Role of Piezo1 Channels Expressed in PVN in Regulating Sympathetic Nerve Activity and Arterial Blood Pressure in Rats

TL;DR

This study shows that Piezo1 channels in a specific brain region control sympathetic nerve activity and blood pressure in rats.

Contribution

The study is the first to demonstrate the role of Piezo1 channels in the paraventricular nucleus in regulating sympathetic and cardiovascular function.

Findings

Blocking Piezo1 channels in the PVN increased renal sympathetic nerve activity and blood pressure in a dose-dependent manner.

Piezo1 channels are abundantly expressed in the PVN and are located in pre-sympathetic neurons projecting to the medulla.

Activating Piezo1 channels in the PVN did not significantly alter sympathetic nerve activity or blood pressure.

Abstract

Mechanosensitive Piezo1 channels participate in regulating pain sensitivity, insulin secretion, and vascular tension; however, their expression in the autonomic paraventricular nucleus (PVN) and role in modulating sympathetic outflow and cardiovascular function remain unstudied. In this study, unilateral PVN microinjection of the Piezo1 channel blocker Dooku1 (0.1, 1, 10, 100, and 200 pmol) administered to anesthetized male rats increased renal sympathetic nerve activity (RSNA) and mean artery pressure (MAP) in a dose-dependent manner, with maximum increases of 93 ± 30% (p < 0.0001) and 21 ± 5 mmHg (p < 0.0001), respectively, elicited by Dooku1 at 100 pmol. Similarly, PVN microinjection of the peptide Piezo1 channel blocker GsMTx4 (1 nmol) significantly increased RSNA (p < 0.001) and MAP (p < 0.0001). Conversely, PVN-microinjected Piezo1 channel activators Yoda1 (5 nmol) and Jedi2 (5 nmol) did not significantly alter RSNA or MAP. Western blot and qRT-PCR analyses of the hypothalamic PVN showed abundant Piezo1 mRNA and protein expression. Immunofluorescence detection showed that Piezo1 was expressed in pre-sympathetic PVN neurons with axons projecting to the rostral ventrolateral medulla. We conclude that Piezo1 channels expressed in the autonomic PVN neurons play an important role in regulating sympathetic outflow and cardiovascular function.

Figures

Full text

1. Introduction

The hypothalamic paraventricular nucleus (PVN) is an important nuclear cluster that maintains the body’s internal environment homeostasis and coordinates the functions of the neuroendocrine and autonomic nervous systems [1,2]. The neuronal activity of the autonomic PVN participates in the regulation of the sympathetic outflow and cardiovascular function [3,4]. In pathophysiological states such as cardiovascular disease, diabetes, and obesity, the increased activity of PVN neurons is closely associated with the hyper-sympathetic outflow [5,6,7,8,9]. Evidence suggests that endogenous active substances such as leptin [8,10], nitric oxide (NO) [11,12,13], carbon monoxide (CO) [12,14], hydrogen sulfide (H_2_S) [15,16], angiotensin II (ANG II) [17,18], and microRNAs (miRNAs) [19,20] in the PVN are involved in the regulation of sympathetic outflow and cardiovascular function. It has also been reported that voltage-gated ion channels such as voltage-gated potassium channel subfamily 4 member 2 (Kv4.2) and voltage-gated potassium channel subfamily 4 member 3 (Kv4.3) [21] are involved in affecting the in vitro activity of pre-sympathetic PVN neurons. Moreover, it has been demonstrated that ligand-gated ion channels in the PVN, including γ-aminobutyric acid type A receptor (GABA_a_R) [22,23], N-methyl-D-aspartate receptor (NMDAR) [9,24], α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) [25,26], and small-conductance Ca^2+^-activated K^+^ (SK) channels [27,28,29], play an important role in regulating both in vitro PVN neuronal activity and in vivo sympathetic nerve activity (SNA). However, little is known about the role of mechanically gated ion channels expressed in the PVN in regulating cardiovascular function [30]. In particular, there is no report regarding how Piezo1 channels expressed in the autonomic PVN neurons influence sympathetic outflow and arterial blood pressure (ABP).

Piezo1 mechanosensitive ion channels could play an important role in regulating the excitability of PVN neurons. Molecular biology and functional studies have revealed that Piezo1 channels are expressed in the central nervous system [31,32], as well as in the cerebellum, the dorsal root ganglia (DRG) sensory neurons [33], and the nucleus tractus solitarius [34]. Intriguingly, a previous study reported that the mechanical pain threshold is increased in Piezo1-Transgenic mice, which suggests that inhibition of Piezo1 channels increases the excitability of the pain-sensitive DRG neurons [35]. Additionally, studies have shown that blocking the Piezo1 channels enhances the traction force of CD^8+^ T cells, thereby strengthening their tumor-killing efficiency [36]. Furthermore, it has been reported that Piezo1 channels expressed in the vagal nodose ganglion (NG) neurons play an important role in the regulation of cardiovascular function via the parasympathetic nerve system [34]. Following increased membrane tension, Piezo1 channels can non-selectively mediate the influx of Na^+^ and Ca^2+^ [33,37,38,39].

So far, there have been no reports on the expression and physiological function of Piezo1 channels expressed in the autonomic PVN neurons. Thus, the present study tests the hypothesis that Piezo1 channels expressed in autonomic PVN neurons are involved in the regulation of sympathetic outflow and cardiovascular function in anesthetized rats.

2. Results

2.1. Piezo1 Channels Gene and Protein Expression in PVN

Protein and mRNA expression analyses confirmed the distribution characteristics of Piezo1 channels across multiple tissues. Western blotting assays detected the 286 kDa Piezo1 protein in the right cardiac ventricles (RVs), right cardiac auricles (RAs), lung tissue, and paraventricular nucleus of the hypothalamus (PVN) (Figure 1A). Quantitative analysis of band grayscale (normalized to GAPDH) revealed no significant inter-group differences in the relative expression of Piezo1 protein among the tissues; specifically, the Piezo1 protein level in the PVN showed no statistical differences compared to that in the RV, RA, or lung (RV vs. PVN: p > 0.05; RA vs. PVN: p > 0.05; lung vs. PVN: p > 0.05) (Figure 1B). Quantitative real-time PCR analysis further detected Piezo1 mRNA expression in the PVN and cerebral cortex (CC) tissue. Quantitative results (normalized to GAPDH) indicated no significant difference in the relative Piezo1 mRNA expression between the cerebral cortex and PVN (p > 0.05) (Figure 1C). Immunofluorescence staining results demonstrated that Piezo1 is localized in PVN pre-sympathetic neurons projecting to the rostral ventrolateral medulla (RVLM) (Figure 1D).

2.2. Effects of PVN-Injected Piezo1 Channel Blocker on RSNA, MAP, and HR

Microinjection of the Piezo1 channel antagonist Dooku1 into the unilateral PVN evoked significant (Figure 2A) dose-dependent elevations in RSNA (Figure 2B) and MAP (Figure 2C). Likewise, Dooku1 administration into the PVN induced dose-dependent increases in both RSNA and MAP across all tested doses (0.1 pmol, n = 6; 1 pmol, n = 6; 10 pmol, n = 7; 100 pmol, n = 9; 200 pmol, n = 9; Figure 3). The maximal response, observed at the 100 pmol dose, comprised a 93 ± 30% (p < 0.0001) rise in RSNA and a 21 ± 5 mmHg (p < 0.0001) increase in MAP but did not affect HR (20 ± 15 beats/min; p > 0.05).

To further substantiate the neuronal excitatory effect resulting from the specific Piezo1 channel blockade, we examined the impact of the peptidic Piezo1 inhibitor GsMTx4 on RSNA and MAP (Figure 4A), and the following results were observed: Unilateral microinjection of GsMTx4 into the PVN elicited pronounced elevations in RSNA (Figure 4B) and MAP (Figure 4C), paralleling the effects of Dooku1. Likewise, administration of GsMTx4 (1 nmol, n = 6) into the PVN produced significant increases in RSNA (85 ± 24%; p < 0.001) and MAP (15 ± 2 mmHg; p < 0.0001) but did not affect HR (5 ± 7 beats/min; p > 0.05). Dose-dependent augmentation of RSNA and MAP was observed following PVN microinjection of GsMTx4 (0.1 nmol, n = 6; 1 nmol, n = 6; Figure 4).

The effects of Dooku1 and GsMTx4 were found to be anatomically specific to the PVN region. For instance, microinjection of Dooku1 (100 pmol, n = 4) and GsMTx4 (1 nmol, n = 4) administered approximately 2.5 mm lateral to the PVN midline failed to significantly modify RSNA, MAP, and HR. To assess potential peripheral contributions, the maximally effective doses of Dooku1 (100 pmol, n = 4) and GsMTx4 (1 nmol, n = 4) were administered intravenously via the femoral vein, and it was found that peripheral administration did not significantly alter baseline RSNA, MAP, or HR (Table 1). Likewise, vehicle control injections (DMSO, 50 nL and saline, 50 nL) failed to evoke significant alterations in RSNA, MAP, or HR (Table 1).

2.3. Effects of PVN-Injected Piezo1 Channel Agonists on RSNA, MAP, and HR

To evaluate neuronal excitability mediated by Piezo1 channels within the PVN, we assessed the impact of selective agonists Yoda1 (Figure 5) and Jedi2 (Figure 6) (5 nmol, n = 6) on RSNA, MAP, and HR, and it was found that neither Yoda1 nor Jedi2 microinjection into the PVN elicited significant alterations in baseline RSNA (p > 0.05), MAP (p > 0.05), or HR (p > 0.05) (Figure 5 and Figure 6 and Table 1).

2.4. Histological Analysis

Histological examinations of brain sections that showed tracings of the outermost distribution of dye were performed by overlying areas from similar rostral–caudal sections taken from different brains (Figure S1A). These areas represent maximal diffusion extents for Dooku1 (100 pmol/50 nL), exceeding distributions observed in individual brains. Figure S1B illustrates a representative unilateral PVN injection site following 50 nl administration. Three animals exhibiting intraventricular tracer deposition were excluded from analysis. Anatomical control injections (n = 6) targeted sites 2.5 mm lateral to the midline without significant PVN encroachment.

3. Discussion

This investigation provides the first in vivo evidence for Piezo1 channel involvement within the PVN in modulating sympathetic outflow and cardiovascular dynamics. Notably, acute PVN Piezo1 blockade elicited significant elevations in RSNA and MAP. Local microinjection of the non-peptide Piezo1 channel blocker Dooku1, a chemically distinct blocker possessing the pharmacological properties commonly known for blocking Piezo1 channels, into the PVN produced dose-dependent increases in RSNA and MAP [40]. In addition, we used the peptide Piezo1 channel blocker GsMTx4, which produced similar sympatho-excitatory and pressor responses to the non-peptide Piezo1 channel blocker, thereby supporting the conclusion that Piezo1 channels expressed in the PVN contribute to tonic inhibition of SNA and ABP. Moreover, through Western blot assay and qRT-PCR techniques, we detected robust expression of the Piezo1 channel protein and its corresponding mRNA within the PVN. Finally, the immunofluorescence CTB staining study showed that Piezo1 was expressed in pre-sympathetic PVN neurons with axon projections to the RVLM. Taken together, these findings provide new evidence that Piezo1 channels are expressed in autonomic PVN neurons, and, furthermore, that they play an important role in regulating sympathetic outflow and cardiovascular function.

Data from the present study showed the following results: Microinjecting Piezo1 channel blockers (Dooku1 and GsMTx4) into the PVN significantly increased RSNA and MAP, indicating a key regulatory role of PVN Piezo1 channels. In anesthetized animals, Piezo1 channels expressed in the PVN appeared to tonically suppress ongoing RSNA and MAP. However, the signaling molecular mechanisms of Piezo1 channel inhibition that induce these sympatho-excitatory and pressor responses remain to be determined.

Currently, there are several possible explanations for why Dooku1 and GsMTx4 actively block piezo1 channels and cause sympatho-excitation and an increase in ABP. One of the possibilities is that either Dooku1 or GsMTx4 inhibits the Piezo1 channels to reduce the intracellular Ca^2+^ concentration in the PVN. In favor of this possibility, there are studies reporting that, in the neuronal cell line CLU199 and primary cultured neurons, GsMTx4 can inhibit the Ca^2+^ influx [41]. Moreover, in HEK293 cells and human umbilical vein endothelial cells, Dooku1 can specifically inhibit the Ca^2+^ influx mediated by the Yoda1-induced opening of Piezo1 channels [40]. Interestingly, our previous studies have demonstrated that inhibiting the endoplasmic reticulum Ca^2+^-ATPase in the PVN and depleting the endoplasmic reticulum calcium stores with thapsigargin significantly increases the in vitro excitability of autonomic PVN neurons [42] and in vivo sympathetic outflow and ABP in anesthetized rats [42]. Therefore, inhibited Ca^2+^ influx caused by either Dooku1 or GsMTx4 could reduce the Ca^2+^-induced Ca^2+^ release (CICR) from the ER. This inhibition of CICR could cause an increase in the PVN neural activity, which may underlie the mechanism of increased sympathetic outflow and ABP observed when blocking Piezo1 channels in the PVN (Figure 7). Clearly, additional studies are needed to clarify these potential mechanisms in the future.

Another potential mechanism to explain the sympathetic excitation caused by the Piezo1 channels blockade could be that the influx of Ca^2+^ via Piezo1 channels is decreased, which could reduce the opening probability of calcium-activated potassium channels and calcium-activated chloride channels. For instance, it has been reported that blockade of SK channels in the PVN leads to an increase in the excitability of autonomic PVN neurons, which contribute to the increased SNA and ABP. In addition, the desensitization of Piezo1 channels could decrease the open probability of big-conductance Ca^2+^-activated K^+^ (BK) channels of atrioventricular fibroblasts in patients with atrial fibrillation [43]. Moreover, blockade of Piezo1 channels to reduce the influx of Ca^2+^ could decrease the conductance of calcium-activated chloride channels in the ganglion cells and reduce the hyperpolarizing chloride current [44], which causes the depolarization and increase in neuronal excitability. This evidence also suggests that the opening of Piezo1 channels in the PVN could increase the activity of both calcium-activated potassium channels and calcium-activated chloride channels, which could contribute to the reduction or inhibition of sympathetic outflow and ABP (Figure 7).

Interestingly, Cui et al. [34] investigated the effects of Yoda1, the activator of Piezo1 channels, microinjected into vagal afferent nodose ganglion (NG) neurons on the ABP of normotensive, fructose drink-induced hypertension (HFD-HTN) and deoxycorticosterone (DOCA)-sensitive hypertensive animals [34]. They found that in vivo microinjection of Yoda1 into the NG decreased the ABP in both normotensive and hypertensive animals [34] and that Yoda1 increased the instant firing frequency of the single Ah-fiber discharge of the aortic depressor nerve and the spontaneous synaptic currents of Ah-baroreceptive neurons of the nucleus tractus solitarius, which could contribute to the increased activity of baroreflex afferent nerves [34]. Therefore, activation of Piezo1 channels expressed in the vagal NG neurons could be able to reduce ABP by the mechanisms of increased parasympathetic outflow [34].

In the present study, we also tested the hypothesis that opening Piezo1 channels can decrease PVN neuronal activity, RSNA, and ABP. However, our data showed that microinjecting either Yoda1 or Jedi2 into the PVN had no effect on RSNA, ABP, or the activity of the PVN neurons. The reason for this failure is not clear, but previous studies have reported the following: Either Yoda1 or Jedi2 selectively activates Piezo1 channels in HEK293T cells, leading to Ca^2+^ influx and depolarization [45,46]. It is supposed that, if the Piezo1 channels expressed in the PVN were activated by Yoda1 or Jedi2 to increase the Ca^2+^ influx and depolarize the neurons, it would increase the PVN neuronal activity and sympathetic outflow. However, increased Ca^2+^ influx not only serves to depolarize the neuron, but also is able to increase the activity of Ca^2+^-activated K^+^ channels, including SK and BK [47,48,49] channels and Ca^2+^-activated Cl^−^ channels [50,51]. Therefore, the increased activity of these channels will be able to reduce the neuronal excitability by activating either Ca^2+^-activated K^+^ channels or Ca^2+^-activated Cl^−^ channels. As a result, either the increased efflux of K^+^ or the influx of Cl^−^ ultimately counteracts the depolarization of the membrane potential caused by the Ca^2+^ influx (Figure 7). This interpretation could be supported by our previous study showing that SK channels expressed in autonomic PVN neurons exert a tonic inhibitory effect on the neuronal excitability and ongoing RSNA and ABP [27,49,52]. The above interpretation is also supported by the fact that, among Drosophila S2 cells and HEK293 cells, changing the spatial configuration of Piezo1 can significantly enhance the K^+^ outflow mediated by K2P channel K2P2.1 (TREK1) [53]. Studies have shown that there is a functional coupling between the Piezo1 channel and BK channel in the right atrial fibroblasts of patients with atrial fibrillation [43], suggesting that there may be functional coupling among the Piezo1 channel, TREK1, and BK channels. The above coupling mechanisms could counteract the Ca^2+^ influx-induced depolarization of the membrane potential caused by PVN injection of Yoda1 or Jedi2 (Figure 7).

In addition, ruling out the possibilities of restricted drug diffusion and insufficient effective concentration. The precision of drug action and the validity of the applied concentrations have been verified through our rigorous experimental design: Firstly, after all microinjection experiments, 5% Chicago Sky Blue solution was administered as a tracer, and only samples with staining confined to the PVN core region were included in the final analysis. The injection volume (50 nL) strictly adhered to the standard protocols for central nucleus microinjection, thus preventing drug diffusion to adjacent brain regions. Secondly, the injection concentrations of Yoda1 and Jedi2 were set at 100 µmol/L (5 nmol/50 nL), which falls within the in vivo effective concentration range (60–6000 µmol/L) reported by Cui et al. [34] and also covers the commonly used concentration spectrum for in vitro Piezo1 channel activation [54]. We speculate that Piezo1 channels in PVN neurons may be maintained at a moderate level of activation under basal physiological conditions, a characteristic linked to the regulatory properties of central mechanosensitive channels. As a central hub for integrating baroreceptive and metabolic signals, PVN neurons are constantly subjected to mechanical stimulation from the local microenvironment [55], which may sustain the basal open state of Piezo1 channels. Under such circumstances, stimulation with exogenous agonists may trigger rapid desensitization-existing studies have demonstrated that Piezo1 channels undergo rapid inactivation via phosphorylation modification of their intracellular domains when exposed to sustained mechanical stimulation or high concentrations of agonists [56]. This hypothesis explains why the blocker experiments (which inhibit basally activated Piezo1) yielded significant effects, whereas no obvious changes were observed in agonist experiments due to channel desensitization.

The present study utilized an anesthetized preparation, which constitutes a potential limitation when evaluating the effect of Piezo1 channel blockers on SNA because anesthesia may inhibit the activity of PVN neurons, thereby altering cardiovascular responses. Studies that measured ABP and RSNA in response to electrical stimulation of the PVN have demonstrated differential responses between conscious [57] and anesthetized rats [58]. However, this study employed a combined anesthesia regimen of urethane and α-chloralose, a protocol widely recognized in autonomic neuroscience research [59,60]. Furthermore, after careful consideration of relevant factors, the combined anesthetic approach of urethane and α-chloralose, which our research group and other teams have used to investigate the intrinsic mechanisms by which the hypothalamic PVN regulates sympathetic nerve activity and cardiovascular function [28,42,61], has been widely accepted by numerous academic journals. Unlike anesthetics such as isoflurane and pentobarbital, which strongly suppress the sympathetic nervous system, neither urethane nor α-chloralose significantly impairs the physiological regulatory capacity of baroreceptors in response to blood pressure fluctuations [62]. Although urethane and α-chloralose exert certain effects on brain function, ABP, and HR, these effects are extremely limited when appropriate doses are used [63]. Nevertheless, it is plausible that anesthesia could potentially cause underestimation of the role of Piezo1 channels in the PVN in regulating SNA and ABP. Importantly for this study, Piezo1 channels in the PVN are known to play a role in the regulation of sympathetic outflow.

4. Materials and Methods

4.1. Animals

Male Sprague-Dawley rats (n = 94; 350–450 g) were obtained from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China), maintained under a constant temperature (22 ± 1 °C), humidity (50 ± 10%) and a 12 h light/dark cycle, and given free access to food and water. All experimental and surgical procedures were approved by the Animal Ethics Committees of Jilin Normal University (KJLL20250401) and Henan University (HUSOM2025-017). This manuscript was prepared following the ARRIVE guidelines [64].

4.2. Recordings of Sympathetic Nerve Activity

Rats were anesthetized via intraperitoneal injection of α-chloralose (80 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) and urethane (800 mg/kg; Sigma-Aldrich, St. Louis, MO, USA). Surgical anesthesia depth was confirmed by absent pedal and corneal reflexes. Aortic access was established by femoral artery cannulation connected to a pressure transducer (Millar Instruments, Houston, TX, USA) for continuous ABP monitoring. The left femoral vein was cannulated for drug delivery. Following tracheal intubation, neuromuscular blockade was induced with intravenous gallamine triethiodide (25 mg·kg^−1^·h^−1^; Tocris Bioscience, Bristol, UK) during mechanical ventilation (oxygen-enriched air; 80–100 breaths/min; tidal volume 2.0–3.0 mL; Kent Scientific Corporation, Torrington, CT, USA). Anesthetic depth was maintained through ABP stability assessment, with supplemental boluses (10% initial dose) administered as required. End-tidal CO_2_ (PetCO_2_) was maintained at 35–40 mmHg through ventilator adjustments (CWE Inc., Ardmore, PA, USA). Core temperature was regulated at 37 °C using a feedback-controlled heating pad (ATC2000, WPI, Sarasota, FL, USA).

A left renal sympathetic nerve was isolated from adjacent tissues via a flank incision. Nerve bundles were then positioned onto silver wire electrodes (MLT185/ST, AD Instruments, Bella Vista, NSW, Australia) and covered with silicon-based impression material (Kwik-sil, WPI, Sarasota, FL, USA). The recorded signal was directed to an AC amplifier (DP 311, WARNER Instruments, Hamden, MA, USA) equipped with half-amplitude filters (band pass 100 to 1000 Hz).

4.3. Microinjection of Drugs

PVN microinjections were performed as previously described. Animals were placed in a stereotaxic head frame, and the skull was leveled between the bregma and lambda. A small section of the skull was removed in order to expose the dura, and a single-barreled glass microinjector pipette was lowered vertically into the PVN (coordinates: 1.2–1.6 mm caudal to bregma, 0.5 mm lateral to midline, and 7.1–7.5 mm ventral to dura). After a 20 min baseline period, Dooku1 (100 pmol/50 nL; Tocris Bioscience, Bristol, UK), GsMTx4 (1 nmol/50 nL; Alomone Labs, Jerusalem, Israel), Yoda1 (5 nmol/50 nl; Tocris Bioscience, Bristol, UK), Jedi2 (5 nmol/50 nL; Tocris Bioscience, Bristol, UK), or the vehicle control (DMSO, 50 nL) was injected unilaterally into the PVN. The above microinjected doses of piezo1 blockers or agonists were based on [34,40,46,65] and our preliminary experimental data. Variables were recorded for ~2 h following microinjection. At the conclusion of each experiment, Chicago blue dye (5% in saline, 50 nL; Sigma-Aldrich, St. Louis, MO, USA) was injected into the PVN to mark the injection sites. Upon completing the experiment, the animals were administered with a high-dose anesthetic combination and decapitated, and the brain was removed, placed in 4% paraformaldehyde, and then transferred to 30% sucrose–PBS. The hypothalamus, including the PVN area, was sliced in coronal sections, and microinjection sites were visualized under bright-field microscopy (Olympus BX53, Olympus Corporation, Tokyo, Japan).

4.4. Experimental Protocols

Following surgery, animals underwent a minimum 2-h stabilization period before assessment of their responses to PVN microinjection of Dooku1, GsMTx4, Yoda1, or Jedi2. Dose-dependent effects of Dooku1 (0.1 pmol, 1 pmol, 10 pmol, 100 pmol, and 200 pmol) were determined in separate animals. MAP and RSNA responses were recorded following each injection. Peripheral Piezo1 blockade was induced via femoral vein injection of Dooku1 (100 pmol) to isolate peripheral contributions to the evoked responses. PVN vehicle controls (50 nl saline or DMSO) were administered to account for non-specific volume effects. To confirm the PVN-specific actions of Dooku1, its effects on MAP and RSNA were tested via microinjection ~2.5 mm lateral to the midline (outside the PVN).

4.5. Punched Brain Tissues from Anesthetized Rats

Rats were deeply anesthetized with 5% isoflurane and decapitated. Brains were rapidly extracted and placed in a liquid nitrogen–pre-frozen rat brain matrix. To isolate hypothalamic PVN tissue, the optic tract was identified, and a 1 mm coronal section was obtained from its rostral tip. The thalamus, cerebral cortex, and hypothalamic PVN were dissected using a 12-gauge needle (1.5 mm inner diameter). Samples were snap-frozen in liquid nitrogen and stored at −80 °C for Western Blot and quantitative real-time polymerase chain reaction (qRT-PCR) analyses.

4.6. Western Blot Analysis of Piezo1 Channels Protein

Frozen brain tissues were homogenized in ice-cold lysis buffer (RIPA:PMSF:cOmplete Mini, EDTA-free = 100:1:5), agitated for 30 min at 4 °C, and centrifuged (12,000 rpm, 5 min, 4 °C). Supernatants were collected for triplicate protein quantification via BCA (562 nm; bovine serum albumin standard) [66], aliquoted, and stored at −80 °C. For immunoblotting, 75 μg total protein/sample was resolved on 10% SDS–polyacrylamide gels and electrophoretically transferred to PVDF membranes. After blocking with 5% non-fat milk (1 h, RT) and TBST washes, membranes were probed overnight at 4 °C with Rabbit anti-Piezo1 (1:200; APC-087, UNIV, Wuhan, Hubei, China) and Mouse anti-GAPDH (1:1000; G8795, Sigma-Aldrich, St. Louis, MO, USA). Following TBST washes, membranes were incubated (1 h, RT) with fluorescence-conjugated secondaries: Goat anti-rabbit 680RD (1:2000; 925-68071; LI-COR Biosciences, Lincoln, NE, USA) and Goat anti-mouse 800CW (1:2000; 925-32210; LI-COR Biosciences, Lincoln, NE, USA). Protein bands were visualized and quantified using an Odyssey DLX-3421 Dual-Color Infrared Imaging System ((LI-COR Biosciences, Lincoln, NE, USA).

4.7. Retrograde Labeling of PVN-RVLM Neurons

Five to seven days prior to the start of the immunofluorescence staining study, PVN neurons were retrogradely labeled from the rostral ventrolateral medulla (RVLM) as previously described. Briefly, rats were anesthetized with isoflurane (3% in O_2_) and placed in a stereotaxic frame (Kopf Instruments, Tujunga, CA, USA). The cerebellum was exposed through a small burr hole, and a glass micropipette (Drummond Scientific, Broomall, PA, USA) was lowered into the pressor region of the RVLM (coordinates: −12.7 mm caudal to bregma, 1.8 mm lateral to midline, and 8.9 mm ventral to dura). Using a microinjection pump (UMP 3, WPI, Sarasota, FL, USA). 50 nl of cholera toxin B subunit (CTB) (CTB-488, Brain VTA, Optics Valley, Wuhan, Hubei, China) was injected bilaterally into the RVLM. Animals received a daily subcutaneous injection of penicillin G (30,000 units; Sigma-Aldrich, St. Louis, MO, USA) and meloxicam (1 mg/kg; Boehringer Ingelheim, Ingelheim am Rhein, Germany) for 3 days post-surgery. Tracer location was verified histologically post-mortem.

4.8. Immunofluorescence Analysis of Piezo1 Channel Protein

Immunofluorescence staining was performed using 25 μm coronal brain sections containing the PVN with the following protocols: Rats were deeply anesthetized and transcardially perfused with 4% paraformaldehyde (PFA; Sigma-Aldrich, St. Louis, MO, USA) (in 1 × PBS). After perfusion, the brain was removed and fixed in 4% PFA overnight at 4 °C. The brain was then transferred to a tube containing 30% sucrose–PBS and kept at 4 °C until the brain sank to the bottom of the tube. Brain coronal slices were then cut from the hypothalamus area using a cryostat (Leica CM1950, Leica Biosystems, Wetzlar, Germany).

The immunofluorescence analysis of Piezo1 channel protein was performed as described previously [35]. For immunostaining, brain sections were first washed in PBS three times for 10 min each. Before undergoing blocking with the blocking solution (V11307, Invitrogen, Carlsbad, CA, USA) for 3 h, the rabbit anti-Piezo1 antibody (1:200) (V11305, Invitrogen, Carlsbad, CA, USA) was diluted with the antibody dilution solution at 4 °C for 72 h. The secondary antibody, goat anti-rabbit IgG H&L (Alexa Fluor^®^555) (1:200) (ab150078, Abcam, Cambridge, UK), was diluted with the antibody dilution solution. After mounting with a DAPI-free mounting medium, the brain slices were photographed using a Zeiss 710 laser-scanning confocal microscope (Carl Zeiss, Oberkochen, Germany).

4.9. qRT-PCR Analysis of Piezo1 Channels mRNA

qRT-PCR analysis of Piezo1 channels mRNA was performed as described previously [67]. Briefly, the total RNA from the PVN and cerebral cortex was extracted using the TRIzol™ Plus RNA Purification Kit (12183555, Invitrogen, Carlsbad, CA, USA). The optical density of the RNA was calculated at 2.066 using an ultra-micro ultraviolet–visible spectrophotometer (NanoDrop One C, Invitrogen, Carlsbad, CA, USA). After the RNA quality was determined, the RNA was incubated at 65 °C for 5 min and then rapidly cooled on ice. A Revertra Ace qPCR RT Kit (FSQ-101) (FSQ-101, TOYOBO, Osaka, Japan) was used to reverse-transcribe the total RNA into template DNA. The reaction solution was prepared and incubated at 37 °C for 15 min to conduct the reverse-transcription reaction. Then, it was incubated at 98 °C for 5 min for the enzyme inactivation reaction. After the reaction was completed, the product was stored at −20 °C (the primers are listed in Table S1). The cDNA was subjected to a three-step qRT-PCR using the SYBR Green Realtime PCR Master Mix fluorescence quantitative kit (QPK-201, TOYOBO, Osaka, Japan) on an Agilent Mx3005P qPCR System (Agilent Technologies, Santa Clara, CA, USA).

4.10. Data Analysis

RSNA was computed as the mean rectified, integrated neurogram. Baseline values were derived from a 10 min pre-treatment data segment. Normalized RSNA alterations were expressed as percentage change from baseline. Peak hemodynamic and sympathetic responses to PVN-administered agents (Dooku1, GsMTx4, Yoda1, Jedi2, or vehicle) were quantified during a 2-min epoch encompassing maximal changes in MAP, HR, and RSNA. Prior to choosing parametric or non-parametric tests, we performed normality tests on all experimental data sets using the Shapiro–Wilk test. For data that conformed to a normal distribution, we used parametric tests. Statistical comparisons employed one-way repeated-measures ANOVA with Tukey’s post hoc testing for significant interactions. Statistical analyses were performed using GraphPad Prism (Version 10.4.0; GraphPad Software, Inc., San Diego, CA, USA). Results are reported as mean ± standard error (SE), with p < 0.05 defining significance.