Alleviating Hypertension by Selectively Targeting Angiotensin Receptor-Expressing Vagal Sensory Neurons.

Cardiovascular homeostasis is maintained, in part, by neural signals arising from arterial baroreceptors that apprise the brain of blood volume and pressure. Here, we test whether neurons within the nodose ganglia that express angiotensin type-1a receptors (referred to as NGAT1aR) serve as baroreceptors that differentially influence blood pressure (BP) in male and female mice. Using Agtr1a-Cre mice and Cre-dependent AAVs to direct tdTomato to NGAT1aR, neuroanatomical studies revealed that NGAT1aR receive input from the aortic arch, project to the caudal nucleus of the solitary tract (NTS), and synthesize mechanosensitive ion channels, Piezo1/2 To evaluate the functionality of NGAT1aR, we directed the fluorescent calcium indicator (GCaMP6s) or the light-sensitive channelrhodopsin-2 (ChR2) to Agtr1a-containing neurons. Two-photon intravital imaging in Agtr1a-GCaMP6s mice revealed that NGAT1aR couple their firing to elevated BP, induced by phenylephrine (i.v.). Furthermore, optical excitation of NGAT1aR at their soma or axon terminals within the caudal NTS of Agtr1a-ChR2 mice elicited robust frequency-dependent decreases in BP and heart rate, indicating that NGAT1aR are sufficient to elicit appropriate compensatory responses to vascular mechanosensation. Optical excitation also elicited hypotensive and bradycardic responses in ChR2-expressing mice that were subjected to deoxycorticosterone acetate (DOCA)-salt hypertension; however, the duration of these effects was altered, suggestive of hypertension-induced impairment of the baroreflex. Similarly, increased GCaMP6s fluorescence observed after administration of phenylephrine was delayed in mice subjected to DOCA-salt or chronic delivery of angiotensin II. Collectively, these results reveal the structure and function of NGAT1aR and suggest that such neurons may be exploited to discern and relieve hypertension.

An Angiotensin-Responsive Connection from the Lamina Terminalis to the Paraventricular Nucleus of the Hypothalamus Evokes Vasopressin Secretion to Increase Blood Pressure in Mice.

Blood pressure is controlled by endocrine, autonomic, and behavioral responses that maintain blood volume and perfusion pressure at levels optimal for survival. Although it is clear that central angiotensin type 1a receptors (AT1aR; encoded by the Agtr1a gene) influence these processes, the neuronal circuits mediating these effects are incompletely understood. The present studies characterize the structure and function of AT1aR neurons in the lamina terminalis (containing the median preoptic nucleus and organum vasculosum of the lamina terminalis), thereby evaluating their roles in blood pressure control. Using male Agtr1a-Cre mice, neuroanatomical studies reveal that AT1aR neurons in the area are largely glutamatergic and send projections to the paraventricular nucleus of the hypothalamus (PVN) that appear to synapse onto vasopressin-synthesizing neurons. To evaluate the functionality of these lamina terminalis AT1aR neurons, we virally delivered light-sensitive opsins and then optogenetically excited or inhibited the neurons while evaluating cardiovascular parameters or fluid intake. Optogenetic excitation robustly elevated blood pressure, water intake, and sodium intake, while optogenetic inhibition produced the opposite effects. Intriguingly, optogenetic excitation of these AT1aR neurons of the lamina terminalis also resulted in Fos induction in vasopressin neurons within the PVN and supraoptic nucleus. Further, within the PVN, selective optogenetic stimulation of afferents that arise from these lamina terminalis AT1aR neurons induced glutamate release onto magnocellular neurons and was sufficient to increase blood pressure. These cardiovascular effects were attenuated by systemic pretreatment with a vasopressin-1a-receptor antagonist. Collectively, these data indicate that excitation of lamina terminalis AT1aR neurons induces neuroendocrine and behavioral responses that increase blood pressure.SIGNIFICANCE STATEMENT Hypertension is a widespread health problem and risk factor for cardiovascular disease. Although treatments exist, a substantial percentage of patients suffer from "drug-resistant" hypertension, a condition associated with increased activation of brain angiotensin receptors, enhanced sympathetic nervous system activity, and elevated vasopressin levels. The present study highlights a role for angiotensin Type 1a receptor expressing neurons located within the lamina terminalis in regulating endocrine and behavioral responses that are involved in maintaining cardiovascular homeostasis. More specifically, data presented here reveal functional excitatory connections between angiotensin-sensitive neurons in the lamina terminals and vasopressin neurons in the paraventricular nucleus of the hypothalamus, and further indicate that activation of this circuit raises blood pressure. These neurons may be a promising target for antihypertensive therapeutics.

Identification of Novel Cross-Talk between the Neuroendocrine and Autonomic Stress Axes Controlling Blood Pressure.

The hypothalamic paraventricular nucleus (PVN) controls neuroendocrine axes and the autonomic nervous system to mount responses that cope with the energetic burdens of psychological or physiological stress. Neurons in the PVN that express the angiotensin Type 1a receptor (PVNAgtr1a) are implicated in neuroendocrine and autonomic stress responses; however, the mechanism by which these neurons coordinate activation of neuroendocrine axes with sympathetic outflow remains unknown. Here, we use a multidisciplinary approach to investigate intra-PVN signaling mechanisms that couple the activity of neurons synthesizing corticotropin-releasing-hormone (CRH) to blood pressure. We used the Cre-Lox system in male mice with in vivo optogenetics and cardiovascular recordings to demonstrate that excitation of PVNAgtr1a promotes elevated blood pressure that is dependent on the sympathetic nervous system. Next, neuroanatomical experiments found that PVNAgtr1a synthesize CRH, and intriguingly, fibers originating from PVNAgtr1a make appositions onto neighboring neurons that send projections to the rostral ventrolateral medulla and express CRH type 1 receptor (CRHR1) mRNA. We then used an ex vivo preparation that combined optogenetics, patch-clamp electrophysiology, and Ca2+ imaging to discover that excitation of PVNAgtr1a drives the local, intra-PVN release of CRH, which activates rostral ventrolateral medulla-projecting neurons via stimulation of CRHR1(s). Finally, we returned to our in vivo preparation and found that CRH receptor antagonism specifically within the PVN lowered blood pressure basally and during optogenetic activation of PVNAgtr1a Collectively, these results demonstrate that angiotensin II acts on PVNAgtr1a to conjoin hypothalamic-pituitary-adrenal axis activity with sympathetically mediated vasoconstriction in male mice.SIGNIFICANCE STATEMENT The survival of an organism is dependent on meeting the energetic demands imposed by stressors. This critical function is accomplished by the CNS's ability to orchestrate simultaneous activities of neurosecretory and autonomic axes. Here, we unveil a novel signaling mechanism within the paraventricular nucleus of the hypothalamus that links excitation of neurons producing corticotropin-releasing-hormone with excitation of neurons controlling sympathetic nervous system activity and blood pressure. The implication is that chronic stress exposure may promote cardiometabolic disease by dysregulating the interneuronal cross-talk revealed by our experiments.

Conditioned social preference and reward value of activating oxytocin-receptor-expressing ventral tegmental area neurons following repeated daily binge ethanol intake.

BACKGROUND: Individuals with alcohol use disorder (AUD) exhibit a disruption of social behavior and dysregulation of oxytocin signaling in the brain, possibly reflecting decreased activation of oxytocin receptors (OxTRs) in reward pathways in response to social stimuli. We hypothesize that daily binge ethanol intake causes a deficit in social reward and oxytocin signaling in the ventral tegmental area (VTA). METHODS: After 9 weeks of daily binge ethanol intake (blood ethanol concentration >80 mg%), OxTR-cre mice underwent conditioned place preference for social reward. Separate groups of mice were tested for the effects of binge ethanol on voluntary social interactions, food reward, locomotion, and anxiety-like behaviors. A subset of mice underwent transfection of OxTR-expressing VTA neurons (VTAOxtr ) with a light-sensitive opsin, followed by operant training to respond to light delivered to VTA. RESULTS: Ethanol-naïve male mice increased the time spent on the side previously paired with novel mice while ethanol-treated mice did not. Binge ethanol did not affect conditioned place preference for food reward in males, but this response was weakened in ethanol-treated females. Ethanol treatment also caused a sex-specific impairment of voluntary social interactions with novel mice. There were minimal differences between groups in measures of anxiety and locomotion. Ethanol-naïve mice had significantly greater operant responding for activation of VTAOxtr than sham-transfected mice but ethanol-treated mice did not. There was no difference in the number of VTAOxtr after binge ethanol. CONCLUSIONS: Daily binge ethanol causes social reward deficits that cannot be explained by nonspecific effects on other behaviors, at least in males. Only ethanol-naïve mice exhibited positive reinforcement caused by activation of VTAOxtr while daily binge ethanol did not alter the number of VTAOxtr in either males or females. Thus, subtle dysregulation of VTAOxtr function may be related to the social reward deficits caused by daily binge ethanol.

Oxytocin and cardiometabolic interoception: Knowing oneself affects ingestive and social behaviors.

Maintaining homeostasis while navigating one's environment involves accurately assessing and interacting with external stimuli while remaining consciously in tune with internal signals such as hunger and thirst. Both atypical social interactions and unhealthy eating patterns emerge as a result of dysregulation in factors that mediate the prioritization and attention to salient stimuli. Oxytocin is an evolutionarily conserved peptide that regulates attention to exteroceptive and interoceptive stimuli in a social environment by functioning in the brain as a modulatory neuropeptide to control social behavior, but also in the periphery as a hormone acting at oxytocin receptors (Oxtr) expressed in the heart, gut, and peripheral ganglia. Specialized sensory afferent nerve endings of Oxtr-expressing nodose ganglia cells transmit cardiometabolic signals via the Vagus nerve to integrative regions in the brain that also express Oxtr(s). These brain regions are influenced by vagal sensory pathways and coordinate with external events such as those demanding attention to social stimuli, thus the sensations related to cardiometabolic function and social interactions are influenced by oxytocin signaling. This review investigates the literature supporting the idea that oxytocin mediates the interoception of cardiovascular and gastrointestinal systems, and that the modulation of this awareness likewise influences social cognition. These concepts are then considered in relation to Autism Spectrum Disorder, exploring how atypical social behavior is comorbid with cardiometabolic dysfunction.

Macrophage angiotensin II type 2 receptor triggers neuropathic pain.

Peripheral nerve damage initiates a complex series of structural and cellular processes that culminate in chronic neuropathic pain. The recent success of a type 2 angiotensin II (Ang II) receptor (AT2R) antagonist in a phase II clinical trial for the treatment of postherpetic neuralgia suggests angiotensin signaling is involved in neuropathic pain. However, transcriptome analysis indicates a lack of AT2R gene (Agtr2) expression in human and rodent sensory ganglia, raising questions regarding the tissue/cell target underlying the analgesic effect of AT2R antagonism. We show that selective antagonism of AT2R attenuates neuropathic but not inflammatory mechanical and cold pain hypersensitivity behaviors in mice. Agtr2-expressing macrophages (MΦs) constitute the predominant immune cells that infiltrate the site of nerve injury. Interestingly, neuropathic mechanical and cold pain hypersensitivity can be attenuated by chemogenetic depletion of peripheral MΦs and AT2R-null hematopoietic cell transplantation. Our study identifies AT2R on peripheral MΦs as a critical trigger for pain sensitization at the site of nerve injury, and therefore proposes a translatable peripheral mechanism underlying chronic neuropathic pain.

Oxytocin Receptors Are Expressed by Glutamatergic Prefrontal Cortical Neurons That Selectively Modulate Social Recognition.

Social recognition, the ability to recognize individuals that were previously encountered, requires complex integration of sensory inputs with previous experience. Here, we use a variety of approaches to discern how oxytocin-sensitive neurons in the PFC exert descending control over a circuit mediating social recognition in mice. Using male mice with Cre-recombinase directed to the oxytocin receptor gene (Oxtr), we revealed that oxytocin receptors (OXTRs) are expressed on glutamatergic neurons in the PFC, optogenetic stimulation of which elicited activation of neurons residing in several mesolimbic brain structures. Optogenetic stimulation of axons in the BLA arising from OXTR-expressing neurons in the PFC eliminated the ability to distinguish novel from familiar conspecifics, but remarkably, distinguishing between novel and familiar objects was unaffected. These results suggest that an oxytocin-sensitive PFC to BLA circuit is required for social recognition. The implication is that impaired social memory may manifest from dysregulation of this circuit.SIGNIFICANCE STATEMENT Using mice, we demonstrate that optogenetic activation of the neurons in the PFC that express the oxytocin receptor gene (Oxtr) impairs the ability to distinguish between novel and familiar conspecifics, but the ability to distinguish between novel and familiar objects remains intact. Subjects with autism spectrum disorders (ASDs) have difficulty identifying a person based on remembering facial features; however, ASDs and typical subjects perform similarly when remembering objects. In subjects with ASD, viewing the same face increases neural activity in the PFC, which may be analogous to the optogenetic excitation of oxytocin receptor (OXTR) expressing neurons in the PFC that impairs social recognition in mice. The implication is that overactivation of OXTR-expressing neurons in the PFC may contribute to ASD symptomology.

Brain Angiotensin Type-1 and Type-2 Receptors in Physiological and Hypertensive Conditions: Focus on Neuroinflammation.

PURPOSE OF REVIEW: To review recent data that suggest opposing effects of brain angiotensin type-1 (AT1R) and type-2 (AT2R) receptors on blood pressure (BP). Here, we discuss recent studies that suggest pro-hypertensive and pro-inflammatory actions of AT1R and anti-hypertensive and anti-inflammatory actions of AT2R. Further, we propose mechanisms for the interplay between brain angiotensin receptors and neuroinflammation in hypertension. RECENT FINDINGS: The renin-angiotensin system (RAS) plays an important role in regulating cardiovascular physiology. This includes brain AT1R and AT2R, both of which are expressed in or adjacent to brain regions that control BP. Activation of AT1R within those brain regions mediate increases in BP and cause neuroinflammation, which augments the BP increase in hypertension. The fact that AT1R and AT2R have opposing actions on BP suggests that AT1R and AT2R may have similar opposing actions on neuroinflammation. However, the mechanisms by which brain AT1R and AT2R mediate neuroinflammatory responses remain unclear. The interplay between brain angiotensin receptor subtypes and neuroinflammation exacerbates or protects against hypertension.

Angiotensin II Triggers Peripheral Macrophage-to-Sensory Neuron Redox Crosstalk to Elicit Pain.

Injury, inflammation, and nerve damage initiate a wide variety of cellular and molecular processes that culminate in hyperexcitation of sensory nerves, which underlies chronic inflammatory and neuropathic pain. Using behavioral readouts of pain hypersensitivity induced by angiotensin II (Ang II) injection into mouse hindpaws, our study shows that activation of the type 2 Ang II receptor (AT2R) and the cell-damage-sensing ion channel TRPA1 are required for peripheral mechanical pain sensitization induced by Ang II in male and female mice. However, we show that AT2R is not expressed in mouse and human dorsal root ganglia (DRG) sensory neurons. Instead, expression/activation of AT2R on peripheral/skin macrophages (MΦs) constitutes a critical trigger of mouse and human DRG sensory neuron excitation. Ang II-induced peripheral mechanical pain hypersensitivity can be attenuated by chemogenetic depletion of peripheral MΦs. Furthermore, AT2R activation in MΦs triggers production of reactive oxygen/nitrogen species, which trans-activate TRPA1 on mouse and human DRG sensory neurons via cysteine modification of the channel. Our study thus identifies a translatable immune cell-to-sensory neuron signaling crosstalk underlying peripheral nociceptor sensitization. This form of cell-to-cell signaling represents a critical peripheral mechanism for chronic pain and thus identifies multiple druggable analgesic targets.SIGNIFICANCE STATEMENT Pain is a widespread health problem that is undermanaged by currently available analgesics. Findings from a recent clinical trial on a type II angiotensin II receptor (AT2R) antagonist showed effective analgesia for neuropathic pain. AT2R antagonists have been shown to reduce neuropathy-, inflammation- and bone cancer-associated pain in rodents. We report that activation of AT2R in macrophages (MΦs) that infiltrate the site of injury, but not in sensory neurons, triggers an intercellular redox communication with sensory neurons via activation of the cell damage/pain-sensing ion channel TRPA1. This MΦ-to-sensory neuron crosstalk results in peripheral pain sensitization. Our findings provide an evidence-based mechanism underlying the analgesic action of AT2R antagonists, which could accelerate the development of efficacious non-opioid analgesic drugs for multiple pain conditions.

Fat-brain connections: Adipocyte glucocorticoid control of stress and metabolism.

Glucocorticoids act via multiple mechanisms to mobilize energy for maintenance and restoration of homeostasis. In adipose tissue, glucocorticoids can promote lipolysis and facilitate adipocyte differentiation/growth, serving both energy-mobilizing and restorative processes during negative energy balance. Recent data suggest that adipose-dependent feedback may also be involved in regulation of stress responses. Adipocyte glucocorticoid receptor (GR) deletion causes increased HPA axis stress reactivity, due to a loss of negative feedback signals into the CNS. The fat-to-brain signal may be mediated by neuronal mechanisms, release of adipokines or increased lipolysis. The ability of adipose GRs to inhibit psychogenic as well as metabolic stress responses suggests that (1) feedback regulation of the HPA axis occurs across multiple bodily compartments, and (2) fat tissue integrates psychogenic stress signals. These studies support a link between stress biology and energy metabolism, a connection that has clear relevance for numerous disease states and their comorbidities.

Angiotensin receptor expression revealed by reporter mice and beneficial effects of AT2R agonist in retinal cells.

The renin-angiotensin system (RAS) plays a vital role in cardiovascular physiology and body homeostasis. In addition to circulating RAS, a local RAS exists in the retina. Dysfunction of local RAS, resulting in increased levels of Angiotensin II (Ang II) and activation of AT1R-mediated signaling pathways, contributes to tissue pathophysiology and end-organ damage. Activation of AT2R on other hand is known to counteract the effects of AT1R activation and produce anti-inflammatory and anti-oxidative effects. We examined the expression of angiotensin receptors in the retina by using transgenic dual reporter mice and by real-time RT-PCR. We further evaluated the effects of C21, a selective agonist of AT2R, in reducing Ang II, lipopolysaccharide (LPS) and hydrogen peroxide induced oxidative stress and inflammatory responses in cultured human ARPE-19 cells. We showed that both AT1Ra and AT2R positive cells are detected in different cell types of the eye, including the RPE/choroid complex, ciliary body/iris, and neural retina. AT1Ra is more abundantly expressed than AT2R in mouse retina, consistent with previous reports. In the neural retina, AT1Ra are also detected in photoreceptors whereas AT2R are mostly expressed in the inner retinal neurons and RGCs. In cultured human RPE cells, activation of AT2R with C21 significantly blocked Ang II, LPS and hydrogen peroxide -induced NF-κB activation and inflammatory cytokine expression; Ang II and hydrogen peroxide-induced reactive oxygen species (ROS) production and MG132-induced apoptosis, comparable to the effects of Angiotensin-(1-7) (Ang-(1-7)), another protective component of the RAS, although C21 is more potent in reducing some of the effects induced by Ang II, whereas Ang-(1-7) is more effective in reducing some of the LPS and hydrogen peroxide-induced effects. These results suggest that activation of AT2R may represent a new therapeutic approach for retinal diseases.

A Unique "Angiotensin-Sensitive" Neuronal Population Coordinates Neuroendocrine, Cardiovascular, and Behavioral Responses to Stress.

Stress elicits neuroendocrine, autonomic, and behavioral responses that mitigate homeostatic imbalance and ensure survival. However, chronic engagement of such responses promotes psychological, cardiovascular, and metabolic impairments. In recent years, the renin-angiotensin system has emerged as a key mediator of stress responding and its related pathologies, but the neuronal circuits that orchestrate these interactions are not known. These studies combine the use of the Cre-recombinase/loxP system in mice with optogenetics to structurally and functionally characterize angiotensin type-1a receptor-containing neurons of the paraventricular nucleus of the hypothalamus, the goal being to determine the extent of their involvement in the regulation of stress responses. Initial studies use neuroanatomical techniques to reveal that angiotensin type-1a receptors are localized predominantly to the parvocellular neurosecretory neurons of the paraventricular nucleus of the hypothalamus. These neurons are almost exclusively glutamatergic and send dense projections to the exterior portion of the median eminence. Furthermore, these neurons largely express corticotrophin-releasing hormone or thyrotropin-releasing hormone and do not express arginine vasopressin or oxytocin. Functionally, optogenetic stimulation of these neurons promotes the activation of the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-thyroid axes, as well as a rise in systolic blood pressure. When these neurons are optogenetically inhibited, the activity of these neuroendocrine axes are suppressed and anxiety-like behavior in the elevated plus maze is dampened. Collectively, these studies implicate this neuronal population in the integration and coordination of the physiological responses to stress and may therefore serve as a potential target for therapeutic intervention for stress-related pathology.SIGNIFICANCE STATEMENT Chronic stress leads to an array of physiological responses that ultimately rouse psychological, cardiovascular, and metabolic impairments. As a consequence, there is an urgent need for the development of novel therapeutic approaches to prevent or dampen deleterious aspects of "stress." While the renin-angiotensin system has received some attention in this regard, the neural mechanisms by which this endocrine system may impact stress-related pathologies and consequently serve as targets for therapeutic intervention are not clear. The present studies provide substantial insight in this regard. That is, they reveal that a distinct population of angiotensin-sensitive neurons is integral to the coordination of stress responses. The implication is that this neuronal phenotype may serve as a target for stress-related disease.

Protective Angiotensin Type 2 Receptors in the Brain and Hypertension.

PURPOSE OF REVIEW: The goal of this review is to assess the evidence that activation of angiotensin type 2 receptors (AT2R) in the brain can lower blood pressure and possibly constitute an endogenous anti-hypertensive mechanism. RECENT FINDINGS: Recent studies that detail the location of AT2R in the brain, particularly within or near cardiovascular control centers, mesh well with findings from pharmacological and gene transfer studies which demonstrate that activation of central AT2R can influence cardiovascular regulation. Collectively, these studies indicate that selective activation of brain AT2R causes moderate decreases in blood pressure in normal animals and more profound anti-hypertensive effects, along with restoration of baroreflex function, in rodent models of neurogenic hypertension. These findings have opened the door to studies that can (i) assess the role of specific AT2R neuron populations in depressing blood pressure, (ii) determine the relevance of such mechanisms, and (iii) investigate interactions between AT2R and depressor angiotensin-(1-7)/Mas mechanisms in the brain.

Angiotensin type 1a receptors in the paraventricular nucleus of the hypothalamus control cardiovascular reactivity and anxiety-like behavior in male mice.

This study tested the hypothesis that deletion of angiotensin type 1a receptors (AT1a) from the paraventricular nucleus of hypothalamus (PVN) attenuates anxiety-like behavior, hypothalamic-pituitary-adrenal (HPA) axis activity, and cardiovascular reactivity. We used the Cre/LoxP system to generate male mice with AT1a specifically deleted from the PVN. Deletion of the AT1a from the PVN reduced anxiety-like behavior as indicated by increased time spent in the open arms of the elevated plus maze. In contrast, PVN AT1a deletion had no effect on HPA axis activation subsequent to an acute restraint challenge but did reduce hypothalamic mRNA expression for corticotropin-releasing hormone (CRH). To determine whether PVN AT1a deletion inhibits cardiovascular reactivity, we measured systolic blood pressure, heart rate, and heart rate variability (HRV) using telemetry and found that PVN AT1a deletion attenuated restraint-induced elevations in systolic blood pressure and elicited changes in HRV indicative of reduced sympathetic nervous activity. Consistent with the decreased HRV, PVN AT1a deletion also decreased adrenal weight, suggestive of decreased adrenal sympathetic outflow. Interestingly, the altered stress responsivity of mice with AT1a deleted from the PVN was associated with decreased hypothalamic microglia and proinflammatory cytokine expression. Collectively, these results suggest that deletion of AT1a from the PVN attenuates anxiety, CRH gene transcription, and cardiovascular reactivity and reduced brain inflammation may contribute to these effects.

Cross talk between AT1 receptors and Toll-like receptor 4 in microglia contributes to angiotensin II-derived ROS production in the hypothalamic paraventricular nucleus.

ANG II is thought to increase sympathetic outflow by increasing oxidative stress and promoting local inflammation in the paraventricular nucleus (PVN) of the hypothalamus. However, the relative contributions of inflammation and oxidative stress to sympathetic drive remain poorly understood, and the underlying cellular and molecular targets have yet to be examined. ANG II has been shown to enhance Toll-like receptor (TLR)4-mediated signaling on microglia. Thus, in the present study, we aimed to determine whether ANG II-mediated activation of microglial TLR4 signaling is a key molecular target initiating local oxidative stress in the PVN. We found TLR4 and ANG II type 1 (AT1) receptor mRNA expression in hypothalamic microglia, providing molecular evidence for the potential interaction between these two receptors. In hypothalamic slices, ANG II induced microglial activation within the PVN (∼65% increase, P < 0.001), an effect that was blunted in the absence of functional TLR4. ANG II increased ROS production, as indicated by dihydroethidium fluorescence, within the PVN of rats and mice (P < 0.0001 in both cases), effects that were also dependent on the presence of functional TLR4. The microglial inhibitor minocycline attenuated ANG II-mediated ROS production, yet ANG II effects persisted in PVN single-minded 1-AT1a knockout mice, supporting the contribution of a non-neuronal source (likely microglia) to ANG II-driven ROS production in the PVN. Taken together, these results support functional interactions between AT1 receptors and TLR4 in mediating ANG II-dependent microglial activation and oxidative stress within the PVN. More broadly, our results support a functional interaction between the central renin-angiotensin system and innate immunity in the regulation of neurohumoral outflows from the PVN.

Role of neurons and glia in the CNS actions of the renin-angiotensin system in cardiovascular control.

Despite tremendous research efforts, hypertension remains an epidemic health concern, leading often to the development of cardiovascular disease. It is well established that in many instances, the brain plays an important role in the onset and progression of hypertension via activation of the sympathetic nervous system. Further, the activity of the renin-angiotensin system (RAS) and of glial cell-mediated proinflammatory processes have independently been linked to this neural control and are, as a consequence, both attractive targets for the development of antihypertensive therapeutics. Although it is clear that the predominant effector peptide of the RAS, ANG II, activates its type-1 receptor on neurons to mediate some of its hypertensive actions, additional nuances of this brain RAS control of blood pressure are constantly being uncovered. One of these complexities is that the RAS is now thought to impact cardiovascular control, in part, via facilitating a glial cell-dependent proinflammatory milieu within cardiovascular control centers. Another complexity is that the newly characterized antihypertensive limbs of the RAS are now recognized to, in many cases, antagonize the prohypertensive ANG II type 1 receptor (AT1R)-mediated effects. That being said, the mechanism by which the RAS, glia, and neurons interact to regulate blood pressure is an active area of ongoing research. Here, we review the current understanding of these interactions and present a hypothetical model of how these exchanges may ultimately regulate cardiovascular function.

Angiotensin type 1a receptors in the paraventricular nucleus of the hypothalamus protect against diet-induced obesity.

Obesity is associated with increased levels of angiotensin-II (Ang-II), which activates angiotensin type 1a receptors (AT1a) to influence cardiovascular function and energy homeostasis. To test the hypothesis that specific AT1a within the brain control these processes, we used the Cre/lox system to delete AT1a from the paraventricular nucleus of the hypothalamus (PVN) of mice. PVN AT1a deletion did not affect body mass or adiposity when mice were maintained on standard chow. However, maintenance on a high-fat diet revealed a gene by environment interaction whereby mice lacking AT1a in the PVN had increased food intake and decreased energy expenditure that augmented body mass and adiposity relative to controls. Despite this increased adiposity, PVN AT1a deletion reduced systolic blood pressure, suggesting that this receptor population mediates the positive correlation between adiposity and blood pressure. Gene expression studies revealed that PVN AT1a deletion decreased hypothalamic expression of corticotrophin-releasing hormone and oxytocin, neuropeptides known to control food intake and sympathetic nervous system activity. Whole-cell patch-clamp recordings confirmed that PVN AT1a deletion eliminates responsiveness of PVN parvocellular neurons to Ang-II, and suggest that Ang-II responsiveness is increased in obese wild-type mice. Central inflammation is associated with metabolic and cardiovascular disorders and PVN AT1a deletion reduced indices of hypothalamic inflammation. Collectively, these studies demonstrate that PVN AT1a regulate energy balance during environmental challenges that promote metabolic and cardiovascular pathologies. The implication is that the elevated Ang-II that accompanies obesity serves as a negative feedback signal that activates PVN neurons to alleviate weight gain.

Optical perturbation of Agtr1a-containing neurons and afferents within the caudal nucleus of the solitary tract modulates sodium intake.

Angiotensin-II (Ang-II) production is driven by deviations in blood volume and osmolality, and serves the role of regulating blood pressure and fluid intake to maintain cardiovascular and hydromineral homeostasis. These actions are mediated by Ang-II acting on its type 1a receptor (AT1aR) within the central nervous system and periphery. Of relevance, AT1aR are expressed on sensory afferents responsible for conveying cardiovascular information to the nucleus of the solitary tract (NTS). We have previously determined that optical excitation of neurons and vagal afferents within the NTS that express AT1aR (referred to as NTSAT1aR) mimics the perception of increased vascular stretch and induces compensatory responses to restore blood pressure. Here, we test whether NTSAT1aR are also involved in the modulation of water and sodium intake. We directed the light-sensitive excitatory channelrhodopsin-2 (ChR2) or inhibitory halorhodopsin (Halo) to Agtr1a-containing neurons and measured water and sodium chloride (NaCl) intake in the presence and absence of optical stimulation within the NTS during various challenges to fluid homeostasis. Optical perturbation of NTSAT1aR modulates NaCl intake, such that excitation attenuates, whereas inhibition increases intake. This effect is only observed in the water-deprived condition, suggesting that NTSAT1aR are involved in the regulation of sodium intake during an imbalance in both the intracellular and extracellular fluid compartments. Furthermore, optical excitation of NTSAT1aR increases c-Fos expression within oxytocinergic neurons of the paraventricular nucleus of the hypothalamus (PVN), indicating that the regulation of sodium intake by NTSAT1aR may be mediated by oxytocin. Collectively, these results reveal that NTSAT1aR are sufficient and necessary to modulate sodium intake relative to perceived changes in vascular stretch.

Angiotensin-(1-5) is a Potent Endogenous Angiotensin AT 2 -Receptor Agonist.

Background: The renin-angiotensin system involves many more enzymes, receptors and biologically active peptides than originally thought. With this study, we investigated whether angiotensin-(1-5) [Ang-(1-5)], a 5-amino acid fragment of angiotensin II, has biological activity, and through which receptor it elicits effects. Methods: The effect of Ang-(1-5) (1µM) on nitric oxide release was measured by DAF-FM staining in human aortic endothelial cells (HAEC), or Chinese Hamster Ovary (CHO) cells stably transfected with the angiotensin AT 2 -receptor (AT 2 R) or the receptor Mas. A potential vasodilatory effect of Ang-(1-5) was tested in mouse mesenteric and human renal arteries by wire myography; the effect on blood pressure was evaluated in normotensive C57BL/6 mice by Millar catheter. These experiments were performed in the presence or absence of a range of antagonists or inhibitors or in AT 2 R-knockout mice. Binding of Ang-(1-5) to the AT 2 R was confirmed and the preferred conformations determined by in silico docking simulations. The signaling network of Ang-(1-5) was mapped by quantitative phosphoproteomics. Results: Key findings included: (1) Ang-(1-5) induced activation of eNOS by changes in phosphorylation at Ser1177 eNOS and Tyr657 eNOS and thereby (2) increased NO release from HAEC and AT 2 R-transfected CHO cells, but not from Mas-transfected or non-transfected CHO cells. (3) Ang-(1-5) induced relaxation of preconstricted mouse mesenteric and human renal arteries and (4) lowered blood pressure in normotensive mice - effects which were respectively absent in arteries from AT 2 R-KO or in PD123319-treated mice and which were more potent than effects of the established AT 2 R-agonist C21. (5) According to in silico modelling, Ang-(1-5) binds to the AT 2 R in two preferred conformations, one differing substantially from where the first five amino acids within angiotensin II bind to the AT 2 R. (6) Ang-(1-5) modifies signaling pathways in a protective RAS-typical way and with relevance for endothelial cell physiology and disease. Conclusions: Ang-(1-5) is a potent, endogenous AT 2 R-agonist.

Pleasurable behaviors reduce stress via brain reward pathways.

Individuals often eat calorically dense, highly palatable "comfort" foods during stress for stress relief. This article demonstrates that palatable food intake (limited intake of sucrose drink) reduces neuroendocrine, cardiovascular, and behavioral responses to stress in rats. Artificially sweetened (saccharin) drink reproduces the stress dampening, whereas oral intragastric gavage of sucrose is without effect. Together, these results suggest that the palatable/rewarding properties of sucrose are necessary and sufficient for stress dampening. In support of this finding, another type of natural reward (sexual activity) similarly reduces stress responses. Ibotenate lesions of the basolateral amygdala (BLA) prevent stress dampening by sucrose, suggesting that neural activity in the BLA is necessary for the effect. Moreover, sucrose intake increases mRNA and protein expression in the BLA for numerous genes linked with functional and/or structural plasticity. Lastly, stress dampening by sucrose is persistent, which is consistent with long-term changes in neural activity after synaptic remodeling. Thus, natural rewards, such as palatable foods, provide a general means of stress reduction, likely via structural and/or functional plasticity in the BLA. These findings provide a clearer understanding of the motivation for consuming palatable foods during times of stress and influence therapeutic strategies for the prevention and/or treatment of obesity and other stress-related disorders.