAS overactivity and restore a normal cardiovascular function. In addition to Ang II generated in the brain, blood-borne Ang peptides can also enter the central nervous system via the circumventricular organs (CVOs) and contribute to the regulation of blood pressure and volume homeostasis.4 The CVOs are represented by the OVLT (organum vasculosum of the lamina terminalis), the subfornical organ (SFO), the median eminence and the neurohypophysis, …

Rationale and objectivesIn conscious mammals the importance of neural control over sympathetic tone in relation to cardiovascular (CV) function is well established. Neuro‐CV dysregulation leads to increased sympathetic activity and neurogenic hypertension. The paraventricular nucleus (PVN) of the hypothalamus is currently viewed as one of the key hubs for BP control and is implicated in producing or relaying the increased sympathetic tone in hypertension. We hypothesize that it is through chronic over excitation of the glutamatergic PVN neurons that this central sympathetic drive is originating. We test this theory by examining how stimulation or lesioning of the excitatory PVN neurons in conscious mice affects blood pressure and sympathetic activity.Results &amp; ConclusionGlutamatergic PVN neurons were unilaterally transduced with channelrhodopsin2 using an adeno‐associated virus (CamKII‐ChR2‐eYFP‐AAV2) in wildtype mice (N = 7). We then measured the effect of acute stimulation of excitatory PVN neurons selectively on resting blood pressure (DSI telemetry) in conscious mice. Stimulation of the PVN glutamatergic population resulted in an immediate frequency dependent (2 Hz, 10Hz, 20Hz) increase in blood pressure from baseline by ~10 mmHg at 20 Hz stimulation (p&lt;0.001). Additionally, in vGlut2‐cre mice glutamatergic neurons of the PVN were bilaterally lesioned utilizing a cre‐dependent caspase (Dio‐Caspase‐AAV). We then recorded resting blood pressure and urinary norepinephrine levels in conscious mice before and after DOCA‐Salt hypertension. Quantified glutamatergic lesions of the PVN (39.3%, p&lt;0.05) resulted in a blunted rise in BP when animals went through the DOCA‐salt protocol (p&lt;0.05 at 7 day time point, N = 8). Finally, we measured nor‐epinephrine levels as a quantification of sympathetic activity between the lesion group and widtype group and observed a significant reduction after DOCA‐salt induced hypertension in the lesioned animals (p&lt;0.05). These experiments demonstrate that stimulation of the PVN is sufficient to cause autonomic dysfunction. Additionally, the dysautonomia observed in neurogenic hypertension is due, in part, to over activation in the PVN.Support or Funding InformationHL093178‐07, HL135635‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neurogenic hypertension is characterized by heightened sympathetic drive to the cardiovascular system from key brain regions, including the hypothalamic paraventricular nucleus (PVN). Dysregulation of Angiotensin II (AngII) during hypertension is linked to elevated synthesis of pro‐inflammatory cytokines in the PVN, suggesting a role for AngII in modulating sympathoexcitation through neuroinflammation. We have recently shown a functional interaction between AngII type‐I receptor (AT1r) and Toll‐like receptor 4 (TLR4) in mediating AngII‐dependent microglial activation and associated oxidative stress within the PVN. Our goal was to elucidate whether a similar AngII‐dependent AT1r‐TLR4 interaction contributes to the maintenance of neurogenic hypertension. Spontaneously hypertensive rats (SHRLos) were treated by oral gavage with an AT1r‐blocker (Losartan; 20mg/kg/day; 4 weeks) or vehicle (SHR), using age‐matched Wistar Kyoto rats (WKY) as a control. Tail‐cuff blood pressure measurements were taken weekly over the 4‐week treatment period. Protein expression of TLR4 and microglia‐marker IBA1 was determined by immunofluorescence (IF) within the PVN. IF skeletal analysis was used to index microglia morphology in the PVN. Real time PCR was performed for TLR4 mRNA in isolated microglia from the hypothalamus. After 4 weeks, SHR mean arterial pressure (MAP) was significantly greater than WKY controls (155±2 vs 100±3), whereas SHRLos MAP showed no significant difference from WKY (104±2 mmHg). IF analysis of PVN slices showed a 41% increase in TLR4 protein density and a 37% increase in TLR4 intensity in SHR compared with WKY, while AT1r‐blockade preserved normative TLR4 protein expression in SHRLos. Co‐localization of TLR4 with IBA1 was significantly elevated in SHR as compared to WKY (66±0.9 vs 1.3±0.2AU) and TLR4 gene expression in isolated microglia was increased 2.56‐fold in SHR compared to WKY. Microglia skeletal analysis showed a significant decrease in end‐points/frame by 21% and a 34% decrease in total branch length/frame in SHR PVN compared to WKY, indicative of increased microglial activation. SHRLos end‐point and branch‐length values were similar to WKY, demonstrating significant attenuation of microglial activation. In summary, blocking AT1r during hypertension ameliorated the elevated TLR4 protein expression seen in SHR, and normalized the extent of both microglial recruitment and activation. These findings implicate a crosstalk interaction between AngII and TLR4, via AT1r, in mediating AngII‐dependent microglial activation in hypertension. This data suggests that the characteristic sympathoexcitation of neurogenic hypertension may result, at least in part, from low‐grade chronic inflammation due to AngII‐AT1r‐TLR4 crosstalk promoting microglial activation during hypertension.Support or Funding InformationThis work was funded by AHA14SDG20400015 to VCB.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In recent years a considerable amount of experimental evidence has suggested that forebrain structures are involved in the pathogenesis of high arterial pressure (AP). However, little is known about the location and function of these supramedullary structures in the hypertensive process. This report reviews a series of studies done to identify the location and to determine the contribution of some forebrain structures to both the development and maintenance of the elevated AP following selective aortic baroreceptor deafferentation (ABD). In the first series of studies, it was demonstrated that the elevated AP resulting from ABD was associated with increased metabolic activity in several forebrain structures: the paraventricular nucleus of the hypothalamus (PVH), supraoptic nucleus, nucleus circularis, median preoptic nucleus, subfornical organ (SFO), and central nucleus of the amygdala. In the second series, bilateral electrolytic lesions of the PVH were shown to prevent the development of and (or) reverse the elevated AP after ABD. Similarly, bilateral microinjections of the neurotoxin kainic acid into the PVH were shown to reverse the increased AP after ABD. In the final series, electrolytic lesions of the SFO were shown to attenuate the rise in AP after ABD and (or) to reduce the elevated AP to a level that remained above control values. Taken together, these data suggest that the PVH and SFO are components of a neuronal circuit involved in the hypertensive process following ABD, and that the SFO likely exerts its effect through the PVH.

Endoplasmic reticulum (ER) stress was previously reported to contribute to neurogenic hypertension while neuronal angiotensin-converting enzyme type 2 (ACE2) overexpression blunts the disease. To assess which brain regions are important for ACE2 beneficial effects and the contribution of ER stress to neurogenic hypertension, we first used transgenic mice harboring a floxed neuronal hACE2 transgene (SL) and tested the impact of hACE2 knockdown in the subfornical organ (SFO) and paraventricular nucleus (PVN) on deoxycorticosterone acetate (DOCA)-salt hypertension. SL and nontransgenic (NT) mice underwent DOCA-salt or sham treatment while infected with an adenoassociated virus (AAV) encoding Cre recombinase (AAV-Cre) or a control virus (AAV-green fluorescent protein) to the SFO or PVN. DOCA-salt-induced hypertension was reduced in SL mice, with hACE2 overexpression in the brain. This reduction was only partially blunted by knockdown of hACE2 in the SFO or PVN, suggesting that both regions are involved but not essential for ACE2 regulation of blood pressure (BP). DOCA-salt treatment did not increase the protein levels of ER stress and autophagy markers in NT mice, despite a significant increase in BP. In addition, these markers were not affected by hACE2 overexpression in the brain, despite a significant reduction of hypertension in SL mice. To further assess the role of ER stress in neurogenic hypertension, NT mice were infused intracerebroventricularlly with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, during DOCA-salt treatment. However, TUDCA infusion failed to blunt the development of hypertension in NT mice. Our data suggest that brain ER stress does not contribute to DOCA-salt hypertension and that ACE2 blunts neurogenic hypertension independently of ER stress.