Meningeal interleukin-17-producing T cells mediate cognitive impairment in a mouse model of salt-sensitive hypertension.

Hypertension (HTN), a disease afflicting over one billion individuals worldwide, is a leading cause of cognitive impairment, the mechanisms of which remain poorly understood. In the present study, in a mouse model of HTN, we find that the neurovascular and cognitive dysfunction depends on interleukin (IL)-17, a cytokine elevated in individuals with HTN. However, neither circulating IL-17 nor brain angiotensin signaling can account for the dysfunction. Rather, IL-17 produced by T cells in the dura mater is the mediator released in the cerebrospinal fluid and activating IL-17 receptors on border-associated macrophages (BAMs). Accordingly, depleting BAMs, deleting IL-17 receptor A in brain macrophages or suppressing meningeal T cells rescues cognitive function without attenuating blood pressure elevation, circulating IL-17 or brain angiotensin signaling. Our data unveil a critical role of meningeal T cells and macrophage IL-17 signaling in the neurovascular and cognitive dysfunction in a mouse model of HTN.

Early life high fructose exposure disrupts microglia function and impedes neurodevelopment.

Despite the success of fructose as a low-cost food additive, recent epidemiological evidence suggests that high fructose consumption by pregnant mothers or during adolescence is associated with disrupted neurodevelopment 1-7 . An essential step in appropriate mammalian neurodevelopment is the synaptic pruning and elimination of newly-formed neurons by microglia, the central nervous system's (CNS) resident professional phagocyte 8-10 . Whether early life high fructose consumption affects microglia function and if this directly impacts neurodevelopment remains unknown. Here, we show that both offspring born to dams fed a high fructose diet and neonates exposed to high fructose exhibit decreased microglial density, increased uncleared apoptotic cells, and decreased synaptic pruning in vivo . Importantly, deletion of the high affinity fructose transporter SLC2A5 (GLUT5) in neonates completely reversed microglia dysfunction, suggesting that high fructose directly affects neonatal development. Mechanistically, we found that high fructose treatment of both mouse and human microglia suppresses synaptic pruning and phagocytosis capacity which is fully reversed in GLUT5-deficient microglia. Using a combination of in vivo and in vitro nuclear magnetic resonance- and mass spectrometry-based fructose tracing, we found that high fructose drives significant GLUT5-dependent fructose uptake and catabolism, rewiring microglia metabolism towards a hypo-phagocytic state. Importantly, mice exposed to high fructose as neonates exhibited cognitive defects and developed anxiety-like behavior which were rescued in GLUT5-deficient animals. Our findings provide a mechanistic explanation for the epidemiological observation that early life high fructose exposure is associated with increased prevalence of adolescent anxiety disorders.

Expanding the horizon of research into the pathogenesis of the white matter diseases: Proceedings of the 2021 Annual Workshop of the Albert Research Institute for White Matter and Cognition.

White matter pathologies are critically involved in the etiology of vascular cognitive impairment-dementia (VCID), Alzheimer's disease (AD), and Alzheimer's disease and related diseases (ADRD), and therefore need to be considered a treatable target ( Roseborough A, Hachinski V, Whitehead S. White matter degeneration - a treatable target? Roseborough et al. JAMA Neurol [Internet]. 2020 Apr 27;77(7):793-4, [1] . To help address this often-missed area of research, several workshops have been sponsored by the Leo and Anne Albert Charitable Trust since 2015, resulting in the incorporation of "The Albert Research Institute for White Matter and Cognition" in 2020. The first annual "Institute" meeting was held virtually on March 3-4, 2021. The Institute provides a forum and workspace for communication and support of the advancement of white matter science and research to better understand the evolution and prevention of dementia. It serves as a platform for young investigator development, to introduce new data and debate biology mechanisms and new ideas, and to encourage and support new research collaborations and directions to clarify how white matter changes, with other genetic and health risk factors, contribute to cognitive impairment. Similar to previous Albert Trust-sponsored workshops (Barone et al. in J Transl Med 14:1-14, [2]; Sorond et al. in GeroScience 42:81-96, [3]), established expert investigators were identified and invited to present. Opportunities to attend and present were also extended by invitation to talented research fellows and younger scientists. Also, updates on institute-funded research collaborations were provided and discussed. The summary that follows is a synopsis of topics and discussion covered in the workshop.

Endothelial S1P1 Signaling Counteracts Infarct Expansion in Ischemic Stroke.

RATIONALE: Cerebrovascular function is critical for brain health, and endogenous vascular protective pathways may provide therapeutic targets for neurological disorders. S1P (Sphingosine 1-phosphate) signaling coordinates vascular functions in other organs, and S1P1 (S1P receptor-1) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P1 also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P1 modulation in stroke. OBJECTIVE: To address roles and mechanisms of engagement of endothelial cell S1P1 in the naive and ischemic brain and its potential as a target for cerebrovascular therapy. METHODS AND RESULTS: Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P1 in the mouse brain. With an S1P1 signaling reporter, we reveal that abluminal polarization shields S1P1 from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar endothelial cells. S1P1 signaling sustains hallmark endothelial functions in the naive brain and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by endothelial cell-selective deficiency in S1P production, export, or the S1P1 receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P1 provides modest protection only in the context of reperfusion. In the ischemic brain, endothelial cell S1P1 supports blood-brain barrier function, microvascular patency, and the rerouting of blood to hypoperfused brain tissue through collateral anastomoses. Boosting these functions by supplemental pharmacological engagement of the endothelial receptor pool with a blood-brain barrier penetrating S1P1-selective agonist can further reduce cortical infarct expansion in a therapeutically relevant time frame and independent of reperfusion. CONCLUSIONS: This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with blood-brain barrier-penetrating S1P1 agonists.

Endothelium-Macrophage Crosstalk Mediates Blood-Brain Barrier Dysfunction in Hypertension.

Hypertension is a leading cause of stroke and dementia, effects attributed to disrupting delivery of blood flow to the brain. Hypertension also alters the blood-brain barrier (BBB), a critical component of brain health. Although endothelial cells are ultimately responsible for the BBB, the development and maintenance of the barrier properties depend on the interaction with other vascular-associated cells. However, it remains unclear if BBB disruption in hypertension requires cooperative interaction with other cells. Perivascular macrophages (PVM), innate immune cells closely associated with cerebral microvessels, have emerged as major contributors to neurovascular dysfunction. Using 2-photon microscopy in vivo and electron microscopy in a mouse model of Ang II (angiotensin II) hypertension, we found that the vascular segments most susceptible to increased BBB permeability are arterioles and venules >10 µm and not capillaries. Brain macrophage depletion with clodronate attenuates, but does not abolish, the increased BBB permeability in these arterioles where PVM are located. Deletion of AT1R (Ang II type-1 receptors) in PVM using bone marrow chimeras partially attenuated the BBB dysfunction through the free radical-producing enzyme Nox2. In contrast, downregulation of AT1R in cerebral endothelial cells using a viral gene transfer-based approach prevented the BBB disruption completely. The results indicate that while endothelial AT1R, mainly in arterioles and venules, initiate the BBB disruption in hypertension, PVM are required for the full expression of the dysfunction. The findings unveil a previously unappreciated contribution of resident brain macrophages to increased BBB permeability of hypertension and identify PVM as a putative therapeutic target in diseases associated with BBB dysfunction.

Publisher Correction: Dietary salt promotes cognitive impairment through tau phosphorylation.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Dietary salt promotes cognitive impairment through tau phosphorylation.

Dietary habits and vascular risk factors promote both Alzheimer's disease and cognitive impairment caused by vascular factors1-3. Furthermore, accumulation of hyperphosphorylated tau, a microtubule-associated protein and a hallmark of Alzheimer's pathology4, is also linked to vascular cognitive impairment5,6. In mice, a salt-rich diet leads to cognitive dysfunction associated with a nitric oxide deficit in cerebral endothelial cells and cerebral hypoperfusion7. Here we report that dietary salt induces hyperphosphorylation of tau followed by cognitive dysfunction in mice, and that these effects are prevented by restoring endothelial nitric oxide production. The nitric oxide deficiency reduces neuronal calpain nitrosylation and results in enzyme activation, which, in turn, leads to tau phosphorylation by activating cyclin-dependent kinase 5. Salt-induced cognitive impairment is not observed in tau-null mice or in mice treated with anti-tau antibodies, despite persistent cerebral hypoperfusion and neurovascular dysfunction. These findings identify a causal link between dietary salt, endothelial dysfunction and tau pathology, independent of haemodynamic insufficiency. Avoidance of excessive salt intake and maintenance of vascular health may help to stave off the vascular and neurodegenerative pathologies that underlie dementia in the elderly.

Dietary salt promotes neurovascular and cognitive dysfunction through a gut-initiated TH17 response.

A diet rich in salt is linked to an increased risk of cerebrovascular diseases and dementia, but it remains unclear how dietary salt harms the brain. We report that, in mice, excess dietary salt suppresses resting cerebral blood flow and endothelial function, leading to cognitive impairment. The effect depends on expansion of TH17 cells in the small intestine, resulting in a marked increase in plasma interleukin-17 (IL-17). Circulating IL-17, in turn, promotes endothelial dysfunction and cognitive impairment by the Rho kinase-dependent inhibitory phosphorylation of endothelial nitric oxide synthase and reduced nitric oxide production in cerebral endothelial cells. The findings reveal a new gut-brain axis linking dietary habits to cognitive impairment through a gut-initiated adaptive immune response compromising brain function via circulating IL-17. Thus, the TH17 cell-IL-17 pathway is a putative target to counter the deleterious brain effects induced by dietary salt and other diseases associated with TH17 polarization.

Brain perivascular macrophages: characterization and functional roles in health and disease.

Perivascular macrophages (PVM) are a distinct population of resident brain macrophages characterized by a close association with the cerebral vasculature. PVM migrate from the yolk sac into the brain early in development and, like microglia, are likely to be a self-renewing cell population that, in the normal state, is not replenished by circulating monocytes. Increasing evidence implicates PVM in several disease processes, ranging from brain infections and immune activation to regulation of the hypothalamic-adrenal axis and neurovascular-neurocognitive dysfunction in the setting of hypertension, Alzheimer disease pathology, or obesity. These effects involve crosstalk between PVM and cerebral endothelial cells, interaction with circulating immune cells, and/or production of reactive oxygen species. Overall, the available evidence supports the idea that PVM are a key component of the brain-resident immune system with broad implications for the pathogenesis of major brain diseases. A better understanding of the biology and pathobiology of PVM may lead to new insights and therapeutic strategies for a wide variety of brain diseases.

Size-selective opening of the blood-brain barrier by targeting endothelial sphingosine 1-phosphate receptor 1.

The vasculature of the central nervous system (CNS) forms a selective barrier termed the blood-brain barrier (BBB). Disruption of the BBB may contribute to various CNS diseases. Conversely, the intact BBB restricts efficient penetration of CNS-targeted drugs. Here, we report the BBB-regulatory role of endothelial sphingosine 1-phosphate (S1P) receptor-1, a G protein-coupled receptor known to promote the barrier function in peripheral vessels. Endothelial-specific S1pr1 knockout mice (S1pr1iECKO ) showed BBB breach for small-molecular-mass fluorescence tracers (<3 kDa), but not larger tracers (>10 kDa). Chronic BBB leakiness was associated with cognitive impairment, as assessed by the novel object recognition test, but not signs of brain inflammation. Brain microvessels of S1pr1iECKO mice showed altered subcellular distribution of tight junctional proteins. Pharmacological inhibition of S1P1 function led to transient BBB breach. These data suggest that brain endothelial S1P1 maintain the BBB by regulating the proper localization of tight junction proteins and raise the possibility that endothelial S1P1 inhibition may be a strategy for transient BBB opening and delivery of small molecules into the CNS.

Perivascular macrophages mediate the neurovascular and cognitive dysfunction associated with hypertension.

Hypertension is a leading risk factor for dementia, but the mechanisms underlying its damaging effects on the brain are poorly understood. Due to a lack of energy reserves, the brain relies on continuous delivery of blood flow to its active regions in accordance with their dynamic metabolic needs. Hypertension disrupts these vital regulatory mechanisms, leading to the neuronal dysfunction and damage underlying cognitive impairment. Elucidating the cellular bases of these impairments is essential for developing new therapies. Perivascular macrophages (PVMs) represent a distinct population of resident brain macrophages that serves key homeostatic roles but also has the potential to generate large amounts of reactive oxygen species (ROS). Here, we report that PVMs are critical in driving the alterations in neurovascular regulation and attendant cognitive impairment in mouse models of hypertension. This effect was mediated by an increase in blood-brain barrier permeability that allowed angiotensin II to enter the perivascular space and activate angiotensin type 1 receptors in PVMs, leading to production of ROS through the superoxide-producing enzyme NOX2. These findings unveil a pathogenic role of PVMs in the neurovascular and cognitive dysfunction associated with hypertension and identify these cells as a putative therapeutic target for diseases associated with cerebrovascular oxidative stress.

Spatio-temporal profile, phenotypic diversity, and fate of recruited monocytes into the post-ischemic brain.

BACKGROUND: A key feature of the inflammatory response after cerebral ischemia is the brain infiltration of blood monocytes. There are two main monocyte subsets in the mouse blood: CCR2+Ly6Chi "inflammatory" monocytes involved in acute inflammation, and CX3CR1+Ly6Clo "patrolling" monocytes, which may play a role in repair processes. We hypothesized that CCR2+Ly6Chi inflammatory monocytes are recruited in the early phase after ischemia and transdifferentiate into CX3CR1+Ly6Clo "repair" macrophages in the brain. METHODS: CX3CR1GFP/+CCR2RFP/+ bone marrow (BM) chimeric mice underwent transient middle cerebral artery occlusion (MCAo). Mice were sacrificed from 1 to 28 days later to phenotype and map subsets of infiltrating monocytes/macrophages (Mo/MΦ) in the brain over time. Flow cytometry analysis 3 and 14 days after MCAo in CCR2-/- mice, which exhibit deficient monocyte recruitment after inflammation, and NR4A1-/- BM chimeric mice, which lack circulating CX3CR1+Ly6Clo monocytes, was also performed. RESULTS: Brain mapping of CX3CR1GFP/+ and CCR2RFP/+ cells 3 days after MCAo showed absence of CX3CR1GFP/+ Mo/MΦ but accumulation of CCR2RFP/+ Mo/MΦ throughout the ischemic territory. On the other hand, CX3CR1+ cells accumulated 14 days after MCAo at the border of the infarct core where CCR2RFP/+ accrued. Whereas the amoeboid morphology of CCR2RFP/+ Mo/MΦ remained unchanged over time, CX3CR1GFP/+ cells exhibited three distinct phenotypes: amoeboid cells with retracted processes, ramified cells, and perivascular elongated cells. CX3CR1GFP/+ cells were positive for the Mo/MΦ marker Iba1 and phenotypically distinct from endothelial cells, smooth muscle cells, pericytes, neurons, astrocytes, or oligodendrocytes. Because accumulation of CX3CR1+Ly6Clo Mo/MΦ was absent in the brains of CCR2 deficient mice, which exhibit deficiency in CCR2+Ly6Chi Mo/MΦ recruitment, but not in NR4A1-/- chimeric mice, which lack of circulating CX3CR1+Ly6Clo monocytes, our data suggest a local transition of CCR2+Ly6Chi Mo/MΦ into CX3CR1+Ly6Clo Mo/MΦ phenotype. CONCLUSIONS: CX3CR1+Ly6Clo arise in the brain parenchyma from CCR2+Ly6Chi Mo/MΦ rather than being de novo recruited from the blood. These findings provide new insights into the trafficking and phenotypic diversity of monocyte subtypes in the post-ischemic brain.

Commensal microbiota affects ischemic stroke outcome by regulating intestinal γδ T cells.

Commensal gut bacteria impact the host immune system and can influence disease processes in several organs, including the brain. However, it remains unclear whether the microbiota has an impact on the outcome of acute brain injury. Here we show that antibiotic-induced alterations in the intestinal flora reduce ischemic brain injury in mice, an effect transmissible by fecal transplants. Intestinal dysbiosis alters immune homeostasis in the small intestine, leading to an increase in regulatory T cells and a reduction in interleukin (IL)-17-positive γδ T cells through altered dendritic cell activity. Dysbiosis suppresses trafficking of effector T cells from the gut to the leptomeninges after stroke. Additionally, IL-10 and IL-17 are required for the neuroprotection afforded by intestinal dysbiosis. The findings reveal a previously unrecognized gut-brain axis and an impact of the intestinal flora and meningeal IL-17(+) γδ T cells on ischemic injury.

Hypertension enhances Aß-induced neurovascular dysfunction, promotes ß-secretase activity, and leads to amyloidogenic processing of APP.

Hypertension (HTN) doubles the risk of Alzheimer's disease (AD), but the mechanisms remain unclear. Amyloid-ß (Aß), a key pathogenic factor in AD, induces cerebrovascular dysfunction. We hypothesized that HTN acts in concert with Aß to amplify its deleterious cerebrovascular effects and to increase Aß production. Infusion of angiotensin II (ANGII; intravenously) elevated blood pressure and attenuated the cerebral blood flow (CBF) response to whisker stimulation or the endothelium-dependent vasodilator acetylcholine (ACh) (P < 0.05). Neocortical application of Aß in mice receiving ANGII worsened the responses to ACh (P < 0.05). The cerebrovascular dysfunction observed in Tg2576 mice, in which Aß is elevated both in blood and in brain due to expression of mutated amyloid precursor protein (APP), was not aggravated by neocortical application of ANGII or by a 2-week administration of 'slow pressor' of ANGII (600 ng/kg per minute; subcutaneously). In contrast, ANGII aggravated the dysfunction in TgSwDI mice, in which Aß is increased only in brain. Slow-pressor ANGII induced microvascular amyloid deposition in Tg2576 mice and enhanced ß-secretase APP cleavage. In Chinese hamster ovary (CHO) cells producing Aß, ANGII increased ß-secretase activity, Aß1-42, and the Aß42/40 ratio. We conclude that HTN enhances amyloidogenic APP processing, effects that may contribute to the pathogenic interaction between HTN and AD.

Dichotomous effects of chronic intermittent hypoxia on focal cerebral ischemic injury.

BACKGROUND AND PURPOSE: Obstructive sleep apnea, a condition associated with chronic intermittent hypoxia (CIH), carries an increased risk of stroke. However, CIH has been reported to either increase or decrease brain injury in models of focal cerebral ischemia. The factors determining the differential effects of CIH on ischemic injury and their mechanisms remain unclear. Here, we tested the hypothesis that the intensity of the hypoxic challenge determines the protective or destructive nature of CIH by modulating mitochondrial resistance to injury. METHODS: Male C57Bl/6J mice were exposed to CIH with 10% or 6% O2 for ≤35 days and subjected to transient middle cerebral artery occlusion. Motor deficits and infarct volume were assessed 3 days later. Intraischemic cerebral blood flow was measured by laser-Doppler flowmetry and resting cerebral blood flow by arterial spin labeling MRI. Ca2+-induced mitochondrial depolarization and reactive oxygen species production were evaluated in isolated brain mitochondria. RESULTS: We found that 10% CIH is neuroprotective, whereas 6% CIH exacerbates tissue damage. No differences in resting or intraischemic cerebral blood flow were observed between 6% and 10% CIH. However, 10% CIH reduced, whereas 6% CIH increased, mitochondrial reactive oxygen species production and susceptibility to Ca2+-induced depolarizations. CONCLUSIONS: The influence of CIH on the ischemic brain is dichotomous and can be attributed, in part, to changes in the mitochondrial susceptibility to injury. The findings highlight a previously unappreciated complexity in the effect of CIH on the brain, which needs to be considered in evaluating the neurological effect of conditions associated with cyclic hypoxia.

Water deprivation induces neurovascular and cognitive dysfunction through vasopressin-induced oxidative stress.

Adequate hydration is essential for normal brain function and dehydration induces cognitive deterioration. In addition, dehydration has emerged as a stroke risk factor. However, it is unknown whether alterations in cerebrovascular regulation are responsible for these effects. To address this issue, C57Bl/6 mice were water deprived for 24 or 48 hours and somatosensory cortex blood flow was assessed by laser-Doppler flowmetry in a cranial window. Dehydration increased plasma osmolality and vasopressin levels, and suppressed the increase in blood flow induced by neural activity, by the endothelium-dependent vasodilator acetylcholine and the smooth muscle relaxant adenosine. The cerebrovascular dysfunction was associated with oxidative stress and cognitive deficits, assessed using the Y maze. The vasopressin 1a receptor antagonist SR49059 improved the dehydration-induced oxidative stress and vasomotor dysfunction. Dehydration upregulated endothelin-1 in cerebral blood vessels, an effect blocked by SR49059. Furthermore, the endothelin A receptor antagonist BQ123 ameliorated cerebrovascular function. These findings show for the first time that dehydration alters critical mechanisms regulating the cerebral circulation through vasopressin and oxidative stress. The ensuing cerebrovascular dysregulation may alter cognitive function and increase the brain's susceptibility to cerebral ischemia.

Circulating endothelin-1 alters critical mechanisms regulating cerebral microcirculation.

Endothelin-1 (ET1) is a potent vasoconstrictor peptide implicated in the cerebrovascular alterations occurring in stroke, subarachnoid hemorrhage, and brain trauma. Brain or circulating levels of ET1 are elevated in these conditions and in risk factors for cerebrovascular diseases. Most studies on the cerebrovascular effects of ET1 have focused on vascular smooth muscle constriction, and little is known about the effect of the peptide on cerebrovascular regulation. We tested the hypothesis that ET1 increases cerebrovascular risk by disrupting critical mechanisms regulating cerebral blood flow. Male C57Bl6/J mice equipped with a cranial window were infused intravenously with vehicle or ET1, and somatosensory cortex blood flow was assessed by laser Doppler flowmetry. ET1 infusion increased mean arterial pressure and attenuated the blood flow increase produced by neural activity (whisker stimulation) or neocortical application of the endothelium-dependent vasodilator acetylcholine but not A23187. The cerebrovascular effects of ET1 were abrogated by the ET(A) receptor antagonist BQ123 and were not related to vascular oxidative stress. Rather, the dysfunction was dependent on Rho-associated protein kinase activity. Furthermore, in vitro studies demonstrated that ET1 suppresses endothelial nitric oxide (NO) production, assessed by its metabolite nitrite, an effect associated with Rho-associated protein kinase-dependent changes in the phosphorylation state of endothelial NO synthase. Collectively, these novel observations demonstrate that increased ET1 plasma levels alter key regulatory mechanisms of the cerebral circulation by modulating endothelial NO synthase phosphorylation and NO production through Rho-associated protein kinase. The ET1-induced cerebrovascular dysfunction may increase cerebrovascular risk by lowering cerebrovascular reserves and increasing the vulnerability of the brain to cerebral ischemia.

Angiotensin II slow-pressor hypertension enhances NMDA currents and NOX2-dependent superoxide production in hypothalamic paraventricular neurons.

Adaptive changes in glutamatergic signaling within the hypothalamic paraventricular nucleus (PVN) may play a role in the neurohumoral dysfunction underlying the hypertension induced by "slow-pressor" ANG II infusion. We hypothesized that these adaptive changes alter production of gp91phox NADPH oxidase (NOX)-derived reactive oxygen species (ROS) or nitric oxide (NO), resulting in enhanced glutamatergic signaling in the PVN. Electron microscopic immunolabeling showed colocalization of NOX2 and N-methyl-D-aspartate receptor (NMDAR) NR1 subunits in PVN dendrites, an effect enhanced (+48%, P < 0.05 vs. saline) in mice receiving ANG II (600 ng·kg⁻¹·min⁻¹ sc). Isolated PVN cells or spinally projecting PVN neurons from ANG II-infused mice had increased levels of ROS at baseline (+40 ± 5% and +57.6 ± 7.7%, P < 0.01 vs. saline) and after NMDA (+24 ± 7% and +17 ± 5.5%, P < 0.01 and P < 0.05 vs. saline). In contrast, ANG II infusion suppressed NO production in PVN cells at baseline (-29.1 ± 5.2%, P < 0.05 vs. saline) and after NMDA (-18.9 ± 2%, P < 0.01 vs. saline), an effect counteracted by NOX inhibition. In whole cell recording of unlabeled and spinally labeled PVN neurons in slices, NMDA induced a larger inward current in ANG II than in saline groups (+79 ± 24% and +82.9 ± 6.6%, P < 0.01 vs. saline), which was reversed by the ROS scavenger MnTBAP and the NO donor S-nitroso-N-acetylpenicillamine (P > 0.05 vs. control). These findings suggest that slow-pressor ANG II increases the association of NR1 with NOX2 in dendrites of PVN neurons, resulting in enhanced NOX-derived ROS and reduced NO during glutamatergic activity. The resulting enhancement of NMDAR activity may contribute to the neurohumoral dysfunction underlying the development of slow-pressor ANG II hypertension.