Paraventricular Nucleus Presympathetic Neurons Research Articles

Microglia in the hypothalamic paraventricular nucleus sense hemodynamic disturbance and promote sympathetic excitation in hypertension

Hypertension is usually accompanied by elevated sympathetic tonicity, but how sympathetic hyperactivity is triggered is not clear. Recent advances revealed that microglia-centered neuroinflammation contributes to sympathetic excitation in hypertension. In this study, we performed a temporospatial analysis of microglia at both morphological and transcriptomic levels and found that microglia in the hypothalamic paraventricular nucleus (PVN), a sympathetic center, were early responders to hypertensive challenges. Vasculature analyses revealed that the PVN was characterized by high capillary density, thin vessel diameter, and complex vascular topology relative to other brain regions. As such, the PVN was susceptible to the penetration of ATP released from the vasculature in response to hemodynamic disturbance after blood pressure increase. Mechanistically, ATP ligation to microglial P2Y12 receptor was responsible for microglial inflammatory activation and the eventual sympathetic overflow. Together, these findings identified a distinct vasculature pattern rendering vulnerability of PVN pre-sympathetic neurons to hypertension-associated microglia-mediated inflammatory insults.

PVN predisposes microglia to be susceptible to hemodynamic signals and promote sympathetic excitation in hypertension

Hypertension is usually accompanied with an elevated sympathetic tonicity, but how sympathetic hyperactivity is triggered is not fully understood. Recent advances reveal that microglia-centered neuroinflammation contributes to sympathetic excitation in hypertension. In this study, we performed a temporospatial analysis of microglia at both morphological and transcriptomic levels, and found that microglia in the hypothalamic paraventricular nucleus (PVN) were early responders to hypertensive challenges. PVN is the central hub for maintaining cardiovascular function via regulation of fluid balance and sympathetic outflow. Comprehensive vasculature analyses unveiled that PVN was characterized by high capillary density, thin vessel diameter, and complex vascular topology among brain regions. As such, PVN is susceptible to the penetration of ATP released from the vasculature in response to hemodynamic disturbance after blood pressure increase. ATP ligation to microglial P2Y12 receptor is responsible for the microglial accumulation and activation in the PVN. Both pharmacological blockade and genetic ablation of microglial P2Y12 could substantially restrain blood pressure increase under hypertensive challenge. Together, these findings disclose that a unique vasculature pattern results in the vulnerability of PVN pre-sympathetic neurons to hypertension-associated inflammatory insults, which is mediated by microglia. NSFC82170441. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Calcineurin regulates synaptic Ca2+-permeable AMPA receptors in hypothalamic presympathetic neurons via α2δ-1-mediated GluA1/GluA2 assembly.

Hypertension is a major adverse effect of calcineurin inhibitors, such as tacrolimus (FK506) and cyclosporine, used clinically as immunosuppressants. Calcineurin inhibitor-induced hypertension (CIH) is linked to augmented sympathetic output from the hypothalamic paraventricular nucleus (PVN). GluA2-lacking, Ca2+-permeable AMPA receptors (CP-AMPARs) are a key feature of glutamatergic synaptic plasticity, yet their role in CIH remains elusive. Here, we found that systemic administration of FK506 in rats significantly increased serine phosphorylation of GluA1 and GluA2 in PVN synaptosomes. Strikingly, FK506 treatment reduced GluA1/GluA2heteromers in both synaptosomes and endoplasmic reticulum-enriched fractions from the PVN. Blocking CP-AMPARs with IEM-1460 induced a larger reduction of AMPAR-mediated excitatory postsynaptic current (AMPAR-EPSC) amplitudes in retrogradely labelled, spinally projecting PVN neurons in FK506-treated rats than in vehicle-treated rats. Furthermore, FK506 treatment shifted the current-voltage relationship of AMPAR-EPSCs from linear to inward rectification in labelled PVN neurons. FK506 treatment profoundly enhanced physical interactions of α2δ-1 with GluA1 and GluA2 in the PVN. Inhibiting α2δ-1 with gabapentin, α2δ-1 genetic knockout, or disrupting α2δ-1-AMPAR interactions with an α2δ-1 C terminus peptide restored GluA1/GluA2heteromers in the PVN and diminished inward rectification of AMPAR-EPSCs in labelled PVN neurons induced by FK506 treatment. Additionally, microinjection of IEM-1460 or α2δ-1 C terminus peptide into the PVN reduced renal sympathetic nerve discharges and arterial blood pressure elevated in FK506-treated rats but not in vehicle-treated rats. Thus, calcineurin in the hypothalamus constitutively regulates AMPAR subunit composition and phenotypes by controlling GluA1/GluA2 interactions with α2δ-1. Synaptic CP-AMPARs in PVN presympathetic neurons contribute to augmented sympathetic outflow in CIH. KEY POINTS: Systemic treatment with the calcineurin inhibitor increases serine phosphorylation of synaptic GluA1 and GluA2 in the PVN. Calcineurin inhibition enhances the prevalence of postsynaptic Ca2+-permeable AMPARs in PVN presympathetic neurons. Calcineurin inhibition potentiates α2δ-1 interactions with GluA1 and GluA2, disrupting intracellular assembly of GluA1/GluA2heterotetramers in the PVN. Blocking Ca2+-permeable AMPARs or α2δ-1-AMPAR interactions in the PVN attenuates sympathetic outflow augmented by the calcineurin inhibitor.

Chronic Intermittent Hypobaric Hypoxia Reduces Hypothalamic N-Methyl-d-Aspartate Receptor Activity and Sympathetic Outflow in Spontaneously Hypertensive Rats.

Guo, Xinqi, Hongyu Ma, Ziye Cui, Qiyue Zhao, Ying Zhang, Lu Jia, Liping Zhang, Hui Guo, Xiangjian Zhang, Yi Zhang, Yue Guan, and Huijie Ma. Chronic intermittent hypobaric hypoxia reduces hypothalamic N-Methyl-d-Aspartate Receptor activity and sympathetic outflow in spontaneously hypertensive rats. High Alt Med Biol. 25:77-88, 2024. Objective: This study aims to determine the role of hypothalamic renin-angiotensin system (RAS) in the antihypertensive effect of chronic intermittent hypobaric hypoxia (CIHH). Methods: Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) received 35 days of hypobaric hypoxia simulating an altitude of 4,000 m, 5 h/day. The levels of RAS, blood pressure, and N-methyl-d-aspartate receptor (NMDAR) activities of hypothalamic paraventricular nucleus (PVN) presympathetic neurons from each group of rats were determined. Results: The systolic blood pressure, diastolic blood pressure, and mean arterial blood pressure (MAP) of SHRs significantly decreased from the third week of CIHH treatment. This blood pressure reduction effect could be maintained for at least 2 weeks after stopping the CIHH treatment. CIHH treatment also attenuated the decrease in MAP and renal sympathetic nerve activity induced by hexamethonium administration in SHRs, but not in WKY rats. Furthermore, CIHH reversed the increase in serum angiotensin (Ang)II concentration and the expression of PVN angiotensin-converting enzyme (ACE) and AngII type 1 (AT1) receptors, as well as the decrease in serum Ang1-7 concentration and the expression of PVN ACE2 and Mas receptors in SHRs. In addition, the administration of CIHH resulted in a reduction in the frequency of miniature excitatory postsynaptic currents and amplitude of NMDAR current in PVN presympathetic neurons of SHRs, which means that CIHH decreased the pre- and postsynaptic NMDAR activity of PVN presympathetic neurons in SHRs. However, pretreatment with A779 (a Mas receptor blocker) or AngII abrogated the above effects. Meanwhile, Ang1-7 pretreatment mimicked the CIHH effect on pre- and postsynaptic NMDAR activity of presympathetic neurons in SHRs. Conclusions: Our data indicate that CIHH reduces pre- and postsynaptic NMDAR activity of PVN presympathetic neurons, sympathetic outflow, and blood pressure by decreasing the activity of the ACE/AngII/AT1 axis and increasing the activity of ACE2/Ang1-7/Mas axis in the hypothalamus in hypertension.

Brain α2δ-1-Bound NMDA Receptors Drive Calcineurin Inhibitor-Induced Hypertension.

Calcineurin is highly enriched in immune T cells and the nervous system. Calcineurin inhibitors, including cyclosporine and tacrolimus (FK506), are the cornerstone of immunosuppressive regimens for preserving transplanted organs and tissues. However, these drugs often cause persistent hypertension owing to excess sympathetic outflow, which is maintained by N-methyl-D-aspartate receptor (NMDAR)-mediated excitatory input to the hypothalamic paraventricular nucleus (PVN). It is unclear how calcineurin inhibitors increase NMDAR activity in the PVN to augment sympathetic vasomotor activity. α2δ-1 (encoded by the Cacna2d1 gene), known colloquially as a calcium channel subunit, is a newly discovered NMDAR-interacting protein. In this study, we determined whether α2δ-1 plays a role in calcineurin inhibitor-induced synaptic NMDAR hyperactivity in the PVN and hypertension development. Immunoblotting and coimmunoprecipitation assays were used to quantify synaptic protein levels and the physical interaction between GluN1 (the obligatory NMDAR subunit) and α2δ-1. Whole-cell patch-clamp recordings of retrogradely labeled, spinally projecting PVN were conducted in perfused brain slices to measure presynaptic and postsynaptic NMDAR activity. Radio-telemetry was implanted in rodents to continuously record arterial blood pressure in conscious states. Prolonged treatment with FK506 in rats significantly increased protein levels of α2δ-1, GluN1, and the α2δ-1-GluN1 complex in PVN synaptosomes. These effects were blocked by inhibiting α2δ-1 with gabapentin or interrupting the α2δ-1-NMDAR interaction with an α2δ-1 C-terminus peptide. Treatment with FK506 potentiated the activity of presynaptic and postsynaptic NMDARs in spinally projecting PVN neurons; such effects were abolished by gabapentin, Cacna2d1 knockout, or α2δ-1 C-terminus peptide. Furthermore, microinjection of α2δ-1 C-terminus peptide into the PVN diminished renal sympathetic nerve discharges and arterial blood pressure that had been increased by FK506 treatment. Remarkably, concurrent administration of gabapentin prevented the development of FK506-induced hypertension in rats. Additionally, FK506 treatment induced sustained hypertension in wild-type mice but not in Cacna2d1 knockout mice. α2δ-1 is essential for calcineurin inhibitor-induced increases in synaptic NMDAR activity in PVN presympathetic neurons and sympathetic outflow. Thus, α2δ-1 and α2δ-1-bound NMDARs represent new targets for treating calcineurin inhibitor-induced hypertension. Gabapentinoids (gabapentin and pregabalin) could be repurposed for treating calcineurin inhibitor-induced neurogenic hypertension.

Hypothalamic Corticotropin-Releasing Hormone Contributes to Hypertension in Spontaneously Hypertensive Rats.

Corticotropin-releasing hormone (CRH) is a neuropeptide regulating neuroendocrine and autonomic function. CRH mRNA and protein levels in the hypothalamic paraventricular nucleus (PVN) are increased in primary hypertension. However, the role of CRH in elevated sympathetic outflow in primary hypertension remains unclear. CRHR1 proteins were distributed in retrogradely labeled PVN presympathetic neurons with an increased level in the PVN tissue in adult spontaneously hypertensive rats (SHRs) compared with age-matched male Wistar-Kyoto (WKY) rats. CRH induced a more significant increase in the firing rate of PVN-rostral ventrolateral medulla (RVLM) neurons and sympathoexcitatory response in SHRs than in WKY rats, an effect that was blocked by preapplication of NMDA receptors (NMDARs) antagonist AP5 and PSD-95 inhibitor, Tat-N-dimer. Blocking CRHRs with astressin or CRHR1 with NBI35965 significantly decreased the firing rate of PVN-RVLM output neurons and reduced arterial blood pressure (ABP) and renal sympathetic nerve activity (RSNA) in SHRs but not in WKY, whereas blocking CRHR2 with antisauvagine-30 did not. Furthermore, Immunocytochemistry staining revealed that CRHR1 colocalized with NMDARs in PVN presympathetic neurons. Blocking CRHRs significantly decreased the NMDA currents in labeled PVN neurons. PSD-95-bound CRHR1 and PSD-95-bound GluN2A in the PVN were increased in SHRs. These data suggested that the upregulation of CRHR1 in the PVN is critically involved in the hyperactivity of PVN presympathetic neurons and elevated sympathetic outflow in primary hypertension.SIGNIFICANCE STATEMENT Our study found that corticotropin-releasing hormone receptor (CRHR)1 protein levels were increased in the paraventricular nucleus (PVN), and CRHR1 interacts with NMDA receptors (NMDARs) through postsynaptic density protein (PSD)-95 in the PVN neurons in primary hypertension. The increased CRHR1 and CRHR1-NMDAR-PSD-95 complex in the PVN contribute to the hyperactivity of the PVN presympathetic neurons and elevated sympathetic vasomotor tone in hypertension in SHRs. Thus, the antagonism of CRHR1 decreases sympathetic outflow and blood pressure in hypertension. These findings determine a novel role of CRHR1 in elevated sympathetic vasomotor tone in hypertension, which is useful for developing novel therapeutics targeting CRHR1 to treat elevated sympathetic outflow in primary hypertension. The CRHR1 receptor antagonists, which are used to treat health consequences resulting from chronic stress, are candidates to treat primary hypertension.

Diet induced obesity alters the excitability of liver-related PVN neurons

Stimulation of hepatic sympathetic nerves increases glucose production and glycogenolysis. Activity of pre-sympathetic neurons in the paraventricular nucleus (PVN) of the hypothalamus and in the ventrolateral and ventromedial medulla (VLM/VMM) largely influence the sympathetic output. Despite the importance of these central circuits, the cellular properties of pre-sympathetic liver-related neurons remain to be determined. Here, we tested the hypotheses that the activity of pre-sympathetic liver-related neurons in the PVN and VLM/VMM is altered in diet induced obese (DIO) mice, as well as their sensitivity to insulin. Whole-cell patch-clamp recordings were conducted from liver-related neurons in male DIO (15–19-week-old) and control mice. During a step protocol, current steps (0-30pA, duration 1s) were applied to reveal the firing activity of neurons. Our data showed increased excitability of liver-related PVN neurons in DIO mice (n=9) compared to Control mice (n=10) (simple linear regression, different elevation, p=0.04). Moreover, in DIO mice, insulin decreased the frequency of action potentials (basal: 0.83±0.28 Hz vs insulin: 0.47±0.22 Hz, n=10; Wilcoxon test, p=0.01), while it had no effect on the firing in control mice (basal: 0.79±0.37 Hz vs insulin: 0.51±0.26 Hz, n=8, Wilcoxon test, p=0.11). Similarly, in DIO mice, insulin decreased the firing of liver-related PVN neurons after current injection (simple linear regression, different elevation, p=0.01, n=9). Next, liver-related PVN neurons were identified based on their projection to the VLM/VMM, and we found that the excitability of pre-sympathetic liver-related PVN neurons (n=7) was increased compared to liver-related PVN neurons (n=10) (simple linear regression, different elevation, p=0.00). Intriguingly, insulin did not alter the excitability of pre-sympathetic liver-related PVN neurons neither in control (n=8) or DIO mice (n=7) (simple linear regression, different elevation (Control: p=0.27; DIO: p=0.21). Since insulin sensitivities of liver-related PVN and pre-sympathetic liver-related PVN neurons were different, recordings were conducted from liver-related neurons in the VLM/VMM, which is well-known for its control over the sympathetic tone. In control mice, during the step protocol insulin had no effect on the excitability (simple linear regression, different elevation, p=0.37 n=7) whereas it decreased the firing rate of liver-related VLM/VMM neurons in DIO mice (nonlinear fit, different curve, p=0.03, n=4). Additionally, insulin receptor expression was confirmed by single cell digital droplet PCR in a subset of liver-related neurons by collecting mRNA from the cytoplasm of the recorded neurons. In summary, these results demonstrate that the firing activity of liver-related neurons are altered in DIO mice as well as their sensitivity to insulin. These data provide further evidence for cellular changes involving neurons regulating the sympathetic output to the liver. This work was supported by the NIH (DK-122842 to AZs and AVD), and Marko Spark Innovation Research Fund to AZs and AVD. We also thank the Tulane Brain Institute Cell and Tissue Imaging Core and the NIH Center for Neuroanatomy and Neurotropic viruses for the PRVs (P40 OD010996). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sympathoexcitation and pressor responses induced by acetate, an ethanol metabolite in the hypothalamic paraventricular nucleus involves activation of NMDA receptors in rats

Alcohol abuse plays a crucial role in the prevalence of cardiovascular and neuronal degenerated diseases. However, the central mechanism that mediates these effects are not fully understood. It has been well know that autonomic hypothalamic paraventricular nucleus (PVN) play an important role in regulating sympathetic outflow and arterial blood pressure (ABP). Moreover, it has been demonstrated that the increased glutamatergic activity among PVN neurons contributed to the sympathoexcitation and development of hypertension. Furthermore, we have reported that ethanol and acetate increase sympathetic outflow and arterial pressure, which may involve the activation of NMDA receptors in autonomic central nucleus of amygdala. Combing with the fact that autonomic PVN neurons are abundantly expressed NMDA receptors, we hypothesize that acetate, the metabolic products from alcohol, activates the PVN neurons through the increase in glutamatergic activity and contributes to the sympathoexcitation and development of chronic diseases, such as hypertension. In anesthetized rats, the effect of acetate microinjected in the PVN on the renal sympathetic nerve activity (RSNA) and arterial blood pressure (ABP) was determined. The PVN acetate microinjection increased sympathoexcitatiory and pressor response in a dose dependent dose-dependent (1.5mm, 0.5mm, 2.0mm, 7.5mm) manner, and 2mm of acetate showed a minimum dose evoked a maximal response. To determine the role of acetate-stimulated glutamatergic receptor activation in driving sympathoexcitatory and pressor responses, either 2mm acetate or pre-treatment of Kynurenic acid (KYN, 7.2mm) ionotropic excitatory amino acid receptor blocker, or D-2-amino-5-phosphopentanoate (AP5, 3.0mm), a N-methyl-D-aspartate (NMDA) receptor antagonist, followed by 2mm acetate were microinjected into the PVN of male Sprague Dawley rats (300-500g, n= 6-9/ group). RSNA and ABP responses were compared among vehicle (saline) and 2mm acetate, pre-treatment of KYN and 2mm acetate, or pre-treatment AP5 and 2mm acetate protocols. 2mm acetate significantly increased RSNA (60-70% baseline, p < 0.001) and mean arterial pressure (MAP, 10-12mmHg, p < 0.05). Non-selective glutamatergic receptor antagonist, KYN significantly blocked the sympathoexcitatory (RSNA, p < 0.05) and pressor response (MAP, p < 0.05) evoked by 2mm acetate in the PVN. Furthermore, selective NMDA receptor antagonist, AP5 significantly attenuates the sympathoexcitatory response induced by PVN 2mm acetate (RSNA, P < 0.05 and MAP, p < 0.01). These data indicate acetate can increase glutamatergic activity and excitability of pre-sympathetic PVN neurons via activation of local NMDA receptors. The metabolism of ethanol to acetate following by alcohol consumption may contribute, in part, to the development of neurogenic hypertension and/or other cardiovascular diseases associated with increased sympathetic outflow. R15HL145655, R15HL150703 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Corticotropin-releasing factor potentiates glutamatergic input and excitability of presympathetic neurons in the hypothalamus in spontaneously hypertensive rats

Hyperactivity of presympathetic neurons in the hypothalamic paraventricular nucleus (PVN) plays a key role in generating excess sympathetic output in hypertension. However, the mechanisms driving hyperactivity of PVN presympathetic neurons in hypertension are unclear. In this study, we determined the role of corticotropin-releasing factor (CRF) in the PVN in augmented glutamatergic input, neuronal excitability and sympathetic outflow in hypertension. The number of CRF or c-Fos immunoreactive neurons and CRF/c-Fos double-labeled neurons in the PVN was significantly greater in spontaneously hypertensive rats (SHRs) than in normotensive Wistar-Kyoto (WKY) rats. Blocking glutamatergic input reduced the CRF-potentiated excitability of spinally projecting PVN neurons. Furthermore, CRF knockdown via Crispr/Cas9 in the PVN decreased the frequencies of spontaneous firing and miniature excitatory postsynaptic currents (mEPSCs) in spinally projecting PVN neurons in SHRs. In addition, the mRNA and protein levels of CRFR1, but not CRFR2, in the PVN were significantly higher in SHRs than in WKY rats. Blocking CRFR1 with NBI-35965, but not blocking CRFR2 with Antisauvagine-30, reduced the frequencies of spontaneous firing and mEPSCs of spinally projecting PVN neurons in SHRs. Also, microinjection of NBI-35965 into the PVN significantly reduced arterial blood pressure (ABP) and renal sympathetic nerve activity (RSNA) in anesthetized SHRs, but not in WKY rats. However, microinjection of Antisauvagine-30 into the PVN had no effect on ABP or RSNA in WKY rats and SHRs. Our findings suggest that endogenous CRF in the PVN potentiates glutamatergic input and firing activity of PVN presympathetic neurons via CRFR1, resulting in augmented sympathetic outflow in hypertension.

Angiotensin II inhibits the A-type K+ current of hypothalamic paraventricular nucleus neurons in rats with heart failure: role of MAPK-ERK1/2 signaling.

Angiotensin II (ANG II)-mediated sympathohumoral activation constitutes a pathophysiological mechanism in heart failure (HF). Although the hypothalamic paraventricular nucleus (PVN) is a major site mediating ANG II effects in HF, the precise mechanisms by which ANG II influences sympathohumoral outflow from the PVN remain unknown. ANG II activates the ubiquitous intracellular MAPK signaling cascades, and recent studies revealed a key role for ERK1/2 MAPK signaling in ANG II-mediated sympathoexcitation in HF rats. Importantly, ERK1/2 was reported to inhibit the transient outward potassium current (IA) in hippocampal neurons. Given that IA is a critical determinant of the PVN neuronal excitability, and that downregulation of IA in the brain has been reported in cardiovascular disease states, including HF, we investigated here whether ANG II modulates IA in PVN neurons via the MAPK-ERK pathway, and, whether these effects are altered in HF rats. Patch-clamp recordings from identified magnocellular neurosecretory neurons (MNNs) and presympathetic (PS) PVN neurons revealed that ANG II inhibited IA in both PVN neuronal types, both in sham and HF rats. Importantly, ANG II effects were blocked by inhibiting MAPK-ERK signaling as well as by inhibiting epidermal growth factor receptor (EGFR), a gateway to MAPK-ERK signaling. Although no differences in basal IA magnitude were found between sham and HF rats under normal conditions, MAPK-ERK blockade resulted in significantly larger IA in both PVN neuronal types in HF rats. Taken together, our studies show that ANG II-induced ERK1/2 activity inhibits IA, an effect expected to increase the excitability of presympathetic and neuroendocrine PVN neurons, contributing in turn to the neurohumoral overactivity that promotes progression of the HF syndrome.

Microglial activation induced by LPS mediates excitation of neurons in the hypothalamic paraventricular nucleus projecting to the rostral ventrolateral medulla

Microglia are known to be activated in the hypothalamic para-ventricular nucleus (PVN) of rats with cardiovascular diseases. However, the exact role of microglial activation in the plasticity of presympathetic PVN neurons associated with the modulation of sympathetic outflow remains poorly investigated. In this study, we analyzed the direct link between microglial activation and spontaneous firing rate along with the underlying synaptic mechanisms in PVN neurons projecting to the rostral ventrolateral medulla (RVLM). Systemic injection of LPS induced microglial activation in the PVN, increased the frequency of spontaneous firing activity of PVN-RVLM neurons, reduced GABAergic inputs into these neurons, and increased plasma NE levels and heart rate. Systemic minocycline injection blocked all the observed LPS-induced effects. Our results indicate that LPS increases the firing rate and decreases GABAergic transmission in PVN-RVLM neurons associated with sympathetic outflow and the alteration is largely attributed to the activation of microglia. Our findings provide some insights into the role of microglial activation in regulating the activity of PVN-RVLM neurons associated with modulation of sympathetic outflow in cardiovascular diseases.

Abstract P201: Negative Regulation Of PVN Pre-sympathetic Neuronal Activity By Resident Microglia

Hypothalamic paraventricular nucleus (PVN) is a critical integrating region in controlling peripheral sympathetic tonicity. While the vast studies have unraveled the regulatory circuits affecting PVN pre-sympathetic neurons, local factors for maintaining the homeostasis of neuronal excitability are barely understood. In the present study we investigated the role of microglia, the primary resident immune cells of the CNS, in this context. By electrophysiological recording, we found that loss of resident microglia induced an increased firing frequency and attenuated outward potassium currents in the PVN pre-sympathetic neurons, tachycardia and impaired heart rate variability. Combining the transcriptomics analysis of the PVN microglia, we identified a releasable factor, which was dominantly expressed in microglia compared to other brain parenchymal cells. ICV infusion of the recombinant peptide restored potassium currents in the PVN pre-sympathetic neurons and autonomic function in microglia-depleted mice. In summary, our results provided a novel intrinsic regulatory mechanism by which microglia suppress neuronal over excitation in physiological condition.

Group III metabotropic glutamate receptors regulate hypothalamic presympathetic neurons through opposing presynaptic and postsynaptic actions in hypertension

The hypothalamic paraventricular nucleus (PVN) plays a major role in generating increased sympathetic output in hypertension. Although group III metabotropic glutamate receptors (mGluRs) are expressed in the hypothalamus, little is known about their contribution to regulating PVN presympathetic neurons in hypertension. Here we show that activating group III mGluRs with L-2-amino-4-phosphonobutyric acid (L-AP4) consistently inhibited the firing activity of spinally projecting PVN neurons in normotensive rats. However, in spontaneously hypertensive rats (SHRs), L-AP4 inhibited 45% of PVN neurons but excited 37%. L-AP4 significantly reduced glutamatergic and GABAergic input to PVN neurons in both groups. Blocking postsynaptic G protein signaling eliminated the excitatory but not the inhibitory effect of L-AP4 on PVN neurons in SHRs. Remarkably, prior activation of group I mGluRs converted the L-AP4 effect from inhibitory to excitatory in PVN neurons, and L-AP4 consistently inhibited PVN neurons when mGluR5 was blocked in SHRs. Furthermore, the expression level of mGluR4 and mGluR6 in the PVN was significantly higher in SHRs than in normotensive rats. Microinjection of L-AP4 into the PVN decreased blood pressure and lumbar sympathetic nerve discharges in normotensive rats and SHRs. Additionally, blocking group I mGluRs in the PVN potentiated L-AP4's sympathoinhibitory effect in SHRs. Therefore, activation of presynaptic group III mGluRs inhibits the excitability of PVN presympathetic neurons to attenuate sympathetic vasomotor activity. Through crosstalk with mGluR5, postsynaptic group III mGluR stimulation paradoxically excites PVN presympathetic neurons in SHRs. Concurrently blocking mGluR5 and activating group III mGluRs in the PVN can effectively reduce sympathetic outflow in hypertension.

GABAergic synaptic inhibition in the hypothalamic paraventricular nucleus (PVN) is strengthened by acute activation of glutamatergic inputs from the median preoptic nucleus

Pre‐sympathetic neurons (PSNs) in the hypothalamic PVN are normally maintained in a state of near discharge quiescence reflecting the resting dominance of GABAergic inhibition. Our studies indicate that synaptic inhibition is actively maintained in the PVN, in part, by EAAT3‐mediated uptake of L‐glutamate (Glu) into local GABAergic synaptic terminals arising from the surrounding peri‐nuclear zone (PNZ). Notably, this Glu‐GABA strengthening (GGS) mechanism relies on apparent increases both in GABA synthesis and packaging into synaptic vesicles. Studies have so far demonstrated that PVN GGS can be elicited either by exposure of brain slices to bath Glu (100 μM) or high K+ (+10 mM). Studies performed in vivo indicate that Glu nanoinjection into the PVN increases GABAergic inhibitory control of renal sympathetic nerve activity (SNA). To investigate whether endogenous synaptic Glu release can drive PVN GGS, glutamatergic EPSCs were evoked in PVN PSNs from the median preoptic nucleus (MnPO). To accomplish this, we created an angled horizontal slice preparation containing the MnPO, PVN and PNZ. Single pulse (0.5 ms, 0.5 to 1.0 mA) electrical stimulation of MnPO evoked amongst PVN PSNs (n=2) mostly unitary EPSCs (ampl.: −139 ± 5 pA, tau: 3.27 ± 0.01). In slices prepared from vGAT‐ChR2(H134R) expressing mice, GABAergic IPSCs were evoked by blue light stimulation (20 ms, 0.4 mW, 0.067 Hz) of PNZ GABA neurons before and after glutamatergic EPSCs were electrically evoked from MnPO (1 Hz, 10 min). The amplitude of photostimulated GABAergic IPSCs at baseline (−152 ± 47 pA) was increased (−220 ± 83 pA) within ~3 minutes following MnPO stimulation (n=2). PNZ eIPSC amplitude was not increased (before: −89.5 ± 9.7 pA, after: −86.5 ± 10.3, pA) when MnPO stimulation was performed in the presence of the pan‐specific Glu uptake inhibitor TBOA (100 μM). Findings indicate that excitatory inputs to PVN from the forebrain lamina terminalis can acutely strengthen GABAergic inhibition through an EAAT3 uptake‐dependent mechanism consistent with PVN GGS. Grading of PVN synaptic inhibition in pace with excitation could help prevent excitotoxic injury of PSNs during prolonged physiological challenges (e.g., water deprivation) and in chronic diseases (e.g., neurogenic hypertension) accompanied by persistently elevated SNA.Support or Funding InformationNIH HL088052 (GMT)

Glutamate strengthening of GABAergic inhibition in the hypothalamic PVN: Dependence on EAAT3‐mediated Glu uptake into local GABA terminals

In unstressed animals, pre‐sympathetic neurons (PSNs) of the hypothalamic paraventricular nucleus (PVN) are held in a state of near discharge quiescence by the dominance of GABAergic inhibition. In brain slice studies we found that the amplitude of PSN GABAergic inhibitory postsynaptic currents (IPSCs) is increased by elevated ECF L‐glutamate (Glu). This Glu‐GABA strengthening (GGS) phenomenon, originally described in the hippocampus, can be elicited in PVN PSNs both by exposure to bath Glu (100 μM) or high K+ (+10 mM) for 10 min. PVN GGS is (1) mediated by a presynaptic mechanism that increases unitary quantal size (more GABA molecules/vesicle), (2) is unaffected by inhibition of glial GLT1‐mediated Glu uptake, and (3) is prevented by global inhibition of Glu uptake. Collectively, these findings indicate that PVN GGS depends on Glu uptake by the neuronal Glu transporter EAAT3. Importantly, bath Glu elicits a delayed GABA‐AR‐mediated suppression of PSN firing, indicating that the Glu‐induced increase of IPSC amplitude (i.e., GGS) is sufficient to regulate PSN discharge quiescence. Moreover, in vivo studies indicate that PVN Glu enhances the increase of renal SNA caused by local blockade of GABA‐AR, consistent with elevated Glu increasing functional GABAergic inhibitory tone in the PVN. Here we performed PVN immunohistochemistry and found EAAT3 localized diffusely within somatodendritic compartments and densely in puncta resembling synaptic terminals. EAAT3+ puncta co‐localized with GAD65 punctate staining, consistent with EAAT3 expression on local GABAergic synaptic terminals. To test dependence of GGS on EAAT3 uptake of Glu specifically by PVN GABA terminals, we used a CRISPR/Cas9 gene editing strategy in which spCas9 was expressed in a Cre‐dependent manner amongst vGAT+ (GABAergic) neurons. To knockdown (KD) EAAT3 specifically amongst GABAergic inputs to PVN, AAV1‐CAG‐EAAT3:sgRNA‐GFP was nanoinjected (400 nl) into the surrounding peri‐nuclear zone (PNZ). Western blot analysis of tissue punches (Cntrl: n=10, CRISPR EAAT3: n=12) revealed a nearly 40% loss of EAAT3 in PVN. Given that EAAT3 amongst non‐GABAergic (i.e., glutamatergic) PVN neurons were not targeted by our CRISPR/Cas9 strategy, loss of EAAT3 specifically amongst PVN‐projecting GABAergic PNZ neurons would likely have been much greater than ~40%. Crucially, strengthening of PNZ evoked GABAergic IPSCs by bath Glu was effectively abolished (before: −238 ± 10 pA, after: −226 ± 12 pA; n=3) by CRISPR EAAT3 KD amongst PNZ GABAergic neurons. Results indicate that EAAT3‐mediated Glu uptake into PVN GABA terminals actively defends and restores PSN quiescence following acute glutamatergic excitation.Support or Funding InformationNIH HL088052 (GMT)

In Female Rats, the Sympathoexcitatory and Pressor Actions of Arcuate Nucleus Angiotensin II Vary During the Estrus Cycle and are Mediated by Inhibition of Neuropeptide Y, and Excitation of α‐Melanocyte Stimulating Hormone, Projections to the Hypothalamic Paraventricular Nucleus

In male rats, nanoinjections of Angiotensin II (AngII) into the arcuate nucleus (ArcN) increases sympathetic nerve activity (SNA) by suppressing tonic sympathoinhibitory Neuropeptide Y (NPY) inputs, and activating excitatory pro‐opiomelanocortin (POMC) inputs that release alpha melanocyte stimulating hormone (α‐MSH), into the hypothalamic paraventricular nucleus (PVN). In female rats, ArcN AngII AT1a receptor (AT1aR) expression is generally higher than in males, but varies with the estrus cycle (highest during estrus; lowest during proestrus). However, whether ArcN AngII increases SNA in females, or if the response varies during the cycle, is unknown. Therefore, we tested bilateral ArcN AngII injections (30 pmol in 30 nL) in α‐chloralose anesthetized female rats throughout the reproductive cycle, and found that 90 min after ArcN AngII injections, LSNA increased (P<0.05) in rats during estrus (by 72±12 %; n=6) and diestrus (by 40±9 %; n=4), but not proestrus (−1±8 %; n=5). ArcN AngII also induced an initial rapid rise (P<0.05) in mean arterial pressure (MAP) in all groups (estrus: 13±2 mmHg; diestrus: 11±3 mmHg; proestrus: 13±3 mmHg), but the increase was sustained only in estrus rats (by 12±2 mmHg; P<0.05). We next tested if ArcN AngII suppresses inhibitory NPY inputs by determining if blockade of PVN NPY Y1 receptors (Y1R) with bilateral nanoinjections of BIBO3304 (60 pmol/60nL) elicits smaller increases in MAP and LSNA 60 min after nanoinjection of AngII into the ArcN. As previously reported, PVN injections of BIBO3304 (without ArcN AngII; n=3) increased (P<0.05) LSNA (by 34±6 %) and MAP (by 10±1 mmHg). However, the increases (P<0.05) in LSNA (to 93±16 %) and MAP (by 19±1 mmHg) (n=4) following ArcN AngII and PVN BIBO3304 were the same as ArcN AngII followed by PVN artificial cerebrospinal fluid (n=3; LSNA by 107±30 % and MAP by 17±4 mmHg). Thus, ArcN AngII markedly attenuated the pressor and sympathoexcitatory effects of blockade of PVN Y1R, indicating that AngII increases LSNA and MAP in part by inhibition of NPY projections to the PVN. Finally, because suppression of tonic NPY inhibition of PVN presympathetic neurons permits increased activation of PVN α‐MSH receptors (MC4R), we tested if blockade of PVN MC4R with SHU9119 decreases LSNA and MAP 90 min after ArcN AngII (n=4). Indeed, bilateral PVN SHU9119 decreased (P<0.05) LSNA (by 30±10 %) and MAP (by 10±1 mmHg). In conclusion, in females as in males, ArcN AngII increases SNA and MAP by inhibiting NPY inputs and increasing α‐MSH inputs into the PVN. However, these effects vary during the estrus cycle in parallel to AngII AT1aR expression: highest during estrus and undetectable during proestrus.Support or Funding InformationHL128181

Endogenous AT1 receptor-protein kinase C activity in the hypothalamus augments glutamatergic input and sympathetic outflow in hypertension.

The angiotensin AT1 receptor expression and protein kinase C (PKC)-mediated NMDA receptor phosphorylation levels in the hypothalamus are increased in a rat genetic model of hypertension. Blocking AT1 receptors or PKC activity normalizes the increased pre- and postsynaptic NMDA receptor activity of hypothalamic presympathetic neurons in hypertensive animals. Inhibition of AT1 receptor-PKC activity in the hypothalamus reduces arterial blood pressure and sympathetic nerve discharges in hypertensive animals. AT1 receptors in the hypothalamus are endogenously activated to sustain NMDA receptor hyperactivity and elevated sympathetic outflow via PKC in hypertension. Increased synaptic N-methyl-d-aspartate receptor (NMDAR) activity in the hypothalamic paraventricular nucleus (PVN) plays a major role in elevated sympathetic output in hypertension. Although exogenous angiotensin II (AngII) can increase NMDAR activity in the PVN, whether endogenous AT1 receptor-protein kinase C (PKC) activity mediates the augmented NMDAR activity of PVN presympathetic neurons in hypertension is unclear. Here we show that blocking AT1 receptors with losartan or inhibiting PKC with chelerythrine significantly decreased the frequency of NMDAR-mediated miniature excitatory postsynaptic currents (mEPSCs) and the amplitude of puff NMDA currents of retrogradely labelled spinally projecting PVN neurons in spontaneously hypertensive rats (SHRs). Also, treatment with chelerythrine abrogated the potentiating effect of AngII on mEPSCs and puff NMDA currents of labelled PVN neurons in SHRs. In contrast, neither losartan nor chelerythrine had any effect on mEPSCs or puff NMDA currents in labelled PVN neurons in Wistar-Kyoto (WKY) rats. Furthermore, levels of AT1 receptor mRNA and PKC-mediated NMDAR phosphorylation in the PVN were significantly higher in SHRs than in WKY rats. In addition, microinjection of losartan or chelerythrine into the PVN substantially reduced blood pressure and renal sympathetic nerve discharges in SHRs but not in WKY rats. Chelerythrine blocked sympathoexcitatory responses to AngII microinjected into the PVN. Our findings suggest that endogenous AT1 receptor-PKC activity is essential for presynaptic and postsynaptic NMDAR hyperactivity of PVN presympathetic neurons and for the augmented sympathetic outflow in hypertension. This information advances our mechanistic understanding of the interplay between angiotensinergic and glutamatergic excitatory inputs in hypertension.

Group III Metabotropic Glutamate Receptors Regulate Excitability of Hypothalamic Presympathetic Neurons and Sympathetic Output in Hypertension

Enhanced glutamatergic input to the hypothalamic paraventricular nucleus (PVN) contributes to the development of hypertension. Group III metabotropic glutamate receptors (mGluRs) are expressed in the PVN. In the present study, we determined the role of group III mGluRs in the control of the excitability of spinally projecting PVN neurons and sympathetic outflow in hypertension. We found that stimulation of group III mGluRs with L‐(+)‐2‐amino‐4‐phosphonobutyric acid (L‐AP4) consistently inhibited the firing activity of PVN neurons in normotensive WKY rats. However, L‐AP4 inhibited 37%, but excited 48%, of spinally projecting PVN neurons in spontaneously hypertensive rats (SHRs). Both the inhibitory and excitatory effects of L‐AP4 on the firing activity were abolished by the group III mGluR antagonist CPPG. Inclusion of GDP‐β‐S in the intracellular recording solution eliminated the excitatory effect of L‐AP4 on the firing activity of PVN neurons in SHRs, but it did not significantly change the inhibitory effect of L‐AP4 on PVN neurons in WKY rats or SHRs. L‐AP4 significantly reduced the frequency of spontaneous excitatory postsynaptic currents of PVN neurons in both WKY rats and SHRs. Interestingly, pretreatment of brain slices with the group I mGluR agonist DHPG converted the L‐AP4 effect on PVN neurons from inhibitory to excitatory in WKY rats. Also, L‐AP4 consistently inhibited PVN neurons in SHRs after treatment with the group I mGluR antagonist MPEP. In addition, microinjection of L‐AP4 into the PVN dose‐dependently decreased arterial Bp and lumbar sympathetic nerve activity (LSNA) in WKY rats and SHRs. Microinjection of L‐AP4 in the presence of MPEP produced a more significant reduction in arterial BP and LSNA than did with microinjection of L‐AP4 alone in SHRs. These findings suggest that group III mGluRs in the PVN regulate the synaptic plasticity and excitability of PVN presympathetic neurons and sympathetic vasomotor activity in hypertension. The signaling of postsynaptic group III mGluRs is influenced by increased group I mGluR activity in PVN presympathetic neurons in hypertension. Supported by NIH grants HL131161 and HL142133.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Combined V1a and V1b Receptor Activation Within the Paraventricular Nucleus Contributes to Sympathoexcitation and Hypertension in Rats Exposed to Chronic Mild Unpredictable Stress

The paraventricular nucleus (PVN) in the brain has been recognized as a major integrative center for neurohumoral regulation which receives a broad array of afferent inputs. As such, the function of the PVN becomes even more important in times of physical or physiological stress. Two distinct neuronal populations we are interested in include the magnocellular and parvocellular neurons. Specifically, we are interested in deciphering how intranuclearly released vasopressin (AVP) from the magnocellular neurons acts upon the parvocellular pre‐sympathetic neurons and glia that are richly endowed with V1a and V1b receptors in a rat model of depression. It has been demonstrated that V1a receptor activation depolarizes PVN pre‐sympathetic neurons and initiates an action potential, which puts forth a signaling cascade that increases sympathetic nerve activity, arterial pressure and heart rate. Additionally, we previously published that renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) responses to AVP microinjected in the PVN is significantly attenuated when the rats were pretreated with V1b receptor antagonist nelivaptan. In this study, we set out to identify differences in baseline RSNA, MAP and HR between rats exposed to the chronic mild unpredictable stress protocol (CMS) and their normal counterparts, as well as their responses to AVP microinjected into the PVN in the presence of the V1a receptor antagonist, Manning compound, and the V1b receptor inhibitor, nelivaptan. We observed that baseline MAP is higher in CMS rats (110 ± 2 mmHg vs. 115 ± 1 mmHg; P < 0.05). Furthermore, in control rats, combined inhibition of AVP with nelivaptan and Manning compound resulted in near complete abolition of the rise in MAP and baroreflex‐mediated decreases in HR and RSNA. CMS rats exhibited exaggerated baseline RSNA, 43.6 ± 9.97 μV.ms compared with control rats, 8.04 ± 3.24 μV.ms (P < 0.025). PVN AVP injection increased RSNA by 52.1 ± 3.3 %baseline in controls and was blocked by the dual blockade. RSNA did not increase significantly after PVN AVP in CMS rats and V1a receptor and V1b receptor blockade did not change this response. CMS rats displayed higher baseline RSNA 43.6 ± 9.9 μV.ms vs control rats 8.0 ± 3.9 μV.ms P < 0.025). Moreover, dual blockade significantly decreased RSNA from baseline values only in CMS rats: −12.6 ± 0.4 μV.ms compared with control rats −1.1 ± 0.6 μV.ms (P < 0.05). These findings are consistent with the concept that arterial pressure and RSNA are increased during chronic stress via dendritic release of AVP within the PVN thereby contributing to sympathoexcitation and hypertension.Support or Funding InformationDepartment of Veterans Affairs Merit Award to NFRThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impaired Hypothalamic Regulation of Sympathetic Outflow in Primary Hypertension.

The hypothalamic paraventricular nucleus (PVN) is a crucial region involved in maintaining homeostasis through the regulation of cardiovascular, neuroendocrine, and other functions. The PVN provides a dominant source of excitatory drive to the sympathetic outflow through innervation of the brainstem and spinal cord in hypertension. We discuss current findings on the role of the PVN in the regulation of sympathetic output in both normotensive and hypertensive conditions. The PVN seems to play a major role in generating the elevated sympathetic vasomotor activity that is characteristic of multiple forms of hypertension, including primary hypertension in humans. Recent studies in the spontaneously hypertensive rat model have revealed an imbalance of inhibitory and excitatory synaptic inputs to PVN pre-sympathetic neurons as indicated by impaired inhibitory and enhanced excitatory synaptic inputs in hypertension. This imbalance of inhibitory and excitatory synaptic inputs in the PVN forms the basis for elevated sympathetic outflow in hypertension. In this review, we discuss the disruption of balance between glutamatergic and GABAergic inputs and the associated cellular and molecular alterations as mechanisms underlying the hyperactivity of PVN pre-sympathetic neurons in hypertension.