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

Abstract

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.

Introduction

The elevated sympathetic vasomotor tone is critically involved in the pathogenesis of primary hypertension (DiBona, 2013), which is a risk factor for stroke, heart failure, and renal failure (Lewington et al., 2002; Ramchandra et al., 2013; Huang et al., 2014). Although many attempts have been made to elucidate the pathogenesis of hypertension, especially primary hypertension, the etiology of primary hypertension remains to be determined. The elevated sympathetic outflow is an important mechanism involved in developing and maintaining essential hypertension (Goncharuk et al., 2002; Li and Pan, 2007; Ramchandra et al., 2013). The heightened sympathetic outflow in hypertension is attributed to the hyperactivity of neurons controlling sympathetic nerve activity in several brain regions including the paraventricular nucleus (PVN) of the hypothalamus (Goncharuk et al., 2002; Ramchandra et al., 2013). The presympathetic PVN neurons provide excitatory drive to sympathetic outflow in hypertension (Li et al., 2017; Dampney et al., 2018; J.J. Zhou et al., 2019) through projections to the rostroventral medulla (RVLM) and intermediolateral (IML) cell column in the spinal cord. Although extensive studies have been performed, the etiology of heightened sympathetic outflow in hypertension remains unknown.

The corticotropin-releasing hormone (CRH) is a neuropeptide which mainly synthesized in the PVN (Bale and Vale, 2004; Aguilera and Liu, 2012) and regulates stress response (Sukhareva, 2021). CRH binds to CRHR1 and CRHR2, which belong to G-protein coupled receptor family and are expressed in peripheral tissues and the brain (Slater et al., 2016) including the PVN (Perrin et al., 1993; Bale and Vale, 2004), CRHR1 is expressed at a higher level than CRHR2 and CRH has a higher affinity for CRHR1 than for CRHR2 in the CNS (Bale and Vale, 2004; Garcia et al., 2016). It has been shown that CRH mRNA expression levels in the PVN are increased in primary hypertension patients (Goncharuk et al., 2002, 2007). Central administration of CRH also elevates arterial pressure, heart rate (HR), and sympathetic outflow (Fisher et al., 1983; Nijsen et al., 2000). CRH-expressing neurons in the PVN activate neighboring interneurons and projecting neurons through CRFR1 (Jiang et al., 2018). However, the role of CRH signals in elevated sympathetic outflow in primary hypertension remains unclear.

The NMDA receptors (NMDARs) are critically involved in regulating neurons excitability, synaptic transmission, and neuronal plasticity in a variety of diseases, including chronic pain (Lau and Zukin, 2007; Li et al., 2008) and neurogenic hypertension (Cull-Candy et al., 2001; H.Y. Zhou et al., 2011). Functional NMDARs mainly compose 2 GluN1 and 2 GluN2 (GluN2A and GluN2B) subunits in PVN neurons (Herman et al., 2000; Furukawa et al., 2005; Sun et al., 2017). The NMDARs activity in PVN is increased in hypertension to support elevated sympathetic outflow (Li and Pan, 2007; Ye et al., 2012; Ma et al., 2018). The postsynaptic density protein-95 (PSD-95) is a member of the membrane-associated guanylate kinase family, which plays an important role in the assembly of receptors and intracellular signaling proteins (Kim and Sheng, 2004; Bender et al., 2015). PSD-95 directly links to NMDAR, promotes NMDAR clustering, and stabilizes the surface and synaptic expression of NMDARs (Lin et al., 2004, 2006; Won et al., 2016). Previous studies have shown that CRHR1 directly interacts with PSD-95 through a PDZ (PSD-95, discs large, zona occludes 1) binding motif located in the C-terminal of CRHR1 to directly affects receptor function (Dunn et al., 2013, 2016). This study identified the role of CRHR1-NMDARs interaction in regulating sympathetic outflow in primary hypertension.

Materials and Methods

Animals

Adult (12–13 weeks) male Wistar–Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs; Envigo) were used in this study. The animal protocol and surgical procedure were approved by the Institutional Animal Care and Use Committee of The University of Missouri (#9439) and conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The rats were housed (two to three rats per cage) in a 12/12 h light/dark cycle and maintained at a controlled temperature (24–25°C) with food and tap water ad libitum.

Immunoblotting

Rats subjected to immunoblotting analysis were decapitated under 5% isoflurane anesthesia in 100% O₂. Quickly removed the brain and placed it in ice-cold artificial CSF (aCSF) saturated with a mixture of 95% O₂ and 5% CO₂. Tissue samples were obtained from rat brain slices using the micro-punch method as described previously (Ma et al., 2018). Total proteins were extracted using RIPA lysis buffer (Thermo Fisher Scientific 89901) containing a mixture of protease inhibitors (Halt Protease Inhibitor Single-Use Cocktail 100×, Thermo Scientific 78430) according to the manufacturer's instruction. Tissue samples were homogenized and centrifuged at 12,000 × g for 30 min at 4°C to obtain the supernatant. The protein concentrations were determined by a Pierce Rapid Gold BCA Protein Assay kit (Thermo Scientific A53225). The proteins were denatured by SDS protein gel loading solution and were subjected to 5−10% SDS PAGE for being separated and transferred to a PVDF membrane (Millipore) followed by blockage with 3% BSA for 1 h and incubation with antibodies overnight at 4°C. Then, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-goat IgG (1:8000; Jackson ImmunoResearch) for 1 h at room temperature. An ECL kit (Thermo Scientific 34580) was used to detect and visualize the protein bands under the Odyssey Fc Imager system (LI-COR Biosciences), which was also used to qualify the densities of protein bands and normalized to the reference band on the same gel. The following antibodies were used during the immunoblotting: anti-CRHR1 (1:500, Invitrogen, catalog #PA5-18801), anti-CRHR2 (1:500, ABclonal Technology, catalog #A7659), anti-PSD-95 (1:500, Abcam, catalog #ab238135), and anti-GAPDH antibody (1:500, Cell Signaling Technology, catalog #5174).

For co-immunoprecipitation (Co-IP), a Pierce Co-IP kit (Thermo Scientific 26 149) was used to pull down the proteins with an anti-PSD-95 antibody. In brief, the tissues were dissected and homogenized in ice-cold IP lysis buffer containing a mixture of protease inhibitors and incubated on ice for 30 min. The lysates were centrifuged at 12,000 × g for 30 min at 4°C to obtain the supernatants. The protein concentrations were determined, and a total of 1 mg proteins were used to incubate at 4°C overnight with Protein-G beads pretreated to rabbit anti-PSD-95 antibody. Protein-G beads prebound to rabbit IgG were used as controls. Samples were washed three times with immunoprecipitation buffer and then immunoblotted with detected the bands with the following antibodies: anti-PSD-95, anti-CRHR1, anti-GluR1-NT (1:500, MilliporeSigma, catalog #MAB2263), anti-NMDAR2B (1:500, Abcam, catalog #ab254356), and anti-NMDAR2A (1:500, Abcam, catalog #ab169873). The mean band density values of in respective groups in untreated groups were considered as 1.0.

Celiac ganglionectomy and blood pressure measurement

We performed celiac ganglionectomy (CGx) or sham surgery on SHRs (Ye et al., 2011; Li et al., 2012). After a midline laparotomy, the celiac ganglion plexus was identified near the superior mesenteric artery. Then the celiac plexus and all visible nerves connected to the celiac ganglion plexus were dissected and stripped completely. The celiac ganglion plexus was exposed in the sham control rats but not disturbed. Two weeks after CGx or sham surgery, the rats' blood pressure was measured via a noninvasive tail-cuff system (CODA, Kent Scientific), and their brain tissues were harvested for immunoblotting analysis.

Retrograde labeling of RVLM-projecting PVN neurons

The RVLM-projecting PVN neurons were retrograded and identified as described in our previous studies (Li et al., 2017). Rats were anesthetized by 2–3% isoflurane, and their heads were fixed in a stereotactic frame. After exposing the skull, two holes (1 mm in diameter) were drilled bilaterally through the skull using a micromotor drill at 13 mm caudal to the bregma and 2 mm lateral to the midline. A microinjection pipette (tip diameter, 20–30 μm) filled with DiI (2–4%) or FluoSpheres (0.04 μm, Invitrogen) was advanced targeting the RVLM (7.5 mm ventral to the surface of the brain) through a micromanipulator. The microinjection was conducted using a calibrated microinjection system (NanojectorIII, Drummond Scientific) in 100-nl injections bilaterally (50 nl in each side) and monitored using a surgical microscope. The rats were returned to their home cages for 14 d to allow the FluoSpheres to be transported into the PVN. The rats were monitored continuously during the surgery, including paw/tail pinch reflex, palpebral reflex, and spontaneous movement. After surgery, Baytril (10 mg/kg) and Carprofen (5 mg/kg) were injected through intraperitoneal immediately after the surgery and in three consecutive days postsurgery.

Brain slices preparation

The rats were decapitated under anesthesia with 5% isoflurane, and the brain was quickly removed and put into the ice-cold artificial CSF (aCSF) containing (in mm) 126.0 NaCl, 3.0 KCl, 1.5 MgCl₂, 2.4 CaCl₂, 1.2 NaH₂PO₄, 10.0 glucose, and 26.0 NaHCO₃ (290–300 mOsmol/l) saturated with 95% O₂ and 5% CO₂. Coronal slices in a thickness of 400 μm containing the PVN were sectioned using a vibrating microtome (VT1200S, Leica Biosystems Inc.). The slices were incubated in the aCSF at 37°C for at least 1 h before recording.

Electrophysiological recordings

The slices were put into the recording chamber and perfused by aCSF (saturated with 95% O₂ and 5% CO₂) at a 3.0 ml/min speed at 37°C maintained by an in-line solution heater. The labeled RVLM projected PVN neurons were identified under an upright microscope equipped with fluorescent illuminance (BX51WI; Olympus). The whole-cell patch-clamp recording electrodes were pulled from borosilicate capillaries (1.2 mm outer diameter, 0.68 mm inner diameter; World Precision Instruments) by a micropipette puller (P-1000; Sutter Instruments). The resistance of the pipette was 3–6 MΩ when it was filled with an internal solution containing (in mm) 140.0 K gluconate, 2.0 MgCl₂, 0.1 CaCl₂, 10.0 HEPES, 1.1 EGTA, 0.3 Na₂-GTP, and 2.0 Na₂-ATP (pH 7.25 adjusted with 1.0 m KOH, 280–290 mOsm). After the whole-cell configuration was established, the spontaneous firing activities were recorded in current-clamp mode with a holding current 0 pA. The miniature EPSCs (mEPSCs) were recorded in the presence of 1 μmol/l tetrodotoxin (TTX) and 20 μmol/l bicuculline at a holding potential of −60 mV. The miniature IPSCs (mIPSC) were recorded in the presence of 1 μmol/l tetrodotoxin (TTX) and 10 μmol/l 6,7-dinitroquinoxaline-2,3-dione (DNQX) at a holding potential of 0 mV. The evoked EPSCs were elicited by electrical stimulation (0.2 ms, 0.8–1.0 mA at 0.2 Hz) through a bipolar tungsten electrode connected to a stimulator as we used before. In brief, the evoked AMPA receptor (AMPAR)-EPSCs were recorded at a holding potential of −60 mV in the presence of 10 μmol/l bicuculline and blocked by the AMPAR antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 μmol/l). The evoked NMDAR-EPSCs were recorded at a holding potential of +40 mV in the presence of 10 μmol/l bicuculline and 10 μmol/l DNQX, and blocked by NMDARs antagonist, AP5 (50 μmol/l). The inward NMDA currents elicited by transient bath application of NMDA (20 μmol/l) were recorded at a holding potential of −60 mV in an Mg²⁺ free aCSF containing glycine (10 μmol/l) and tetrodotoxin (1.0 μmol/l; Naskar and Stern, 2014). The NMDA current was quantified as the change in the holding current and verified by AP5. The electrophysiology signals were recorded using a Multiclamp 700B amplifier (Molecular Devices), filtered at 1–2 kHz, and digitized at 20 kHz by a Digidata 1550B (Molecular Devices).

Immunohistochemical staining

Immunohistochemical staining was performed to determine the expression of CRHR1 in RVLM-projecting PVN neurons and the colocalization between CRHR1 and NMDAR subunit GluN2A in the PVN. Rats with DiI injected into the RVLM were rapidly perfused with 4% paraformaldehyde, and the brain was quickly removed and fixed by 4% paraformaldehyde overnight. Brain slices were sectioned to 30 μm thick by a cryostat. After blocking with 10% serum solution, brain slices were incubated with the primary anti-CRHR1 and anti-GluN2A antibodies for 2 h at room temperature and overnight in 4°C. Subsequently, sections were incubated with the secondary antibodies (IgG conjugated to Alexa Fluor 488 or 594, dilution 1:200) and viewed using a confocal microscope and the PVN areas were photographed.

PVN microinjection and recording of hemodynamics and renal sympathetic nerve activity (RSNA)

We performed PVN microinjection and recording of arterial blood pressure (ABP), RSNA, and heart rate (HR), as described previously (Li et al., 2017; Sheng et al., 2022). Briefly, rats were anesthetized by a mixture of urethane (800 mg/kg, i.p.) and α-chloralose (60–75 mg/kg, i.p.) mechanically ventilated with 100% O₂ using a rodent ventilator through the trachea. The CO₂ concentration in exhale was monitored with a CO₂ analyzer and maintained at 4% to 5% by adjusting the ventilation rate (50–70 breaths per minute) or tidal volume (2–3 ml). The pulsatile ABP was monitored through a catheter inserted into the femoral artery, and HR was extracted from the pulsatile pressure signal. A small branch of the isolated left renal postganglionic sympathetic nerve was put on a bipolar silver recording electrode to record the nerve activity, which was amplified, bandpass filtered (100–3000 Hz) using a differential high-impedance amplifier (AM3000H, A-M Systems), and monitored via an audio amplifier. The RSNA and ABP were digitally recorded at 10 kHz by an analog-to-digital interface and LabChart Pro (ADInstruments). At the end of the experiment, the electrical background noise was determined by an intravenous bolus injection of phenylephrine (4 μg/kg), which raised the ABP and led to complete inhibition of RSNA. The noise level was subtracted from the integrated RSNA values. The percentage change in RSNA from the baseline was calculated. ABP, RSNA, and HR were continuously recorded throughout the experiment.

Then, the stereotaxic surgery for PVN microinjection was started following the surgical preparation of recording of ABP, HR, and RSNA. The dorsal surface of the skull was exposed, and a small hole was drilled to expose the brain, when the rat heads were fixed on a stereotactic apparatus. The agents in the volume of 100 nl for each injection were bilaterally microinjected into the PVN through a 31-gauge needle connected to a 1-μl microsyringe (Hamilton). The following coordinates were used for microinjection in the PVN: 1.7–1.9 mm caudal to the bregma; 0.5 mm lateral to the midline; and 7.2–7.5 mm ventral to the dura. To determine the location of the injection site and diffusion of the drugs in the PVN, we included 5% rhodamine-labeled fluorescent microspheres (0.04 μm; Invitrogen) in the injection solution. After completion of each experiment, the rat brain was removed and fixed in a 4% paraformaldehyde solution. Frozen coronal sections (50 μm thick) were cut, and FluoSpheres fluorescent regions were identified under a microscope and plotted on standardized sections from the atlas of Paxinos and Watson. In the case where the microinjection site was outside the PVN, the data from this rat were excluded from the analysis.

After completion of the surgical procedures of PVN microinjection, ABP, HR, and RSNA were continually monitored for at least 20–30 min. An observation that no significant fluctuation of these parameters during a 5- to 10-min period indicated that these cardiovascular measures reached a stable baseline. Then, a baseline of ABP, HR, and RSNA were recorded for 10–15 min followed by microinjections of vehicles (aCSF) or agents into the PVN. Each microinjection took 30 s, and the glass injection needle was left in place for 1 min to ensure the agents were adequately delivered to the injection site. The injection needle was withdrawn and immediately placed into the contralateral PVN based on the respective stereotactic coordinates for injection and followed by 30 min recording of ABP, HR, and RSNA. The response latency of ABP, LSNA, and HR to each agent injection was 3–5 min. All the agents used for microinjection were dissolved in ddH₂O and diluted into the final concentration in aCSF. Microinjection of aCSF into the PVN did not affect ABP, HR, and RSNA. Full-length CRH was purchased from Phoenix Pharmaceuticals. Astressin was purchased from MedChemExpress. NBI35965 and antisauvagine-30 were purchased from R&D Systems. AP5 was obtained from Hello Bio and Tat-N-Dimer was purchased from Millipore Sigma.

Statistical analysis

Data were presented as means ± SEM. Normal distribution was assessed for statistical analysis by using the Shapiro–Wilk test (p < 0.05 was considered as not conforming to a normal distribution). A two-tailed Student's t test was used to compare values between two groups and repeated-measure (RM) ANOVA (RM one-way ANOVA followed by Tukey's post hoc test, and RM two-way ANOVA followed by Bonferroni's multiple-comparison test) was used to determine differences between more than two groups. The amplitude and frequency of mEPSCs were analyzed offline with peak detection software (MiniAnalysis; Synaptosoft Inc.). Clampfit, version 10.2 (Molecular Devices) was used to calculate the electrophysiological recording data. The mean ABP, RSNA, and HR were analyzed using Spike2 software. Statistical analyses were performed using Prism, version 7 (GraphPad Software Inc.); p < 0.05 was considered statistically significant.

Data availability

The data underlying this article are available in the article and in its online supplementary material.

Results

CRHR1 protein expression in PVN was increased in SHRs

Using western blot analysis, we first determined CRHR expression levels in the PVN in WKY and SHR. The CRHR1 protein levels in the PVN were significantly higher in SHRs than in WKY rats (SHRs n = 6 rats, WKY n = 6 rats, unpaired t test, t₍₁₀₎ = 2.608, p = 0.0261). The CRHR1 protein levels in the hippocampus did not differ between WKY and SHRs (SHRs n = 6 rats, WKY n = 6 rats, unpaired t test, t₍₁₀₎ = 0.4673, p = 0.6503; Fig. 1A,B). The CRHR2 protein levels in the PVN and hippocampus did not differ between WKY rats and SHRs (Fig. 1A,B). Moreover, CRHR1 protein expression levels did not differ in the frontal cortex and the RVLM between SHR and WKY rats (Extended Data Fig. 1-1A and B). Next, we determined the distribution of CRHR1 in retrogradely labeled PVN-RVLM neurons. PVN-RVLM neurons were retrogradely labeled by DiI injected into the RVLM. Brain sections containing DiI-labeled PVN neurons were immunostained with antibody against CRHR1. All negative controls displayed no detectable staining (Extended Data Fig. 1-1). Almost all DiI-labeled PVN neurons (red) were CRHR1-positive (green) in the PVN (Fig. 1C).

Figure 1.

CRHR1 protein level was significantly increased in the PVN in SHRs. A, Representative blot images and summary data show that CRHR1 protein expression levels were higher in SHRs than in WKY rats, while CRHR2 expression levels in the PVN did not differ between SHRs and WKY rats. B, CRHR1 and CRHR2 expressions in the hippocampus were similar between WKY rats and SHRs. C, Immunocytochemical staining images show that CRHR1 immunoreactivities were distributed on retrogradely labeled PVN-RVLM neurons. D, E, Celiac ganglionectomy (CGX) significantly decreased blood pressure in SHRs, (D) but the CRHR1 expression levels were not changed in the PVN in SHRs subjected to CGX (E). *p < 0.05, ***p < 0.001 compared with the baseline value within the group or in WKY rats. Extended Data Figure 1-1 contains more information.

Extended Data Figure 1-1

CRHR1 protein expression in frontal cortex and RVLM in SHRs and WKY rats. A and B: Representative blot images (upper panel) and summary data (lower panel) show that CRHR1 protein expression levels did not differ in frontal cortex (A) and RVLM (B) between SHR and WKY rats. C: Immunocytochemical staining images show that slice incubation with secondary antibody (IgG conjugated to Alexa fluor 488, dilution 1:200) without primary antibody did not label any neuron. Download Figure 1-1, docx file ^{(656.8KB, docx)} .

To determine whether the increased expression of CRHR1 in the PVN in SHRs was a secondary response to the higher blood pressure in SHRs, celiac ganglionectomy (CGx) was used to lower the blood pressure in SHRs, and the CRHR1 protein levels in the PVN were determined. We found that CGx surgery significantly decreased the blood pressure in SHRs (baseline: 145.60 ± 4.39 mmHg, CGx: 113.57 ± 1.95 mmHg, n = 6 rats, paired t test, t₍₅₎ = 8.127, p = 0.0005) and lasted for at least two weeks. The CRHR1 protein levels in the PVN were not significantly different between sham and CGx (n = 6 rats in each group, unpaired t test, t₍₁₀₎ = 0.3983, p = 0.6988; Fig. 1D,E). These results suggest that the upregulation of CRHR1 in PVN of SHRs is independent of blood pressure change.

The role of CRHR1 in mediating hyperactivity of presympathetic PVN neurons in SHRs

We next determined the role of CRHR1 in controlling the activity of presympathetic PVN neurons. PVN-RVLM output neurons were retrogradely labeled by Fluorspheres injected into the RVLM (Fig. 2A). The membrane properties, including input resistance, cell capacitance, and access resistance of labeled PVN neurons, were not significantly different between WKY rats and SHRs (Extended Data Table 1-1). Bath application of CRH (100 nm) significantly increased the firing rate of PVN-RVLM neurons in both WKY rats (n = 8 neurons from three rats) and SHRs (n = 7 neurons from 3 rats; RM two-way ANOVA, CRH effect: F_(2,26) = 73.79, p < 0.0001, baseline comparison: F_(1,13) = 179.4, p < 0.0001; Fig. 2B,C, left). CRH induced increases in firing rate in WKY rats were significantly smaller than those in SHRs (unpaired t test, t₍₁₃₎ = 4.973, p = 0.0003; Fig. 2C, right panel). In addition, the CRH significantly depolarized membrane potentials in WKY rats and SHRs (RM two-way ANOVA, CRH effect: F_(2,26) = 128.6, p < 0.0001, baseline value comparison: F_(1,13) = 49.07, p < 0.0001; Fig. 2D, left panel). CRH-induced a significantly greater membrane depolarization in SHRs than in WKY rats (unpaired t test, t₍₁₃₎ = 2.19, p = 0.0473; Fig. 2D, right panel). On the other hand, a CRHR antagonist, astressin, or a CRHR1 specific antagonist, NBI35965, decreased the firing rate of PVN-RVLM neurons in SHRs without changing the firing activity in WKY rats (n = 7–10 neurons from three rats, RM two-way ANOVA; astressin effect: F_(2,30) = 13.04, p < 0.0001, baseline value comparison: F_(1,15) = 6.062, p = 0.0264; NBI35965 effect: F_(2,26) = 60.27, p < 0.0001, baseline value comparison: F_(1,13) = 101, p < 0.0001; Fig. 2E). A CRHR2 specific antagonist, antisauvagine-30, did not alter the firing rate in both WKY rats and SHRs (RM two-way ANOVA, antisauvagine-30 effect: F_(2,26) = 2.242, p = 0.1264, baseline value comparison: F_(1,13) = 35.75, p < 0.0001; Fig. 2E). In addition, bath application of CRH did not change the frequency and amplitude of miniature EPSCs or miniature IPSCs of labeled PVN neurons in WKY rats and SHRs (Extended Data Fig. 2-1).

Figure 2.

CRHR1 plays an important role in regulating the activity of PVN-RVLM neurons. A, Left, diagram of microinjection of tracer into the RVLM and representative image showing the microinjection site of Fluospheres. Right, Images show the retrogradely labeled neuron in PVN under light and fluorescent illuminant. B–D, Representative traces and histograms (B) and summary data (C) show that bath application of CRH induced a greater increase in the firing rate and depolarization of membrane potentials (D) in retrogradely labeled PVN-RVLM neurons in SHRs than in WKY rats. E, Blocking CRHRs by astressin, a nonspecific CRHRs antagonist, significantly decreased the firing rate in SHRs but not in WKY rats (upper). Blocking CRHR1 with NBI35965 (middle panel), a selective CRHR1 antagonist, rather than antisauvagine-30 (low panel), a selective CRHR2 antagonist, (lower) significantly decreased the firing rate in SHRs. Please note that blockade of CRHR1 or CRHR2 had no effects on the baseline firing rate of PVN neurons in WKY rats. ***p < 0.001, ****p < 0.0001 compared with the baseline values in each group. #p < 0.05, #p < 0.05, ###p < 0.001, ####p < 0.0001 compared with the corresponding values in WKY rats. Extended Data Figure 2-1 contains more information.

Extended Data Table 1-1

Electrophysiological membrane properties of labelled PVN neurons in WKY and SHR. Download Table 1-1, DOCX file ^{(12.1KB, docx)} .

Extended Data Figure 2-1

CRH had no effects on synaptic inputs to spinally projecting PVN-RVLM neurons. Representative traces (A and B) and cumulative curves (C and D), and summary data (E and F) show CRH did not change the frequency and amplitude of miniature excitatory postsynaptic currents of labeled PVN neurons in WKY rats and SHRs. Representative traces (G and H) and cumulative curves (I and J), and summary data (K and L) show CRH did not change the frequency and amplitude of miniature inhibitory postsynaptic currents of labeled PVN neurons in WKY rats and SHRs. Download Figure 2-1, docx file ^{(367.4KB, docx)} .

CRHR1 played an essential role in regulating blood pressure and sympathetic outflow in SHRs

Since CRH-CRHR1 critically regulates the firing activity of PVN-RVLM neurons, we determine whether CRH-CRHR1 in the PVN are involved in regulating blood pressure and sympathetic outflow in vivo. Bilateral microinjection of CRH at low (0.15 nmol/100 nl) and high (0.3 nmol/100 nl) doses into the PVN increased mean arterial blood pressure (ABP, RM two-way ANOVA, CRH effect: F_{(2.396,28.75)} = 27.88 p < 0.0001, baseline value comparison: F_(1,12) = 57.44, p < 0.0001), heart rate (HR, RM two-way ANOVA, CRH effect: F_{(2.125,25.50)} = 16.40 p < 0.0001, baseline value comparison: F_(1,12) = 40.02, p < 0.0001), and renal sympathetic nerve activity (RSNA, RM one-way ANOVA, F_{(1.867,11.20)} = 47.55, p < 0.0001) in SHRs (n = 7 rats; Fig. 3A). In WKY rats, bilateral microinjection of CRH at a lower dose (0.15 nmol/100 nl) did not alter the mean ABP, HR, and RSNA, and a higher dose (0.3 nmol/100 nl) significantly increased these variables (n = 7 rats; Fig. 3A). The baseline mean ABP and heart rate were significantly higher in SHRs than in WKY rats (Fig. 3A). We also checked the injection site and excluded the data if the microinjections were misplaced outside the PVN (Fig. 3B). We then found that the bilateral microinjection of CRHR nonspecific antagonist, astressin at a dose of 0.12 nmol/100 nl, significantly decreased the mean ABP (n = 6 rats, RM two-way ANOVA, astressin effect: F_(2,20) = 25.13 p < 0.0001, baseline value comparison: F_(1,10) = 55.89, p < 0.0001), HR (RM two-way ANOVA, astressin effect: F_(2,20) = 3.973, p = 0.0352, baseline value comparison: F_(1,10) = 36.67, p = 0.0001) and RSNA (RM one-way ANOVA, F_{(1.324,6.621)} = 18.28, p = 0.0031) in SHRs but did not change these variables in WKY rats (Fig. 3C). In addition, bilateral microinjection of CRHR1 specific antagonist, NBI 35 965 (30 pmol/100 nl; Robinson et al., 2019) also significantly decreased the mean ABP (n = 6 rats, RM two-way ANOVA, NBI35965 effect: F_(2,20) = 21.93, p < 0.0001, baseline value comparison: F_(1,10) = 8.14, p = 0.0172), HR (RM two-way ANOVA, NBI35965 effect: F_(2,20) = 17.67, p < 0.0001, baseline value comparison: F_(1,10) = 3.478, p = 0.0918) and RSNA (RM one-way ANOVA, F_{(1.774,8.872)} = 50.42, p < 0.0001) in SHRs but not in WKY rats (Fig. 3D,E). However, bilateral microinjection of CRHR2 antagonist, antisauvagine-30 (50 pmol/100 nl; Gao et al., 2016) did not alter mean ABP (RM two-way ANOVA, antisauvagine-30 effect: F_(2,20) = 1.241, p = 0.3104, baseline value comparison: F_(1,10) = 46.34, p < 0.0001), HR (RM two-way ANOVA, antisauvagine-30 effect: F_(2,20) = 0.7022, p = 0.5073, baseline value comparison: F_(1,10) = 9.240, p = 0.0125), and RSNA in WKY rats or SHRs (n = 6 rats per group; Fig. 3F). These data suggested that the increased expression level of CRHR1 in PVN is critically involved in elevated sympathetic outflow in primary hypertension.

Figure 3.

CRHR1 in the PVN was critical in regulating blood pressure and sympathetic outflow in SHRs. A, Representative traces (left), and summary data (right) show that microinjection of CRH into the PVN increased blood pressure (BP), heart rate (HR), and renal sympathetic nerve activity (RSNA) in WKY rats and SHRs. Please note that microinjection of CRH produced greater pressor responses in SHRs than in WKY rats. B, Microphotography and brain atlas depict the microinjection sites in the PVN in WKY rats and SHRs. C, Representative traces (upper) and summary data (lower) show that microinjection of a CRHR antagonist, astressin, into the PVN decreased the BP, RSNA, and HR in SHRs but not in WKY rats. D, E, Representative traces (D) and summary data (E) show that microinjection of CRHR1-selective antagonist NBI 35 965 into the PVN decreased the BP, RSNA, and HR in SHRs but not in WKY rats. F, Summary data show that microinjection of CRHR2 antagonist antisauvagine-30 did not affect BP, RSNA, or HR in either WKY rats or SHRs. *p < 0.05, **p < 0.01, ****p < 0.0001 compared with the baseline values in each group. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 compared with the corresponding values in the WKY rats.

PSD-95 and NMDARs were involved in the CRHRs signal pathway in PVN in SHRs

NMDAR is critically involved in regulating neurons excitability, and its expression levels and activity of NMDAR subunits in the PVN are increased in hypertension (Vyklicky et al., 2014; Li and Pan, 2017). PSD-95 is closely associated with NMDARs through binding to the PDZ-binding motif of the GluN2A subunit (Dong et al., 2004; Lin et al., 2006; Stanic et al., 2015). It has been shown that PSD-95 is colocalized with CRHR1 via the PDZ-binding motif CRHR1 (Dong et al., 2004; Bender et al., 2015). We determined whether NMDAR-PSD-95 signals mediated the action of CRHR1 in the PVN in controlling blood pressure and sympathetic vasomotor tone in hypertension. First, we performed double-immunostaining using antibodies against CRHR1 and NMDAR subunit GluN2A. We found that the majority of CRHR1-immunoreactive neurons (green) were GluN2A positive (red) in the PVN (Fig. 4A). The co-localization of CRHR1 and GluN2A connotes a functional interaction to regulate neuronal activity. We then determined whether CRHRs regulates NMDARs function by recording the evoked NMDAR-EPSCs and NMDA-induced currents in PVN-RVLM neurons in WKY rats and SHRs. In brain slice preparation, blocking the CRHRs with astressin at a concentration of 200 nm significantly decreased the amplitude of evoked NMDAR-EPSCs (n = 8 neurons from 3 rats; RM two-way ANOVA, astressin effect: F_(2,24) = 18.33, p < 0.0001, baseline value comparison: F_(1,12) = 5.972, p = 0.0309; Fig. 4B,C) and NMDA currents (n = 7 neurons from three rats, two-way ANOVA followed by unpaired t test, vehicle comparison: F_(1,11) = 0.9098, p = 0.3607, astressin effect: t₍₁₂₎ = 4.115, p = 0.0014; Fig. 4D,E) in SHRs. However, astressin did not alter evoked NMDAR-EPSCs and NMDA currents in PVN-RVLM neurons in WKY rats (Fig. 4B–E). The evoked NMDA-EPSCs and NMDA currents were eliminated by bath application of 50 μm AP5 (Extended Data Fig. 4-1).

Figure 4.

Relationship between CRHR1 and NMDARs. A, Double immunostaining shows that CRHR1-immunoreactivities (green) were colocalized with GluN2A immunoreactivities (red) in the majority of PVN neurons. B, C, Representative traces (B) and summary data (C) show that CRHRs antagonist (astressin, 200 nm) significantly decreased evoked NMDAR-EPSCs in retrogradely labeled PVN-RVLM neurons in SHRs but not in WKY rats. D, Representative raw traces show NMDA currents elicited by bath application of 20 μm NMDA in labeled PVN neurons in the vehicle-treated group and in brain slices treated with astressin (200 nm) in SHR and WKY rats. E, Summary data shows that NMDA induced a smaller current in astressin pretreated group than vehicle-treated group in SHRs but not in WKY rats. **p < 0.01, ****p < 0.0001 compared the baseline values in each group. #p < 0.05, ##p < 0.01 compared with the corresponding values in WKY rats. Extended Data Figure 4-1 contains more information.

Extended Data Figure 4-1

Protocols used to record the evoked NMDA-EPSCs and NMDA currents in PVN-RVLM neurons. A: The evoked NMDA-EPSCs were recorded at a holding potential of +40 mV in the presence of 10 μM DNQX, an antagonist for non-NNDA receptors, and 20 μM bicuculline, an antagonist for GABAA receptors. B: NMDA currents elicited by bath application of 20 μM NMDA were blocked by 50 μM AP5. Download Figure 4-1, docx file ^{(138.2KB, docx)} .

Furthermore, we determined whether CRHR1 directly interacts with NMDARs by conducting co-immunoprecipitation. The anti-PSD-95 antibody was used to pull down protein complexes from the PVN tissues obtained from WKY rats and SHRs. The anti-CRHR1, anti-GluN2A, anti-GluN2B and anti-GluN1 antibodies were used to measure the PSD-95-bound CRHR1, GluN2A, GluN2B, or GluN1 by immunoblotting. The results revealed that the PSD-95 co-precipitated with CRHR1, GluN2A, and GluN2B but not GluN1 (Fig. 5A; Extended Data Fig. 5-1). Both PSD-95-bound CRHR1 and PSD-95-bound GluN2A levels were significantly increased in SHRs compared with those in WKY rats (CRHR1: n = 5 rats, unpaired t test, t₍₈₎ = 2.423, p = 0.0417, GluN2A: n = 5 rats, unpaired t test, t₍₈₎ = 2.965, p = 0.018; Fig. 5A). However, PSD-95-bound GluN2B levels were not significantly different between WKY rats and SHRs (n = 6 rats in each group, unpaired t test, t₍₁₀₎ = 0.6383, p = 0.5376; Extended Data Fig. 5-1). The PSD-95 protein expression level was significantly increased in the PVN tissue obtained from SHRs compared with that in WKY rats (n = 5 rats, unpaired t test, t₍₈₎ = 2.544, p = 0.0345; Fig. 5B).

Figure 5.

PSD-95 interacts with CRHR1 and NMDARs. A, Proteins from hypothalamic tissues of WKY and SHR rats were immunoprecipitated by anti-PSD-95 or anti-IgG antibody, and immunoblotting (IB) was performed using anti-GluN2A, anti-GluN1, and anti-CRHR1 antibodies. The representative blot images show that PSD-95 pulled down GluN2A and CRHR1 but not GluN1. Furthermore, PSD-95 bound GluN2A subunit, and PSD-95-bound CRHR1 were increased in SHRs compared with those in WKY rats. B, PSD-95 expression level was increased in SHRs. *p < 0.05 compared with values in WKY rats. C, Representative traces and summary data show that NMDAR antagonist AP5 eliminated CRH-induced excitatory effect, or astressin-induced inhibitory effect, on the firing rate in retrogradely labeled PVN-RVLM neurons in SHRs. AP5 also significantly decreased the baseline firing rate of PVN-RVLM neurons in SHRs. D, PSD-95 inhibitor, Tat-N-dimer, eliminated CRH-induced excitatory, or astressin-induced inhibitory effect, on the firing rate in PVN-RVLM neurons in SHRs, and significantly decreased the baseline firing rate of PVN-RVLM neurons in SHRs. *p < 0.05 and ***p < 0.001 compared with the baseline values in each group, ns: no significant. Extended Data Figure 5-1 contains more information.

Extended Data Figure 5-1

PSD-95 interacts with NMDARs-GluN2B. Proteins from hypothalamic tissues of WKY and SHR rats were immunoprecipitated by anti-PSD-95 or anti-IgG antibody, and immunoblotting (IB) was performed by using anti-PSD-95 and anti-GluN2B antibodies. The representative blot images show that PSD-95 pulled down GluN2B (upper panel). Furthermore, PSD-95 bound GluN2B subunit had no significant different between WKY and SHR rats (lower panel). Download Figure 5-1, docx file ^{(271.1KB, docx)} .

We then determined whether NMDAR and PSD-95 mediate the effect of CRHR1 by using NMDAR antagonist AP5 or PSD-95 inhibitor Tat-N-dimer. Bath application of AP5 or Tat-N-dimer decreased the baseline firing rate of PVN-RVLM neurons in SHRs. Bath application of AP5 (50 μm) or Tat-N-dimer (0.5 μm) eliminated CRH-induced increases in the firing activity of PVN-RVLM neurons in SHRs. Furthermore, astressin did not alter the firing activity of PVN-RVLM neurons in the presence of AP5 (50 μm) or Tat-N-dimer (0.5 μm; RM one-way ANOVA, AP5 + CRH: F_{(1.911,15.29)} = 6.428, p = 0.0101, AP5 + Astressin: F_{(1.349,9.442)} = 2.615, p = 0.1343, Tat-N-dimer + CRH: F_(1.704,8.52) = 5.662, p = 0.0306, Tat-N-dimer + Astressin: F_{(2.127,12.76)} = 5.801, p = 0.015; Fig. 5C,D).

Finally, we determined whether NMDARs mediated the depressor and sympathoinhibitory responses induced by the antagonism of CRHRs in SHRs. AP5 and astressin were subsequently microinjected into the PVN and ABP, HR, and RSNA were measured in SHRs. Bilateral microinjection of AP5 (7.5 nmol/100 nl) significantly decreased the means ABP (RM one-way ANOVA, F_{(1.213,6.066)} = 14.35, p = 0.0076), HR (RM one-way ANOVA, F_{(1.607,8.036)} = 12.55, p = 0.0044) and RSNA (RM one-way ANOVA, F_{(1.113,5.566)} = 13.29, p = 0.0114). Following AP5 injection, bilateral microinjection of astressin 0.12 nmol/100 nl) did not alter the mean ABP, HR, and RSNA in SHRs (n = 6 rats; Fig. 6). These data suggested that CRHRs interact with NMDARs through binding to PSD-95 and affect NMDAR activity in the PVN-RVLM neurons to support elevated blood pressure and sympathetic outflow in SHRs.

Figure 6.

Blockade of NMDARs in the PVN eliminated depressor and sympathoinhbitory responses induced by antagonizing CRHRs in SHRs. Representative traces (A) and summary data (B) depict that bilateral microinjection of NMDARs antagonist AP5 into the PVN significantly decreased BP, RSNA, and HR in SHRs. Subsequent microinjection of CRHR antagonist astressin did not decrease BP, RSNA, and HR in these SHRs. n = 6 rats, *p < 0.05, **p < 0.01compared with the baseline values in each group.

Discussion

This study determined the role of CRHR1 in controlling sympathetic vasomotor tone in primary hypertension and the underlying mechanism. We found that CRHR1 protein level is higher in the PVN in SHRs than in WKY rats. Compared with WKY rats, activation of CRHR1 with CRH produced more significant increases in firing activity of PVN presympathetic neurons and sympathetic vasomotor tone, while blocking CRHR1 decreased the firing activity of PVN presympathetic neurons and sympathetic vasomotor tone in SHRs. These data suggest that CRHR1 is tonically activated to support hyperactivity of the PVN presympathetic neurons and elevated sympathetic vasomotor tone. Furthermore, we found that PSD-95 interacted with CRHR1 and GluN2A in the PVN tissue, and the PSD-95 bound CRHR1 and PSD-95 bound GluN2A were significantly increased in SHRs compared with WKY rats. CRHR antagonist decreased evoked NMDAR-EPSCs and NMDA currents in the PVN-RVLM neurons in SHRs. Finally, we found that the NMDAR antagonist eliminated depressor and sympathoinhibitory effects induced by blocking CRHRs in the PVN in SHRs. Our findings provide insights into understanding the role of CRHR1 in regulating blood pressure and the underlying mechanisms.

SHR strain was established from outbred Wistar rats selected for high blood pressure and is an often-used hypertension rodent model that may resemble, to a certain extent, essential human hypertension. WKY strain was established from the same parental Wistar stock as the SHR and has been commonly used as a control strain for the SHR. Because of the complicated breeding history of the SHR and WKY rats and possible genetic contamination, considerable genetic divergence and complex relationships exist among the SHR and WKY substrains (Zhang-James et al., 2013). Genomic studies have shown that SHRs and WKY rats are as genetically dissimilar as SHR and normotensive Sprague Dawley (SD) rats (Zimdahl et al., 2004). We realize that SHRs are not a bona fide model of human essential hypertension. In this study, we used SHRs to study the neurobiology of hypertension. Our previous studies used SD rats and WKY rats as normotensive controls to determine the hypothalamus GABAergic plasticity in controlling sympathetic vasomotor tone (Li and Pan, 2006; Li et al., 2008). No difference was noticed between SD and WKY rats in our studies. Thus, we used only WKY rats as normotensive control in the current study.

CRHR1 and CRHR2 are distributed in the PVN (Hashimoto et al., 2001; Bale and Vale, 2004; Slater et al., 2016). We found that the CRHR1 expression levels were increased in the PVN, but not other brain regions, such as hippocampus and RVLM, in SHRs compared with WKY rats. Our immunostaining revealed that CRHR1 was expressed on retrogradely labeled PVN-RVLM neurons. These data suggest that upregulated CRHR1 play an important role in regulating PVN-RVLM neuron activity in SHRs. However, CRHR2 expression levels in the PVN did not differ between SHR and WKY rats. Although CRHR2 is indicated in the regulation of blood pressure and autonomic nerve activity in other brain regions, such as the nucleus of the solitary tract (NTS; Wang et al., 2018, 2019) and RVLM (Bardgett et al., 2014), blocking CRHR2 with its specific antagonist did not alter that firing activity of PVN-RVLM neurons from either SHRs or WKY rats. Furthermore, microinjection of CRHR2 antagonist into the PVN did not change blood pressure and sympathetic outflow in either SHRs or WKY rats. We determined that the upregulation of CRHR1 is not because of high blood pressure since lowing blood pressure with CGx surgery did not alter CRHR1 expression level in the PVN. Instead, the increased CRHR1 level in the PVN may contribute to hypertension. Our finding supports this notion that blocking CRHR1 with astressin or NBI decreased the firing activity of PVN-RVLM neurons and reduced the elevated sympathetic outflow in SHRs but not WKY rats. These data suggest that CRHR1 is tonically activated in hypertension while playing a minor role in regulating PVN presympathetic activity and sympathetic outflow in normotensive conditions. Thus, the application of CRH produces a higher stimulatory effect on the firing activity of PVN-RVLM neurons in SHRs than in WKY rats. However, the elevation of blood pressure induced by microinjection of CRH into the PVN of WKY rats did not increase blood pressure to the level of SHRs. Although our findings suggest that a greater density of CRHRs in the PVN supports the higher sympathetic outflow in SHRs, it is unclear whether the source of CRH that generate exaggerated CRH-CRHR1 activity in SHRs. Previous studies have shown that CRH contents in the hypothalamus do not differ between adult SHRs and WKY rats (Hattori et al., 1986), while the CRH mRNA level is higher in SHRs than in WKY rats (Krukoff et al., 1999). The CRH measured in hypothalamus tissue includes extracellular CRH and CRH within the CRH neurons, which project to other brain regions such as median eminence, locus coeruleus the NTS (Valentino et al., 1992; Aguilera and Liu, 2012). Although activation of CRH neurons leads to sequential CRH release and synthesis and CRH neurons are distributed in many brain regions (Aguilera and Liu, 2012), it is not clear whether CRH neuron activity in SHRs is increased and the local extracellular CRH concentrations in the PVN are higher in SHRs than in WKY rats. It has been shown that glucocorticoid negative feedback on adrenocorticotropic hormone release is reduced in SHRs (Gómez et al., 1998), however, it remains unknown whether the glucocorticoid negative feedback on CRH neuron activity is defective in SHRs.

The most salient finding of our present study was revealing an interaction between CRHR1 and NMDARs. Previous studies have shown that CRHR1 interacts with PSD-95 by binding to N-terminal tandem PSD-95/discs large/zona occludens-1 (PDZ) domain 1 or 2 alone, or a combination of 1–2 (Bender et al., 2015). NMDARs are heteromeric protein complexes containing 2 GluN1 subunits and two GluN2 subunits (GluN2A and GluN2B; Cull-Candy et al., 2001; H.Y. Zhou et al., 2011). The PDZ domain 1 and 2 of PSD-95 bind to GluN2A and GluN2B subunits to regulate NMDAR function (Niethammer et al., 1996; Zhao et al., 2015). This GluN2A-PSD-95 association is critical for the postsynaptic expression of GluN2A-containing NMDARs (Lin et al., 2004; Lin et al., 2006). Thus, these data suggest that CRHR1 interacts with NMDARs through PSD-95, and this interaction regulates the firing activity of the PVN presympathetic neurons. Given the potential overlap of binding in PDZ domains of PSD-95, one would expect that CRHR1 and NMDARs compete for binding to PSD-95. However, CRHR1, GluN2A, and GluN2B were detected in the complex pulled down using PSD-95 antibody. It is likely that PSD-95 tandem PDZ domains 1 and 2 simultaneously bind to CRHR1 and GluN2A or GluN2B of NMDARs. Thus, although both CRHR1-bound PSD-95 and GluN2A-bound PSD-95 were increased in SHRs compared with WKY rats, the PSD-95 expression level was slightly increased, rather than a superposed increase, in SHRs. The greater PSD-95 expression level may account for the greater pull-down of CHR1 and GluN2A protein levels in SHR compared with WKY rats. Furthermore, we found that CRH-induced excitation of firing activity of PVN-RVLM neurons was eliminated by pretreatment of the brain slice with NMDAR antagonist AP5 or PSD-95 inhibitor Tat-N-dimer. Tat-N-Dimer was designed from GluN2B9c interference peptide binding PSD-95 PDZ1–2 domains with high affinity to uncouple PSD-95 binding to GluN2B subunit of NMDAR (Bach et al., 2012). It has been shown that Tat-NR2B9c uncouples bindings between PSD-95 to GluN2A, GluN2B, or GluN2C. Furthermore, Tat-NR2B9c induces a more potent inhibition of PSD-95 binding to NR2A than NR2B (Cui et al., 2007). Thus, in addition to the uncoupling interaction between PSD-95 and GluN2B, Tat-A-Dimer also uncouples the binding of PSD-95 to GluN2A.

In this study, we found that blocking NMDARs with AP5, or inhibiting PSD-95 with Tat-N-Dimer, decreased the basal firing activity, blocked the CRH-induced increase in firing activity, and occluded the decrease in firing activity of PVN-RVLM neurons produced by blocking CRHR1 with astressin. These data suggest that CRHR interacts with NMDARs to support the hyperactivity of PVN-RVLM neurons in SHRs. Furthermore, our in vivo data showed that microinjection of AP5 into the PVN occluded the reduction of sympathetic outflow produced by blocking CRHRs with Astressin in the PVN in SHRs. We acknowledge that this study did not include in vivo experiment testing whether microinjection of AP5 into the PVN blocks CRH-induced increases in blood pressure and sympathetic outflow. AP5 competitively binds to the glutamate binding site of NMDAR to block its gating (Olverman et al., 1988), while Tat-N-Dimer dissociates PSD-95 with NMDARs (Bach et al., 2012) rather affects NMDAR channel gating (Lin et al., 2004). Because PSD-95 suppresses the CRHR1 endocytosis in the dendrites of cortical neurons in a PDZ motif-dependent manner (Dunn et al., 2016), and stabilizing membrane NMDARs (Lin et al., 2004; Won et al., 2016), Tat-N-Dimer promotes a rapid internalization of NMDARs or CRHR1 through removing PSD-95-mediated stabilization of NMDARs or CRHR1 (Lin et al., 2004; Dunn et al., 2016; Won et al., 2016). Furthermore, Tat-N-Dimer can interfere with NMDA-stimulated reactive oxygen species and nitric oxide production (Bach et al., 2012), and uncouple interaction between NMDAR and K⁺ channels (Naskar and Stern, 2014; Zhang et al., 2017). Thus, the Tat-N-Dimer-induced reduction of neuronal firing activity cannot be only attributed to uncoupling the interaction between CRHR1 and PSD-95.

Our Co-IP data showed that CRHR1, GluN2A, and GluN2B were detected in complex pull-down by PSD-95 antibody. Furthermore, compared with WKY rats, PSD-95-bound CRHR1 and PSD-95-bound GluN2A were increased in the PVN tissue in SHRs, while the PSD-95-bound GluN2B did not differ between SHR and WKY rats. These data are consistent with previous findings that GluN2A-containing NMDAR are increased more in the PVN in SHRs (Ye et al., 2012). Also, we found that CRHR1 immunoreactivities were colocalized with GluN2A in the PVN neurons, providing morphologic evidence at the molecular level that CRHR1 directly interacts with NMDARs. We further observed that astressin reduced the amplitude of evoked NMDAR-EPSCs, suggesting that CRHR1 links to GluN2A-containing NMDAR, which is increased in the PVN neurons in SHR (Ye et al., 2012). Astressin also reduced the currents induced by bath application of NMDA, which suggests that CRHR1 links to GluN2B-containing NMDARs (Zhang et al., 2017). These data suggest that CRHR1 interacts with both GluN2A-containing and GluN2B-containing NMDARs. Among the NMDAR subunits, the GluN2 subunit determines many biophysical and pharmacological properties of NMDARs, including downstream signaling, trafficking, and synaptic targeting (Cull-Candy et al., 2001). In the PVN, NMDARs comprise two GluN1 subunits and two GluN2A or GluN2B subunits (Herman et al., 2000). The subunit composition of NMDARs is not static but dynamically changes in response to neuronal activity or sensory experience and during development (Carmignoto and Vicini, 1992; Barria and Malinow, 2002; Bellone and Nicoll, 2007; Matta et al., 2011). A switch from GluN2B to GluN2A at the synaptic site has been observed in the PVN and many other brain regions during postnatal development (Monyer et al., 1994). Although both GluN2A and GluN2B protein levels in the PVN are increased in SHRs, the GluN2A-NMDAR currents are increased more than GluN2B-NMDAR currents in PVN neurons in SHRs (Ye et al., 2012).

Our previous studies have shown that glutamatergic inputs, especially postsynaptic NMDA receptor activity in the PVN is augmented and led to heightened sympathetic vasomotor tone in SHR (Li and Pan, 2007; Li et al., 2008). Many studies have been performed to seek the mechanism underlying enhanced hypothalamic NMDAR activity in SHRs. In this regard, increases in NMDAR phosphorylation in the hypothalamus are attributed to increases in kinase activity such as casein kinase I, casein kinase II, Src kinase, Ca²⁺/calmodulin dependent protein kinase (Li and Pan, 2017; J.J. Zhou et al., 2019). In addition, altering protein phosphatase 1 and protein phosphatase 2B increases NMDAR phosphorylation. Our recent study found that increased NMDAR bounding protein α2δ1 in the PVN contributes to the enhanced NMDAR activity and hyperactivity of PVN neurons in SHRs (Ma et al., 2018). In this study, we found that an increased expression level of CRHR1 enhances NMDAR activity in the presympathetic PVN neurons in SHR. It has been shown that CRHR1 interacts with PSD-95, a postsynaptic protein that determines NMDAR activity. PSD-95 likely links CRHR1 and NMDAR, and an increase in the CRHR1-PSD-95-NMDAR interaction contributes to the hyperactivity of presympathetic PVN neurons and elevated sympathetic outflow in SHRs. These findings indicate a new mechanism responsible for increased NMDAR activity in the hypothalamus in SHRs. Enhanced CRHR1-NMDAR interaction in the PVN presympathetic neurons is involved in high blood pressure of SHRs, a model of hypertension. It is unclear whether enhanced CRHR1-NMDAR interaction in the PVN is also increased in nongenetic models of hypertension such as AngII-induced hypertension, renal hypertension, or hypertension in humans, which are warranted to be determined in future studies.

A limitation of this study is that a high lipophilic dye DiI was used to retrogradely label PVN neurons in our immunohistochemical staining. Because of its lipophilic nature and the large injection volume of DiI, DiI might diffuse through a certain distance and reach brain regions outside of the RVLM. This raises concern that the retrogradely labeled PVN neurons may not exclusively represent neurons that specifically project to the RVLM. However, based on the injection site and diffusion size, DiI was deposited within the RVLM region with a limited amount diffused outside the RVLM (Fig. 2A). Thus, the DiI-labeled PVN neurons represent the majority of PVN neurons projecting to the RVLM. Another concern is that DiI potentially enters axonal terminals and passing-by fibers, labeling PVN neurons not projecting to the injection site. A similar pattern of labeled neurons in the PVN was observed when using a nonlipophilic tracer Fluospheres, which is confined to its injection sites. Therefore, DiI-labeled PVN neurons likely represent presympathetic PVN neurons innervating the RVLM.

Perspectives

This study found that the upregulation of CRHR1 in the PVN is involved in the hyperactivity of the PVN presympathetic neurons, which drive elevated sympathetic vasomotor tone in hypertension through interaction with NMDARs in SHRs. Therefore, blockade of CRHR1 decreased the firing activity of PVN presympathetic neurons and sympathetic vasomotor tone in SHRs. Our findings provide information on developing novel therapeutics targeting CRHR1 to treat elevated sympathetic vasomotor tone and hypertension. We expect that the downregulation of hypothalamic CRHR1 decreases sympathetic outflow and blood pressure. In addition, we propose reducing sympathetic outflow and blood pressure by using CRHR1 antagonist, which has been used to treat the health consequences of chronic stress. Future studies are warranted to delineate the mechanisms involved in the upregulation of CRHR1 in the PVN in hypertension.