The PVN enhances cardiorespiratory responses to acute hypoxia via input to the nTS
Abstract
Chemoreflex neurocircuitry includes the paraventricular nucleus (PVN), but the role of PVN efferent projections to specific cardiorespiratory nuclei is unclear. We hypothesized that the PVN contributes to cardiorespiratory responses to hypoxia via projections to the nucleus tractus solitarii (nTS). Rats received bilateral PVN microinjections of adeno-associated virus expressing inhibitory designer receptor exclusively activated by designer drug (GiDREADD) or green fluorescent protein (GFP) control. Efficacy of GiDREADD inhibition by the designer receptor exclusively activated by designer drug (DREADD) agonist Compound 21 (C21) was verified in PVN slices; C21 reduced evoked action potential discharge by reducing excitability to injected current in GiDREADD-expressing PVN neurons. We evaluated hypoxic ventilatory responses (plethysmography) and PVN and nTS neuronal activation (cFos immunoreactivity) to 2 h hypoxia (10% O2) in conscious GFP and GiDREADD rats after intraperitoneal C21 injection. Generalized PVN inhibition via systemic C21 blunted hypoxic ventilatory responses and reduced PVN and also nTS neuronal activation during hypoxia. To determine if the PVN-nTS pathway contributes to these effects, we evaluated cardiorespiratory responses to hypoxia during selective PVN terminal inhibition in the nTS. Anesthetized GFP and GiDREADD rats exposed to brief hypoxia (10% O2, 45 s) exhibited depressor and tachycardic responses and increased sympathetic and phrenic nerve activity. C21 was then microinjected into the nTS, followed after 60 min by another hypoxic episode. In GiDREADD but not GFP rats, PVN terminal inhibition by nTS C21 strongly attenuated the phrenic amplitude response to hypoxia. Interestingly, C21 augmented tachycardic and sympathetic responses without altering the coupling of splanchnic sympathetic nerve activity to phrenic nerve activity during hypoxia. Data demonstrate that the PVN, including projections to the nTS, is critical in shaping sympathetic and respiratory responses to hypoxia.
INTRODUCTION
Peripheral chemoreflex activation during hypoxia leads to compensatory increases in autonomic, cardiorespiratory, and neuroendocrine output. The central neurocircuitry involved in regulating appropriate adjustments to chemoreflex stimulation is not completely understood. Chemoafferent input first terminates in the nucleus tractus solitarii (nTS), where it is integrated and information is then transmitted to other nuclei to produce cardiorespiratory responses (2, 19). The classical chemoreflex pathway involves a projection from the nTS to the rostral ventrolateral medulla (RVLM) and ventral respiratory group (1, 20, 29). However, it is also clear that the hypothalamic paraventricular nucleus (PVN) is an integral component of chemoreflex neurocircuitry (41, 45).
The PVN is a highly integrative nucleus that regulates neuroendocrine and cardiorespiratory function. The PVN is required for full expression of autonomic and cardiorespiratory responses to peripheral chemoreflex stimulation by potassium cyanide (38, 41). However, although the PVN is important in producing long-term facilitation following bouts of intermittent hypoxia (4), much less is known regarding its role in responses during acute hypoxic stimuli. Acute hypoxia activates neurons in the nTS and ventrolateral medulla (VLM) that send projections to the PVN, a substantial portion of which are catecholaminergic (25, 26, 47). These catecholaminergic ascending projections to the PVN are critical for hypoxia-induced PVN neuronal activation (47, 48) and contribute to cardiorespiratory chemoreflex responses (27).
The PVN projects to multiple brainstem and spinal nuclei, including the nTS, VLM, and intermediolateral cell column (IML) and phrenic motor nucleus in the spinal cord (43, 46, 56). RVLM-projecting PVN neurons are activated by chemoreflex stimulation using potassium cyanide (38). However, our group found that PVN projections to the RVLM and IML are not activated by acute hypoxia (10). In contrast, we recently showed that nTS-projecting PVN neurons exhibit increased cFos immunoreactivity induced by hypoxia (42). This raises the possibility that the PVN, likely via a descending PVN-to-nTS (PVN-nTS) pathway, contributes to chemoreflex responses to hypoxia. However, the physiological effects of the PVN and this pathway, or the manner in which they affect cardiorespiratory responses to chemoreflex activation by hypoxia, are not known. We postulated that the PVN increases nTS neuronal activation during hypoxia and contributes to hypoxic ventilatory responses. Specifically, we hypothesized that activation of a descending PVN-nTS projection enhances cardiorespiratory responses during hypoxia.
This study used a chemogenetic approach to evaluate the ability of the PVN and neurons in the PVN-nTS pathway to facilitate hypoxia-induced nTS neuronal activation and modulate cardiorespiratory chemoreflex responses. An adeno-associated virus (AAV) carrying inhibitory designer receptors exclusively activated by designer drugs (GiDREADDs) was bilaterally microinjected into the PVN. GiDREADDs are mutated human muscarinic receptors that are coupled to Gi signaling (hM4D; GiDREADD) and have been designed to produce neuronal or synaptic inhibition in response only to synthetic ligands, such as the designer receptor exclusively activated by designer drug (DREADD) agonist Compound 21 (C21) (30, 52, 57). Stimulation of these inhibitory receptors via C21 was used to produce global silencing of neurons within the PVN or selective inhibition of PVN terminals located in the nTS. We found that DREADD-mediated inhibition of PVN neurons blunted hypoxic ventilatory responses in conscious animals and was associated with a reduction in hypoxia-induced cFos immunoreactivity in the nTS. Furthermore, selective DREADD-mediated inhibition of PVN terminal fields in the nTS blunted phrenic nerve activity and augmented tachycardic and sympathoexcitatory responses to hypoxia. Taken together, our data support the concept that the PVN, likely via a PVN-nTS pathway, is a key component of chemoreflex neurocircuitry, and activation of this pathway is required to produce full compensatory responses to peripheral chemoreflex activation by acute hypoxia.
METHODS
Ethical Approval
All experiments were conducted on male Sprague-Dawley rats (Envigo, Indianapolis, IN; 225–350 g; number of animals, n = 56). Rats were housed in a 12-h light/dark cycle at a temperature of 22°C and 40% humidity, with food and water provided ad libitum. All experimental procedures were performed in accordance with the American Physiological Society’s Guiding Principles for the Care and Use of Vertebrate Animals in Research and Training National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of Missouri Animal Care and Use Committee.
PVN Microinjection of Adeno-Associated Virus Vectors
All recovery surgical procedures were performed using aseptic technique. Rats were given dexamethasone (2 mg/kg) and anesthetized with isoflurane (5% for induction and 2%–2.5% for maintenance, Aerane; Baxter, Deerfield, IL). They were placed in a stereotaxic apparatus, and the skull was exposed via a midline incision along the dorsal surface. The position of the head was adjusted so that bregma and lambda were oriented in the same horizontal plane. The surface of the brain was exposed via a small drill hole in the skull. A single-barrel glass micropipette was filled with an adeno-associated viral vector 2 (AAV2) containing the inhibitory DREADD hM4D (GiDREADD) expressed under the human synapsin promoter (hSyn, to target neurons) with a fluorescent mCherry reporter (AAV2-hSyn-hM4D(Gi)-mCherry; Addgene, 50475-AAV2; GiDREADD rats, n = 37). Control rats received a virus lacking the GiDREADD sequence and containing green fluorescent protein (GFP) as reporter (AAV2-hSyn-GFP; Addgene, 50465-AAV2; GFP rats, n = 14). The glass micropipettes were lowered into the PVN using the following target stereotaxic coordinates: 1.8–2.0 mm caudal to bregma, ±0.5 mm lateral from the midline, and 7.6–7.8 mm ventral to the dura. Rats received bilateral microinjections (180 nL/side) of viral vectors containing either GiDREADD or GFP. The vector was injected over 1 min, and the volume of the vector injected into the PVN was verified by monitoring the movement of the meniscus within the pipette, as previously described (25, 26). The pipette remained in the brain for at least 5 min after injection to minimize movement up the injection tract. Following bilateral microinjections, the pipette was removed, and the injection site was closed. Rats were treated postoperatively with fluids (3 mL sc, 0.9% saline), enrofloxacin (5 mg/kg im; Bayer, Shawnee Mission, KS), and buprenorphine (0.05 mg/kg sc; Reckitt Benckiser Pharmaceuticals, Richmond, VA) for hydration, prevention of infection, and pain management, respectively. Rats were allowed 3–5 wk to recover to maximize viral expression in PVN neurons and allow for expression in fibers projecting to the nTS.
PVN Slice Preparation to Verify GiDREADD Effects on Neuronal Function
GFP (n = 3) and GiDREADD (n = 3) rats were anesthetized using 5% isoflurane and rapidly decapitated. The forebrain was removed and placed in ice-cold N-Methyl-d-glucamine (NMDG)-HEPES cutting solution (in mM: 93 NMDG, 93 HCl, 2.5 KCl, 1.2 NaH2PO4, 10 MgSO4, 30 NaHCO3, 20 HEPES, 25 d-glucose, 5 l-ascorbic acid, 2 thiourea, 3 sodium pyruvate, and 0.5 CaCl2, aerated with 95% O2-5% CO2, pH 7.4, and 300–310 mosM). Coronal slices containing the PVN (280 µm) were generated using a vibratome (VT 1200S, Leica, Germany). Slices were allowed to equilibrate before use for 12 min in the NMDG-HEPES solution and then for 30 min in standard recording artificial cerebrospinal fluid (aCSF; in mM: 124 NaCl, 3 KCl, 1.2 NaH2PO4, 1.2 MgSO4, 25 NaHCO3, 11 d-glucose, and 2 CaCl2, saturated with 95% O2/5% CO2, pH 7.4, ~300 mosM) at 31°C–33°C. Tissue sections were secured via nylon mesh in a superfusion recording chamber and superfused at ~3 mL/min with the standard recording aCSF (described above).
Neuronal recordings.
Electrodes (resistance = 3.0–6.0 MΩ) were prepared using a Flaming-Brown micropipette puller (P97, Sutter Instrument) and filled with (in mM) 10 NaCl, 130 K+-gluconate, 11 EGTA, 1 CaCl2, 10 HEPES, 1 MgCl2, 2 Mg-ATP, 0.2 Na-GTP, pH 7.3, and ∼280 mosM. Recording pipettes were guided toward the caudal PVN using a piezoelectric manipulator (Burleigh, PCS-6000). PVN neurons expressing GFP or mCherry were identified in GFP and GiDREADD rats using a Prime 95B CMOS scientific camera (Photometrics) and either FITC Cube Set (3540B, Semrock) or Texas Red Cube Set (4040B, Semrock), respectively. In addition, unlabeled neurons (negative for mCherry) in caudal PVN slices from GiDREADD rats were recorded. Following initial membrane rupture cell capacitance (GFP, 16.9 ± 3.0 pF; GiDREADD, 18.4 ± 2.7 pF; unlabeled, 23.4 ± 0.1 pF; one-way ANOVA, P = 0.34) and resting membrane potential (GFP, −64.7 ± 3.0 mV; GiDREADD, −57.5 ± 2.8 mV; unlabeled, −63.8 ± 4.4 mV; one-way ANOVA, P = 0.47) were measured and were not different among groups. Neurons were rejected if the holding current was more negative than −30 pA (GFP, −9.5 ± 7.2 pA; GiDREADD, −14.0 ± 5.1 pA; unlabeled, −20.5 ± 2.2 pA; one-way ANOVA, P = 0.53) upon membrane rupture or if the series resistance changed 20% or more during the experiment. Resting membrane potential, spontaneous action potentials, and evoked action potentials were recorded in current clamp mode. In most cells a small amount of depolarizing current (<10 pA) was injected to induce action potential discharge; the amount of current injected was not different among groups. Spontaneous action potential frequency was measured during the last 30 s of aCSF control, application of the DREADD agonist Compound 21 (C21, Tocris, 5548C21; 10 µm), and aCSF wash (10 min each). C21 is a potent activator of DREADDs and does not produce off-target behavioral effects (52, 53) that have been observed with other DREADD agonists, such as clozapine-N-oxide (32). PVN neuron action potentials were evoked through depolarizing current step injection (−20 pA to 100 pA; 10 pA steps, 100 ms duration) from the neuron’s resting potential, and the number of action potentials evoked at each current step was determined. Data obtained from PVN slice experiments were acquired in pClamp 10.7 using a Multiclamp 700B amplifier (Molecular Devices), filtered at 2 kHz and sampled at 20 kHz. Data were analyzed using Clampfit 10.7.
Conscious Animal Experiments
Evaluation of hypoxic ventilatory responses.
Ventilation was assessed in a subset of conscious, freely moving GFP (n = 5) and GiDREADD (n = 7) rats using whole body plethysmography, as previously described (27). Flow-through plethysmography chambers (Data Sciences International) were used with inlet and outlet ports to allow airflow. The animal chamber and a reference chamber were connected to a differential pressure transducer (Validyne MP45; Validyne Engineering). The recorded pressure signal is proportional to volume changes, providing a measurement of tidal volume by integrating the area under the inspiratory pressure curve (tidal volume index). For experiments examining hypoxic ventilatory responses, mixtures of gases were regulated by a gas blender that provided precise control of oxygen concentrations (Hypoxydial; Starr Life Sciences), which were confirmed by an O2 analyzer. All ventilatory measurements were acquired using a PowerLab data acquisition system (version 7, ADInstruments, Colorado Springs, CO), and respiratory rate (breaths/min), tidal volume index (normalized to body weight), and minute ventilation index (the product of respiratory rate and tidal volume index) were determined. Oxygen saturation was assessed using a pulse oximeter collar (MouseOx; Starr Life Sciences).
All animals were acclimatized to the plethysmography chambers for 3 days (2 h in room air/day) before experiments. On the day of the experiment, rats were placed in the chambers and allowed 60 min in room air for adaptation. To examine peripheral chemoreflex responses, rats were subjected to an experimental protocol consisting of 5-min sequential episodes of 21%, 14%, 12%, 10%, and 8% O2-balanced N2. In addition, to evaluate central chemoreflex function, animals were exposed to a 5-min episode of hyperoxia (100% O2) followed by exposure to hyperoxic hypercapnia (95% O2-5% CO2, 5 min). The chamber was then switched back to room air. After 5 min, rats received intraperitoneal injections (1 mL) of the selective DREADD agonist C21 (1 mg/kg). Ventilation was assessed for 60 min in room air following injection to allow for C21-mediated DREADD activation (21) and subsequent inhibition of GiDREADD-expressing PVN neurons and their projections. The hypoxia and hyperoxia/hypercapnia protocols were repeated and ventilatory parameters measured. Preliminary experiments in rats expressing control vector or GiDREADD indicate that intraperitoneal injection of saline had no effect on baseline ventilation or responses to hypoxia or hyperoxic hypercapnia in either group. Ventilatory parameters were measured from an average of ~20 consecutive breaths in the absence of sniffs, sighs, or movement artifacts within the last minute of exposure to each level of oxygen or hyperoxic hypercapnia. At the end of the experiment, rats were returned to their home cages and allowed 7–10 days to recover. A subset of GiDREADD animals (n = 5) was subsequently used for additional experiments.
Assessment of hypoxia-evoked activation of nTS neurons.
GiDREADD rats (n = 14) in their home cages were acclimated to a hypoxia chamber (Biospherix, Redfield, NY) for 3 days (2 h/day in room air). The following day, rats were placed in the hypoxia chamber. After a 60-min acclimation period, rats were treated with intraperitoneal injection of saline (1 mL) or an equivalent volume of C21 (1 mg/kg) and 60 min allowed for C21-mediated inhibition of GiDREADD-expressing neurons. The gas mixture was then adjusted to 21% O2 (normoxia; saline, n = 3; C21, n = 3) or 10% O2 (hypoxia: saline, n = 4; C21, n = 4) for 2 h using a negative feedback control system, as reported previously (25, 28). Immediately following normoxic or hypoxic exposure, rats were deeply anesthetized (5% isoflurane) and transcardially perfused with oxygenated, heparinized Dulbecco’s modified Eagle medium (125 mL, pH 7.4, Sigma) followed by 4% paraformaldehyde (Sigma, 250 mL, pH 7.4) in 0.01 M phosphate-buffered saline solution (PBS). Brains were removed, postfixed overnight in 4% paraformaldehyde, and stored in cryoprotectant at 4°C.
Anesthetized Animal Experiments
In vivo surgical preparation.
Rats (n = 29) were anesthetized using isoflurane (5% induction, 2%–3% maintenance, in 100% O2). Femoral venous and arterial catheters (PE-10 fused to PE-50, A-M Systems) were inserted to allow administration of drugs and measurement of arterial pressure, respectively. The trachea was cannulated, and rats were mechanically ventilated (60–65 breaths per min; 683 Harvard Apparatus) with O2-enriched room air. Bilateral cervical vagotomy was performed to prevent entrainment of phrenic motor output with the ventilator. Arterial blood gases were measured (Osmetech OPTI CCA) periodically throughout the experiment and tidal volume was adjusted for each animal as needed. Rectal temperature was monitored and maintained at ~38°C (Tele-Thermometer, Yellow Springs Instrument). Rats were assigned to groups as described in individual protocols.
The left splanchnic nerve was isolated using a retroperitoneal approach and placed on a bipolar silver wire electrode (0.005 in. bare, 0.0070 in. coated, A-M Systems). The nerve was covered in silicone elastomer (Kwik-Cast, World Precision Instruments), which was allowed to harden, and the wound closed. Similarly, the left phrenic nerve was isolated via a ventral cervical approach, placed on a bipolar recording electrode, and covered in silicone elastomer. The nerve was crushed distally, and the contralateral phrenic nerve was cut. Ground wires were inserted in muscle tissue near the electrodes. Once the elastomer hardened, the incision site was closed. Nerve activity was amplified (×1,000), filtered (30–3,000 Hz, P511, Grass Technologies), rectified, and integrated using a root mean square converter (time constant: phrenic = 100 ms; splanchnic = 28 ms; PowerLab, ADInstruments). Background noise in the nerves was determined from the signal between bursts of activity during periods of low activity; we have verified previously that this value is equivalent to that observed following euthanasia (35). The recorded nerve activity minus noise was defined as splanchnic sympathetic nerve activity (sSNA) or phrenic nerve activity (PhrNA).
Rats were placed in a stereotaxic apparatus (Kopf Instruments) and the brainstem exposed via a partial occipital craniotomy, as previously described (27). After surgery was completed, isoflurane was gradually withdrawn, and rats were progressively converted to inactin anesthesia [100 mg/kg iv, with supplements (10–20 mg/kg iv) administered as needed]. Animals were paralyzed using gallamine (10–15 mg/kg iv, 2–3 mg/h iv maintenance). Adequate plane of anesthesia was verified every 10–15 min by evaluation of the cardiovascular response to firm tail pinch [<5 mmHg increase in mean arterial pressure (MAP)]. Cardiorespiratory parameters were allowed to stabilize for at least 60 min before subsequent experimental manipulation. Ventilation with O2-enriched room air was established above apneic threshold by adjusting tidal volume and breathing frequency. All cardiorespiratory parameters, including MAP, heart rate (HR), sSNA and PhrNA [phrenic frequency (Phr Freq), phrenic amplitude (Phr Amp), and minute phrenic nerve activity (Min PhrNA) (Phr Freq × Phr Amp), as indices of neural respiration], were recorded continuously using PowerLab software. At the conclusion of all nTS microinjection experiments, rats were transcardially perfused as described above. Brains were removed, postfixed overnight in 4% paraformaldehyde, and stored in cryoprotectant at 4°C.
Assessment of chemoreflex cardiorespiratory responses.
Baseline cardiorespiratory parameters were evaluated in oxygen-enriched room air in GFP (n = 6) or GiDREADD (n = 12) animals. A separate group of naïve rats that did not receive AAV microinjections (n = 5) served as an additional control group. Results in the naïve animals were not different from the GFP rats, and data were combined with the GFP group for analysis. Rats were then exposed to a brief period of hypoxia (control hypoxia, 10% O2; 45 s), and cardiorespiratory parameters were measured. Thirty minutes later, rats were exposed to an additional hypoxic episode. All hypoxic episodes were separated by at least 30 min to prevent long-term facilitation of sSNA and PhrNA that occurs during acute intermittent hypoxia (15). Rats were exposed to 2–3 hypoxic episodes under baseline conditions to verify that cardiorespiratory chemoreflex responses were similar.
We then examined changes in hypoxia-evoked cardiorespiratory responses during inhibition of PVN terminals in the nTS. A glass micropipette filled with C21 (0.1 mM) was advanced into the nTS using dorsal brainstem surface landmarks. C21 was bilaterally microinjected into the nTS (90 nL/side: 0.3 mm anterior, 0.4 mm lateral, and 0.4 mm ventral relative to calamus scriptorius). The amount of C21 microinjected into nTS was ~105 less than that given intraperitoneally, and thus, the effects were not likely to be due to leakage systemically. Cardiorespiratory parameters were measured during a 60-min equilibration period to allow for C21-mediated inhibition of GiDREADD-expressing PVN terminals in the nTS. Rats were then exposed to an additional episode of hypoxia (10% O2, 45 s), and changes in cardiorespiratory parameters were evaluated. The time course of hypoxic responses within a group was examined by taking a 15-s average of each parameter before initiation of hypoxia (baseline) and 5-s averages immediately before and every 5 s for 5 min after the initiation of exposure to 10% O2. In addition, to compare responses between groups and because the precise time course of responses varied among rats, we also evaluated the peak change (5 s average) in each parameter during the response to hypoxia.
Cardiorespiratory coupling.
A subset of GFP (n = 5) and GiDREADD (n = 5) animals with confirmed activity in both splanchnic and phrenic nerves was evaluated for changes in cardiorespiratory coupling following nTS microinjection of C21. The nerve activity was integrated as described, smoothed using the Triangular (Bartlett) window and zero subtracted (PowerLab, ADInstruments). To evaluate cardiorespiratory coupling, cycle-triggered averages of sSNA were triggered from the onset of PhrNA inspiration, which was defined as 10% above the baseline value on the positive slope of integrated PhrNA. The end of inspiration and beginning of expiration was defined as the point in the PhrNA cycle at which the peak negative slope of PhrNA occurred. Onset of the following Phr burst was defined as end of expiration. We analyzed sSNA during the first and second half of inspiration (I1, I2) and expiration (E1, E2) (15, 33). Because hypoxia altered the absolute level of sSNA in both groups and C21 influenced sSNA at baseline and during hypoxia in GiDREADD but not GFP animals, we examined specifically the pattern of cardiorespiratory coupling by normalizing Phr triggered averages of sSNA to the total activity within a cycle. sSNA during I1, I2, E1, and E2 is presented as a percentage of the total cycle activity. Data were analyzed at the following points: 1) baseline before hypoxia (Pre-Hx; GFP: 19 ± 0.9; GiDREADD: 17 ± 0.7 Phr cycles), 2) during control hypoxia (Hx; at peak increase of sSNA; GFP: 14 ± 0.4; GiDREADD: 17 ± 1 Phr cycles), 3) 60 min after nTS microinjection of C21 before hypoxia (C21 Pre-Hx; GFP: 15 ± 0.7; GiDREADD: 14 ± 0.6 Phr cycles), and 4) during post-C21 hypoxia (C21 Hx, GFP: 14 ± 0.4; GiDREADD: 14 ± 1.5 Phr cycles).
Confirmation of nTS-specific effects of C21 on cardiorespiratory responses to hypoxia.
In a separate group of GiDREADD animals (n = 3), cardiorespiratory responses to hypoxia (10% O2, 45 s) were evaluated before [control (Ctrl)] and 60 min after (C21) bilateral microinjection of C21 (0.1 mM, 90 nL/side) in an area lateral to the nTS (0.3 mm anterior, 3–4 mm lateral, and 0.4 mm ventral relative to calamus scriptorius). Peak changes in cardiorespiratory responses to hypoxia were compared before and after C21 microinjection.
Effect of nTS C21 on glutamate-evoked cardiorespiratory responses.
Microinjection of C21 into the nTS of GiDREADD rats blunted respiratory responses to chemoreflex activation by hypoxia. To verify that this was not due to potential nonspecific effects of C21 on nTS neuronal excitability, we examined the effects of C21 on responses to nTS glutamate. In a separate group of GiDREADD rats (n = 3), cardiorespiratory responses to unilateral nTS microinjection of glutamate (5 mM, 30 nL) were determined before and after bilateral nTS microinjection of C21 (0.1 mM, 90 nL/side) using the same coordinates for nTS injections as described for nTS experiments above.
Immunohistochemistry
Coronal sections (30 µm) of the forebrain and hindbrain containing the PVN or nTS, respectively, were cut using a vibrating microtome (1000S, Leica). Immunohistochemical procedures were performed on PVN or nTS sections, as previously described (26, 42). Briefly, sections were rinsed with 0.01 M PBS (3 times for 10 min), blocked with 10% normal donkey serum (Millipore, S30) in 0.03% Triton in 0.01 M PBS, and incubated overnight in 3% normal donkey serum and 0.03% Triton in 0.01 M PBS containing the primary antibodies. The following day, sections were rinsed in PBS and incubated in appropriate secondary antibodies. PVN and nTS sections from GiDREADD rats were processed for mCherry [1:500; rabbit anti-mCherry; ab167453, Abcam, Cambridge, MA; Research Resource Identifier (RRID): AB_2571870] followed by donkey anti-rabbit cyanine fluorescent dye Cy3 secondary antibody (1:200; Jackson ImmunoResearch). Tissue from GFP rats was processed for GFP (1:500; rabbit anti-GFP; ab290, Abcam, Cambridge, MA; RRID: AB_303395) followed by donkey anti-rabbit Alexa Fluor (AF) 488 secondary antibody (1:200; Jackson ImmunoResearch). In addition, a separate immunohistochemistry protocol was performed on tissue from GiDREADD rats that were exposed to acute normoxia or hypoxia. PVN and nTS sections were incubated with a primary antibody against cFos (1:5,000; rabbit anti-cFos; ab190289, Abcam, Cambridge, MA; RRID: AB_2737414) followed by donkey anti-rabbit AF647 secondary antibody (1:200; Jackson ImmunoResearch). Sections were coverslipped with Prolong Diamond (Thermo Fisher, P36970) and sealed with nail polish.
Microscopy and Image Analysis
The PVN and nTS were examined using a fluorescence microscope (BX51; Olympus) equipped with a digital monochrome camera (ORCA-ER; Hamamatsu) and a spinning disk confocal unit (Olympus). Images were imported into ImageJ (version 1.48v; RRID: SCR_003070) and adjusted for contrast and brightness only.
Verification of bilateral PVN injection sites.
Forebrain sections were evaluated for bilateral expression of GFP- or mCherry-immunoreactivity (IR) in the PVN of GFP and GiDREADD rats, respectively. A rat brain atlas (39) was used to confirm that the AAV microinjection site was located within the PVN. The region of the dorsal medulla was evaluated for the presence of GFP-IR and mCherry-IR fibers.
Analysis of neuronal activation in the PVN and nTS.
Image stacks of 11 consecutive optical planes (2 µm per plane) were taken from 2 coronal sections of the caudal PVN [−1.9 and −2.1 mm, relative to bregma; regions that contain the majority of nTS-projecting neurons (18, 42)] and 3 sections of the nTS (−14.46, −14.28, and −14.1 mm relative to bregma, which corresponds to −180, 0, +180 µm relative to calamus scriptorius). Using ImageJ software, the regions containing the PVN and nTS were outlined, and bilateral counts of cFos-IR neurons were performed in all experimental groups. cFos-IR neurons were defined as displaying round or ovoid-shaped staining restricted to the nucleus. Within each brain region, cFos-IR was quantified as the total (sum) of counts from all sections.
Statistical Analysis
All statistical analyses were performed using GraphPad Prism or SigmaPlot. Post hoc analysis (Fisher’s least-significant difference) was used when appropriate. One-way RM ANOVA was used to evaluate spontaneous action potential frequency in GFP and GiDREADD PVN neurons. Two-way ANOVA was used to examine the total number of cFos-IR neurons in the PVN and nTS of normoxic and hypoxic GiDREADD rats. Two-way RM ANOVA was used to examine differences in evoked action potential discharge in GFP and GiDREADD PVN neurons, the effect of systemic or nTS C21 on baseline cardiorespiratory parameters, and oxygen saturation and hypoxic or hyperoxic hypercapnia ventilatory responses before and after C21. Two-way RM ANOVA was also used to examine the effects of C21 on the time course of hypoxia-evoked cardiorespiratory parameters to compare peak cardiorespiratory responses to hypoxia after saline and C21 in GFP versus GiDREADD rats and examine the effects of hypoxia on cardiorespiratory coupling in the absence and presence of C21. Paired t tests were used to evaluate cardiorespiratory responses to hypoxia before and after microinjection of C21 outside the nTS, and cardiorespiratory responses to glutamate before and after nTS C21. For nTS microinjection experiments, cardiorespiratory responses in naïve rats (n = 5) were similar to those observed in GFP rats (n = 6). Therefore, data from these animals were included with the GFP group. Differences were considered significant if P ≤ 0.05. All values are expressed as means ± SE.
RESULTS
AAV-Mediated Expression of GFP and mCherry in the PVN and nTS
PVN microinjection sites were verified immunohistochemically in animals that received bilateral PVN microinjections of either AAV2-hSyn-GFP or AAV2-hSyn-hM4D(Gi)-mCherry. Figure 1A shows representative photomicrographs of coronal forebrain sections from GFP (top) and GiDREADD (bottom) rats demonstrating high expression of GFP immunoreactivity (IR) and mCherry-IR, respectively, in the PVN. Higher magnification of boxed areas depicts robust expression of GFP- and mCherry-IR in the somas and processes of PVN neurons, which was observed in all rats. In some animals, GiDREADD-mCherry and GFP labeling extended beyond the boundaries of the PVN. However, the only commonality among all animals was high expression in the PVN. Representative photomicrographs of coronal brainstem sections from GFP and GiDREADD rats demonstrate high expression of GFP- or mCherry-IR in fibers in the dorsal vagal complex (Fig. 1B). The majority of labeled fibers was located in the nTS, although GFP- and mCherry-IR were also observed in the dorsal motor nucleus of the vagus and area postrema. Higher magnification of boxed areas depicts GFP- and mCherry-IR in fibers in the nTS. Overall, expression of GFP (in GFP rats) and mCherry (in GiDREADD rats) in fibers in the nTS appeared similar.
Fig. 1.Viral expression of green fluorescent protein (GFP) and mCherry in paraventricular nucleus (PVN) neurons and fibers in the nucleus tractus solitarii (nTS) of GFP and inhibitory designer receptor exclusively activated by designer drug (GiDREADD) rats. A: photomicrographs of coronal PVN sections from individual rats showing expression of adeno-associated virus (AAV) 2-human synapsin promoter (hSyn)-GFP (top, GFP) or AAV2-hSyn-hM4D(Gi)-mCherry (bottom, GiDREADD-mCherry). Insets: higher magnification of boxed areas showing GFP- and mCherry-immunoreactivity (IR) present in neurons and processes. Scale bars = 100 µm. Inset scale bars = 50 µm. B: photomicrographs of coronal brainstem sections from GFP (top) and GiDREADD (bottom) rats showing GFP- and mCherry-IR in the dorsal vagal complex. Insets: higher magnification of boxed areas showing dense GFP- and mCherry-IR expression in fibers in the nTS. GFP- and mCherry-IR fibers were primarily localized to the nTS, although some fibers were present in the area postrema (AP) and dorsal motor nucleus of the vagus (DMX). Overall, GFP- and mCherry-IR was similar between GFP and GiDREADD rats. Scale bars = 100 µm. Inset scale bars = 10 µm. PVN and nTS levels are relative to bregma. 3V, third ventricle; TS, solitary tract.
C21 Markedly Attenuates Action Potential Discharge in GiDREADD-Expressing PVN Neurons
We verified the efficacy of C21 to inhibit PVN neurons from GFP and GiDREADD rats. Fig. 2A includes representative examples of coronal slices of the caudal PVN containing neurons that express GFP (left) or mCherry (indicating presence of GiDREADDs, right). There was no significant difference among groups in resting membrane potential under control conditions immediately before C21 (GFP: −58.0 ± 4.8 mV; GiDREADD, −57.8 ± 3.5 mV; unlabeled, −54.9 ± 1.8 mV; one-way ANOVA, P = 0.86). Fig. 2B shows original recordings of spontaneous action potential discharge observed in GFP (left) and GiDREADD (right) PVN cells under control conditions (Ctrl) during application of C21 (10 µM; 10 min) and during aCSF wash. C21 did not significantly alter membrane potential in GFP (Ctrl: −58.0 ± 4.8; C21: −51.2 ± 3.4; wash: −59.3 ± 1.8 mV; one-way RM ANOVA, P = 0.43) or GiDREADD-expressing PVN neurons (Ctrl: −57.8 ± 3.5; C21: −54.4 ± 3.0; wash: −54.7 ± 3.4 mV; one-way RM ANOVA, P = 0.80). In cells (n = 3) from GFP rats, C21 had no effect on spontaneous action potential discharge (P = 0.86; Fig. 2C). However, C21 nearly eliminated spontaneous action potential discharge in GiDREADD-expressing PVN neurons (P = 0.0013; Fig. 2C, right; n = 4). This inhibitory effect persisted after an aCSF wash, consistent with sustained inhibition of these cells by C21. Figure 2D shows representative examples of current-evoked action potential discharge in GFP and GiDREADD PVN neurons. Mean data (Fig. 2E) indicate that C21 reduced the number of evoked action potentials in GiDREADD-expressing PVN neurons during injection of depolarizing current (40–90 pA) but had no effect in GFP neurons except at the maximum current injected. We also evaluated the effects of C21 in unlabeled (mCherry-negative; n = 3) neurons located in the caudal PVN of GiDREADD rats. In these cells, we observed no inhibitory effect of C21 on membrane potential (Ctrl: −54.9 ± 1.8; C21: −53.4 ± 0.8; wash: −54.2 ± 2.0 mV; one-way RM ANOVA, P = 0.86), spontaneous action potential discharge (Ctrl: 1.2 ± 0.2; C21: 1.2 ± 0.1; wash: 1.0 ± 0.1 Hz; one-way RM ANOVA, P = 0.77) or total number of evoked action potentials across all current steps (number of action potentials: Ctrl, 70 ± 6.1; C21, 73.3 ± 8.4; wash, 68.7 ± 7.2, P = 0.93). Together, in vitro data confirm that C21 suppresses the activity of GiDREADD-expressing PVN neurons.
Fig. 2.Compound 21 (C21) decreases action potential discharge in inhibitory designer receptor exclusively activated by designer drug (GiDREADD)-expressing paraventricular nucleus (PVN) neurons. A: merged fluorescent and DIC composite images of coronal PVN slices showing neurons that express green fluorescent protein (GFP) (left) and mCherry (indicating presence of GiDREADDs, right). A pipette (dashed lines) was placed on labeled cells for patch clamp recording. B: representative traces show spontaneous action potential discharge in GFP (left) and GiDREADD (right) PVN neurons at baseline [control (Ctrl)], following application of C21 (10 µM; 10 min) and during an artificial cerebrospinal fluid (aCSF) wash. C: mean spontaneous action potential discharge in neurons expressing GFP (n = 3 rats) and GiDREADD-mCherry (n = 4 rats). In GFP cells, action potential discharge was not significantly different before, during, or after bath application of C21. In GiDREADD-mCherry neurons, C21 nearly eliminated action potential discharge, an effect that persisted during aCSF wash (one-way RM ANOVA with post hoc analysis, P < 0.05). D: overlay of evoked action potentials induced by injection of depolarizing current (40 pA) into GFP (left) and GiDREADD (right) PVN neurons under control conditions, during C21 application, and following aCSF wash. E: mean data showing the effects of C21 on the number of action potentials induced by depolarizing current (−20 to 100 pA) in GFP (left) and GiDREADD (right) PVN neurons. C21 produced sustained inhibition of GiDREADD-expressing PVN neurons in vitro (two-way RM ANOVA with post hoc analysis, *P < 0.05; **P < 0.01, C21, wash vs. Ctrl).
PVN Neuronal Inhibition Attenuates Ventilatory Responses to Hypoxia in Conscious GiDREADD Rats
Ventilatory responses during normoxia (21% O2, 5 min) and four intensities of hypoxia (14%, 12%, 10%, 8% O2, 5 min each) were evaluated in conscious GFP and GiDREADD rats. Figure 3 includes representative recordings from individual GFP (A) and GiDREADD (B) rats under control conditions (Ctrl, top traces) and 60 min after systemic injection of the DREADD agonist C21 (C21, 1 mg/kg ip; bottom traces) to inhibit PVN neurons. Hypoxic ventilatory responses appear reduced in the GiDREADD animal after injection of C21. Mean data demonstrate that baseline ventilation was similar between GFP and GiDREADD rats, and C21 did not alter baseline ventilatory parameters in either group (Table 1). In both groups, increasing hypoxia progressively decreased oxygen saturation and increased respiratory rate, tidal volume, and minute ventilation. Under control conditions (Ctrl, before C21), there were no significant differences between GFP and GiDREADD rats in the effects of hypoxia on oxygen saturation or any ventilatory parameter (P = 0.1–0.9, two-way RM ANOVA). In addition, in GFP rats, responses to hypoxia were similar under control conditions and following C21 (Fig. 3C). However, in GiDREADD rats (Fig. 3D), PVN neuronal inhibition with C21 blunted the increase in minute ventilation at all hypoxic intensities examined. The attenuated ventilatory response was due to a reduction in both respiratory rate and tidal volume. In addition, oxygen saturation was significantly lower at 10% and 8% O2 following C21 in GiDREADD rats. Responses to hyperoxic hypercapnia before and after C21 were measured in GFP and GiDREADD animals. Under control conditions, the effects of hypercapnia to increase ventilation were similar in both groups. In GFP rats, C21 did not alter the effect of hypercapnia on any ventilatory parameter (not shown, P = 0.12–0.99). Although not significant, in GiDREADD rats, there was a trend for C21 to decrease the maximum minute ventilation in response to hypercapnia (Ctrl: 11.3 ± 1.3; C21 9.2 ± 0.9, P = 0.083, two-way RM ANOVA). These results indicate that PVN activation contributes to ventilatory responses during hypoxia and possibly also during hypercapnia.
Fig. 3.Inhibitory designer receptor exclusively activated by designer drug (GiDREADD)-mediated inhibition of paraventricular nucleus (PVN) neurons blunts ventilatory responses to hypoxia. Plethysmography traces (5 s each) showing changes in breathing in conscious green fluorescent protein (GFP) (A) and GiDREADD (B) rats at the end of 5-min episodes of normoxia (21% O2) and 4 intensities of hypoxia (14%, 12%, 10%, and 8% O2, 5 min each) under control (Ctrl) conditions (top traces) and 60 min after intraperitoneal injection of Compound 21 (C21; 1 mg/kg; bottom traces). Group data showing the intensity-dependent effect of graded hypoxia on oxygen saturation and ventilation in GFP (n = 5 rats) (C) and GiDREADD (n = 7) (D) rats. Rats were exposed to hypoxia under control conditions before C21 microinjection (Ctrl; GFP, open circles; GiDREADD, light gray circles) and after C21 (C21, 1 mg/kg ip; GFP, dark gray circles; GiDREADD, closed circles). In GFP rats, hypoxic ventilatory responses were not affected by C21. In GiDREADD rats, C21 produced a greater reduction in oxygen saturation at severe hypoxic intensities. In addition, respiratory rate and minute ventilation were significantly reduced following C21 at all hypoxic intensities examined (Ctrl vs. C21; two-way RM ANOVA treatment vs. %O2, with post hoc analysis, P < 0.05). C21 also decreased tidal volume in GiDREADD rats (two-way RM ANOVA, treatment; P < 0.05). † vs. 21% O2; †† vs. 14%, 21% O2; ††† vs. 12%, 14%, 21% O2; # vs. 10%, 12%, 14%, 21% O2. For GiDREADD rats, *C21 vs. Ctrl.
| GFP | GiDREADD | |||
|---|---|---|---|---|
| Baseline | C21 | Baseline | C21 | |
| Oxygen saturation, % | 95 ± 1 | 95 ± 0.5 | 96 ± 0.4 | 95 ± 0.5 |
| Respiratory rate | 84 ± 7 | 82 ± 9 | 79 ± 5 | 78 ± 6 |
| Tidal volume index | 0.04 ± 0.003 | 0.04 ± 0.003 | 0.05 ± 0.001 | 0.04 ± 0.003 |
| Minute ventilation index | 3.5 ± 0.3 | 3.3 ± 0.4 | 3.9 ± 0.5 | 3.5 ± 0.4 |
DREADD-Mediated PVN Neuronal Inhibition Limits Hypoxia-Induced cFos Expression in the nTS
Neuronal activation (cFos-IR) in response to acute hypoxia was evaluated in the PVN and nTS of conscious normoxic and hypoxic GiDREADD rats that received pretreatment with saline (Sal, 1 mL; 21% O2, n = 3; 10% O2, n = 4) or an equivalent volume of C21 (1 mg/kg; 21% O2, n = 3; 10% O2, n = 4). Figure 4 shows representative photomicrographs of coronal forebrain and brainstem sections displaying cFos-IR in the PVN (A) and nTS (B) of GiDREADD rats exposed to 2 h of normoxia (left) or hypoxia (10% O2, right) following pretreatment with saline or C21. Mean data show the total number of cFos-IR cells counted in two caudal PVN sections (−1.9 and −2.1 mm, relative to bregma) (C) and three nTS sections (−14.46, −14.28, and −14.1 mm relative to bregma, which corresponds to −180, 0, and +180 μm, relative to calamus scriptorius) (D). In both brain regions, normoxic GiDREADD rats displayed relatively few cFos-IR cells independent of saline or C21 pretreatment. After hypoxia, both groups exhibited robust cFos-IR throughout the PVN and nTS. Consistent with neuronal inhibition, hypoxic GiDREADD rats that received C21 injection had significantly fewer cFos-IR cells in the PVN compared with saline-treated rats. This was reflected in a similar decrease in cFos-IR in the nTS. Taken together, the data demonstrate that activation of GiDREADDs on PVN neurons reduced neuronal activation in the PVN, and this was associated with diminished activation of nTS neurons by hypoxia.
Fig. 4.Compound 21 (C21) decreases hypoxia-induced cFos immunoreactivity (IR) in the paraventricular nucleus (PVN) and nucleus tractus solitarii (nTS) of inhibitory designer receptor exclusively activated by designer drug (GiDREADD) rats. Representative photomicrographs of coronal sections of the PVN (A) and nTS (B) of GiDREADD rats. Rats received intraperitoneal injection of saline (Sal, 1 mL, top) or an equivalent volume of C21 (1 mg/kg, bottom) and, 60 min later, were exposed to 2 h of normoxia (21% O2; left) or hypoxia (10% O2; right). White outlined area represents the regions of the PVN and nTS where cFos-IR counts were performed. Mean data showing the total number of cFos-IR cells counted bilaterally in two caudal PVN sections (−1.9 to −2.1 mm, relative to bregma) (C) and three nTS sections (−14.46, −14.28, and −14.1 mm relative to bregma, which corresponds to −180, 0, and +180 μm, relative to calamus scriptorius) (D) from rats exposed to normoxia (Sal, n = 3 rats; C21, n = 4) or hypoxia (Sal, n = 3 rats; C21, n = 4 rats). In normoxic rats, the number of cFos-IR cells was independent of systemic pretreatment in both the PVN and nTS. In comparison, rats exposed to hypoxia exhibited robust increases in cFos-IR in both brain regions. However, C21-treated GiDREADD rats displayed significantly fewer cFos-IR neurons compared with Sal-treated rats in both the PVN and nTS (two-way RM ANOVA with post hoc analysis, P < 0.05). 3V, third ventricle; DMX, dorsal motor nucleus of the vagus; TS, solitary tract. P < 0.05 for Sal- and C21-treated rats, *normoxia vs. hypoxia. For hypoxic GiDREADD rats, †C21 vs. Sal.
Bilateral Inhibition of PVN Terminals in the nTS Alters Chemoreflex Cardiorespiratory Responses
To evaluate whether PVN inputs specifically to the nTS influence chemoreflex output, we compared cardiorespiratory responses to hypoxia before and after bilateral nTS microinjection of C21 in GFP and GiDREADD rats. Baseline cardiorespiratory parameters before experimental manipulation were generally similar between groups, although GiDREADD rats displayed higher baseline MAP versus GFP rats (Table 2). Figure 5 illustrates representative recordings from a GFP rat and a GiDREADD rat during exposure to hypoxia (10% O2, 45 s) before and 60 min after bilateral nTS microinjection of C21. In the GFP rat (Fig. 5A), cardiorespiratory responses to an initial bout of hypoxia appeared similar before and after C21 microinjection. In the GiDREADD rat, however, C21 blunted the PhrNA response but enhanced the sSNA response to hypoxia (Fig. 5B).
| GFP | GiDREADD | |||
|---|---|---|---|---|
| Ctrl | C21 | Ctrl | C21 | |
| MAP, mmHg | 100 ± 3 | 101 ± 3 | 111 ± 3† | 106 ± 2* |
| HR, beats/min | 286 ± 9 | 289 ± 10 | 307 ± 8 | 309 ± 8 |
| sSNA, mV·s | 0.030 ± 0.006 | 0.032 ± 0.006 | 0.026 ± 0.003 | 0.031 ± 0.004* |
| Min PhrNA, mV·s·min−1 | 12 ± 2 | 12 ± 3 | 9 ± 2 | 9 ± 2 |
| Phr Freq, breaths/min | 33 ± 2 | 31 ± 3 | 34 ± 2 | 33 ± 2 |
| Phr Amp, mV·s | 0.43 ± 0.12 | 0.42 ± 0.12 | 0.27 ± 0.09 | 0.24 ± 0.06 |
Fig. 5.Inhibition of paraventricular nucleus (PVN) terminals in the nucleus tractus solitarii (nTS) alters cardiorespiratory responses to acute hypoxia in inhibitory designer receptor exclusively activated by designer drug (GiDREADD) rats. Original recordings from a representative green fluorescent protein (GFP) (A) or GiDREADD (B) rat showing the changes in cardiorespiratory parameters during an acute hypoxic episode (10% O2, 45 s) under control (Ctrl) conditions (left) and 60 min after nTS microinjection of Compound 21 (C21; right). Insets: splanchnic sympathetic nerve activity (sSNA) at baseline before hypoxia (a), during Ctrl 10% O2 (b), post-C21 before hypoxia (a’), and post-C21 during 10% O2 (b’). Under control conditions, hypoxia evoked depressor and tachycardic responses and increased sSNA and phrenic nerve activity (PhrNA) in both animals. In the GFP rat, cardiorespiratory responses to hypoxia appeared unaltered after C21. In the GiDREADD rat, hypoxia-evoked heart rate (HR) and sSNA effects appeared enhanced after C21, whereas PhrNA responses were blunted. AP, arterial pressure (mean AP in blue superimposed on AP trace); ∫sSNA, integrated splanchnic sympathetic nerve activity; ∫PhrNA, integrated phrenic nerve activity.
Average data depicting the time course of cardiorespiratory chemoreflex responses before and after C21 in GFP and GiDREADD rats are shown in Fig. 6. Under control conditions, hypoxia decreased MAP and increased HR and sSNA in both groups. Hypoxia increased Min PhrNA because of both a transient increase in Phr Freq and a more sustained elevation of Phr Amp. Bilateral nTS microinjection of C21 (0.1 mM, 90 nL/side) had no effect on baseline cardiorespiratory parameters in GFP rats and evoked a small but statistically significant decrease in MAP and increase in sSNA in GiDREADD rats (Table 2). Following C21 microinjection in GFP animals, hypoxia-induced cardiorespiratory responses were unaltered (Fig. 6A). In GiDREADD rats, however, C21-mediated inhibition of PVN terminals in the nTS blunted Min PhrNA responses to hypoxia, primarily because of a reduction in Phr Amp (Fig. 6B). In contrast to phrenic responses, C21 augmented tachycardic and sympathoexcitatory responses to hypoxia in GiDREADD rats. Hypoxia-evoked depressor responses and Phr Freq were not altered by C21.
Fig. 6.DREADD-mediated paraventricular nucleus (PVN) terminal inhibition alters cardiorespiratory responses to acute hypoxia. A and B: group data showing the average time course of cardiorespiratory effects of hypoxia (10% O2, 45 s) before [green fluorescent protein (GFP), open circles; inhibitory designer receptor exclusively activated by designer drug (GiDREADD), light gray circles] and after Compound 21 (C21; GFP, dark gray circles; GiDREADD, closed circles). Under control conditions, hypoxia produced significant decreases in mean arterial pressure (MAP) and increases in heart rate (HR), splanchnic sympathetic nerve activity (sSNA), and phrenic nerve activity (PhrNA) in both groups. Cardiorespiratory chemoreflex responses were similar after nucleus tractus solitarii (nTS) microinjection of C21 in GFP rats. In GiDREADD rats, C21 blunted the hypoxia-evoked increases in minute phrenic nerve activity (Min PhrNA) and phrenic amplitude (Phr Amp). In addition, the HR and sSNA responses to hypoxia were enhanced after C21. (MAP/HR: GFP, n = 11 rats; GiDREADD, n = 12 rats; sSNA: GFP, n = 10 rats; GiDREADD, n = 11 rats; PhrNA: GFP, n = 6 rats; GiDREADD, n = 6 rats) Two-way RM ANOVA with post hoc analysis, P < 0.05, *C21 vs. Ctrl. Phr Freq, phrenic frequency.
Comparison of Peak Responses to Hypoxia in GFP and GiDREADD Rats
To compare responses between groups, and because the time of the maximum response varied among animals, we compared peak cardiorespiratory changes because of hypoxia in GFP and GiDREADD rats, before and after nTS C21 microinjection (Fig. 7). Under control conditions, MAP, HR, sSNA, and PhrNA responses to hypoxia were similar in both groups, indicating that DREADD expression alone did not affect responses to hypoxia. In GFP rats, peak cardiorespiratory responses were not affected by C21. In GiDREADD rats, however, inhibition of PVN terminals by C21 blunted the peak hypoxia-induced increases in Min PhrNA and Phr Amp without affecting Phr Freq. Furthermore, comparison between groups indicated that the peak Min PhrNA and Phr Amp responses to hypoxia after C21 were significantly attenuated compared with those in GFP rats. The peak tachycardic and sympathoexcitatory responses following C21 were greater in GiDREADD rats compared both to their own control responses and to effects in GFP rats. Together, these data indicate that GiDREADD-mediated PVN terminal inhibition alters autonomic and cardiorespiratory chemoreflex responses.
Fig. 7.Nucleus tractus solitarii (nTS) Compound 21 (C21) alters peak cardiorespiratory responses to acute hypoxia in inhibitory designer receptor exclusively activated by designer drug (GiDREADD) rats. Mean data comparing peak changes in cardiorespiratory chemoreflex responses before and after C21 in green fluorescent protein (GFP) and GiDREADD rats. Under control (Ctrl) conditions, peak responses were similar between groups. In GiDREADD rats, C21 decreased peak minute phrenic nerve activity (Min PhrNA) and phrenic amplitude (Phr Amp) and increased peak heart rate (HR) and splanchnic sympathetic nerve activity (sSNA) responses relative to their own control. Responses were also altered compared with both control and C21 responses in GFP rats. (Two-way RM ANOVA with post hoc analysis, P < 0.05.) [Mean arterial pressure (MAP)/HR: GFP, n = 11 rats; GiDREADD, n = 12 rats; sSNA: GFP, n = 10 rats; GiDREADD, n = 11 rats; PhrNA: GFP, n = 6 rats; GiDREADD, n = 6 rats] *C21 vs. Ctrl. For C21: †GiDREADD vs. GFP rats. Phr Freq, phrenic frequency.
nTS C21 Does Not Alter Cardiorespiratory Coupling During Hypoxia
Inhibition of PVN inputs to the nTS produced blunted PhrNA but enhanced sSNA responses to hypoxia in GiDREADD rats. We thus examined whether these changes were associated with altered cardiorespiratory coupling in GiDREADD rats. We analyzed the coupling pattern of sSNA to PhrNA during early and late inspiration (I1, I2) and expiration (E1, E2) in GFP (n = 5) and GiDREADD (n = 5) rats. Because the absolute level of sSNA was altered differently in GiDREADD and GFP rats, the pattern of coupling was assessed by evaluating the value of sSNA during each phase of the respiratory cycle, expressed as a percentage of the total amount of sSNA during the cycle. Responses were examined during baseline conditions before hypoxia (Pre-Hx) and at the peak increase in sSNA during hypoxia (Hx, 10% O2) under control conditions and after nTS microinjection of C21. In both GFP and GiDREADD rats, the pattern of sSNA under baseline conditions before hypoxia was characterized by similar activity in both phases of inspiration, and most activity occurred during expiration, particularly the late phase of expiration. During hypoxia under control conditions, sSNA increased significantly (Fig. 6). In both groups, this was associated with a greater percentage of activity during E1 and less during E2 relative to the Pre-Hx condition (Fig. 8). We then evaluated the effect of nTS microinjection of C21 on cardiorespiratory coupling before hypoxia (C21 Pre-Hx) and during hypoxia (C21 Hx). In GFP rats, C21 did not change the pattern of sSNA during normoxia or hypoxia. In GiDREADD rats, C21 increased the percentage of sSNA occurring during E1 in normoxia. However, the pattern of sSNA during hypoxia post-C21 was similar to that observed during control hypoxia.
Fig. 8.Nucleus tractus solitarii (nTS) Compound 21 (C21) does not alter cardiorespiratory coupling during hypoxia. Mean data showing the coupling patterns of splanchnic sympathetic nerve activity (sSNA) to phrenic nerve activity (PhrNA) in green fluorescent protein (GFP; n = 5, left) and inhibitory designer receptor exclusively activated by designer drug (GiDREADD) rats (n = 5, right). sSNA (expressed as percent total activity within a given cycle) is shown during inspiration (I1, I2) and expiration (E1, E2). In both groups, before hypoxia (Pre-Hx), sSNA primarily occurred during expiration (I1 = I2 < E1 < E2). The sSNA response to hypoxia (Hx, 10% O2) under control conditions was characterized by an elevation in E1 and a reduction in E2 compared with Pre-Hx. In GFP rats, C21 did not change the pattern of sSNA before (C21 Pre-Hx) or during hypoxia (C21 Hx). In GiDREADD rats, C21 increased sSNA during E1 under normoxic conditions before Hx. However, the sSNA response to hypoxia was not altered by nTS microinjection of C21. Two-way RM ANOVA with post hoc analysis, P < 0.05 *Pre-Hx vs. Hx, C21 Pre-Hx vs. C21 Hx. †C21 Pre-Hx vs. Pre-Hx.
Microinjection of C21 Lateral to nTS Has No Effect on Cardiorespiratory Responses to Hypoxia
To examine whether the effects of C21 on chemoreflex cardiorespiratory responses observed in GiDREADD animals were due to PVN terminal inhibition specifically within the nTS, cardiorespiratory responses to hypoxia were evaluated in GiDREADD rats (n = 3) before and after bilateral microinjection of C21 lateral to the nTS. Figure 9 demonstrates that, as in initial experiments, hypoxia decreased arterial pressure and increased heart rate, sSNA, and all PhrNA parameters. C21 microinjection outside the nTS had no effect on these responses to hypoxia. Thus, our data suggest that the effects of nTS C21 to modulate cardiorespiratory chemoreflex responses in GiDREADD rats were due to selective inhibition of PVN terminals located within the nTS.
Fig. 9.Compound 21 (C21) injected lateral to nucleus tractus solitarii (nTS) has no effect on cardiorespiratory responses to hypoxia. Mean data from inhibitory designer receptor exclusively activated by designer drug (GiDREADD) rats (n = 3 rats) showing cardiorespiratory responses to hypoxia (10% O2, 45 s) before and after bilateral microinjection of C21 (0.1 mM, 90 nL/side) lateral to the nTS (0.3 mm anterior, 3–4 mm lateral, and 0.4 mm ventral relative to calamus scriptorius). As above, hypoxia decreased mean arterial pressure (MAP) and increased heart rate (HR), splanchnic sympathetic nerve activity (sSNA), and all phrenic nerve activity (PhrNA) parameters. C21 microinjection outside the nTS had no effect on hypoxia-evoked cardiorespiratory output (paired t test). Phr Amp, phrenic amplitude; Phr Freq, phrenic frequency.
nTS Microinjection of C21 Does Not Attenuate Cardiorespiratory Responses to Glutamate
The effect of nTS C21 to modulate chemoreflex function in GiDREADD rats could possibly be due to a nonspecific effect of C21 to alter nTS neuronal excitability. To test this, we evaluated cardiorespiratory responses to unilateral nTS microinjection of glutamate (5 mM, 30 nL) before and after bilateral nTS microinjection of C21 in GiDREADD rats (n = 3). Peak responses to glutamate before and after C21 are shown in Fig. 10. nTS glutamate produced a transient decrease in MAP, HR, and sSNA and Phr Freq, Phr Amp, and Min PhrNA. These responses were largely unaffected by nTS microinjection of C21, although the decrease in Phr Freq was significantly augmented. Taken together, our data indicate that C21 acts specifically on PVN terminals in the nTS and has minimal nonspecific effects to decrease intrinsic responsiveness to excitatory stimuli of nTS neurons that influence cardiorespiratory function.
Fig. 10.Nucleus tractus solitarii (nTS) microinjection of Compound 21 (C21) does not restrain cardiorespiratory responses to glutamate. Mean data from inhibitory designer receptor exclusively activated by designer drug (GiDREADD) rats (n = 3 rats) showing cardiorespiratory responses to unilateral microinjection of glutamate (5 mM, 30 nL) into the nTS before and after bilateral nTS microinjection of C21 (0.1 mM, 90 nL/side). Under control conditions, glutamate evoked a transient decrease in all cardiorespiratory parameters examined [mean arterial pressure (MAP), heart rate (HR), splanchnic sympathetic nerve activity (sSNA), and phrenic nerve activity (PhrNA)]. Paired t test analyses revealed that C21 had no significant effect on MAP, HR, sSNA, minute phrenic nerve activity (Min PhrNA), or phrenic amplitude (Phr Amp) responses to glutamate. The decrease in Phr Freq was significantly augmented following nTS C21. Data indicate that C21 does not diminish nTS neuronal excitability. Paired t test, P < 0.05, *C21 vs. Ctrl. Phr Freq, phrenic frequency.
DISCUSSION
This study tested the hypothesis that activation of the PVN, and specifically an efferent projection from PVN to the nTS, increases nTS neuronal activation and enhances cardiorespiratory responses to hypoxia. In conscious animals, generalized PVN inhibition decreased hypoxia-induced cFos expression in the PVN, blunted ventilatory responses to hypoxia, and reduced oxygen saturation at more severe hypoxic intensities. In addition, inhibition of PVN neurons was associated with a reduction in the number of activated (cFos-IR) nTS neurons following hypoxia, suggesting that the PVN contributes to hypoxia-induced nTS neuronal excitation. Furthermore, silencing of PVN inputs to the nTS blunted increases in phrenic nerve activity amplitude and minute phrenic nerve activity during hypoxia in anesthetized rats. These results demonstrate that PVN projections to the nTS enhance respiratory responses to chemoreflex stimulation by hypoxia, likely because of enhanced hypoxia-induced activation of nTS neurons. Interestingly, PVN terminal inhibition enhanced tachycardic and sympathoexcitatory responses to hypoxia. Taken together, our data support the concept that the PVN is integral to the regulation of sympathetic and cardiorespiratory responses to chemoreflex stimulation (45, 56), and activation of the PVN-nTS pathway differentially modulates respiratory and cardiovascular adjustments to hypoxia.
In this study, inhibitory DREADDs (GiDREADDs) were used to constrain activation of PVN neurons and their terminals in the nTS. We observed robust expression of GiDREADD and control GFP-labeled neurons throughout the PVN and in fibers in the dorsal medulla, particularly in the nTS. Expression of GiDREADDs alone appeared to have minimal effects. Basal membrane potential in nTS neurons and cardiorespiratory chemoreflex responses were similar in GFP and GiDREADD rats in the absence of C21, although there was a small but significant increase in resting arterial pressure. Our electrophysiological data support previous work (3) confirming the efficacy of GiDREADDs to suppress neuronal activity. Bath application of the synthetic agonist C21 produced sustained reduction of spontaneous and evoked action potential discharge in GiDREADD-mCherry PVN neurons. This inhibitory effect was not observed in GFP-expressing PVN neurons. In addition, intraperitoneal injection of C21 blunted the increase in PVN neuronal activation (i.e., cFos) in conscious GiDREADD rats exposed to acute hypoxia. Taken together, these findings support studies (52, 53) demonstrating that C21-mediated activation of GiDREADDs is effective in inhibiting neuronal activity both in vivo and in vitro.
The PVN plays an important role in respiratory modulation, and PVN activation increases respiratory frequency and tidal volume (17, 44, 56). Furthermore, PVN lesions or inhibition blunts responses to cyanide-evoked chemoreceptor activation and intermittent hypoxia (4, 38, 41). The current data confirm and extend these studies by showing that inhibition of PVN neurons also attenuates the increase in respiratory frequency and tidal volume because of progressive acute hypoxia. This blunting of respiratory responses to both cyanide (41) and hypoxia (current data) occurs despite different arterial pressure responses to these stimuli, suggesting the effects of PVN inhibition are mediated by altered chemoreflex function rather than some other cardiorespiratory afferent system (e.g., arterial baroreflex) that may be secondarily involved in the response. Importantly, PVN inhibition was associated with decreased oxygen saturation at more severe hypoxic intensities, indicating that reduced hypoxic ventilatory responses were physiologically relevant, impairing the ability to maintain arterial oxygen levels in response to a hypoxic challenge. The PVN also may be involved in central chemoreflex function, since PVN inhibition tended to blunt ventilatory responses to hyperoxic hypercapnia. These data differ from previous work (41), showing no effect of PVN inhibition on hypercapnic responses, but are consistent with our previous study showing that removal of catecholaminergic inputs to PVN blunted central chemoreflex effects (27). Overall, our data show for the first time that the PVN is required for full expression of compensatory ventilatory responses to acute hypoxia, the natural stimulus of carotid chemoreceptors, and possibly to central chemoreflex function as well.
The PVN sends projections to cardiorespiratory nuclei throughout the brainstem and spinal cord (18, 43), and respiratory responses to PVN activation likely utilize these pathways (31, 56). Previous work (12) suggests that chemoreflex stimulation by intermittent administration of KCN induces activation of RVLM-projecting but not nTS-projecting neurons in the PVN. Nevertheless, hypoxia-induced chemoreflex stimulation does not activate PVN projections to the ventrolateral medulla or IML (10). Rather, PVN neurons projecting to the nTS are activated by hypoxia (42), consistent with the concept that the PVN contributes to cardiorespiratory responses to KCN and hypoxia via different neuronal pathways, and a descending PVN-to-nTS projection plays a role in chemoreflex hypoxic responses. This concept is supported by our immunohistochemistry data, demonstrating that, in association with blunted ventilatory responses, GiDREADD-mediated PVN inhibition diminished nTS neuronal activation (cFos-IR) in response to hypoxia. These data indicate that the PVN facilitates excitation of chemosensitive nTS neurons during hypoxia and thus may enhance hypoxic cardiorespiratory responses.
To further examine the functional relevance of the descending PVN-nTS projection in modulating cardiorespiratory chemoreflex function, we evaluated responses to hypoxia during selective inhibition of this pathway. Activation of presynaptic GiDREADDs reduces synaptic release probability and synaptic current amplitude without disrupting somatic or axonal membrane potentials, producing synaptic silencing (49, 57). Thus, we microinjected the DREADD agonist C21 directly into the nTS to selectively silence PVN inputs to the nTS. Hypoxia produced an increase in phrenic nerve activity that was characterized by elevations in both phrenic frequency and amplitude, similar to previous reports (9, 33). Microinjection of C21 into the nTS did not alter baseline phrenic activity, suggesting only a minor tonic role for the PVN-nTS pathway in basal respiratory control. However, this pathway is important for chemoreflex ventilatory stimulation, as PVN terminal inhibition in the nTS of GiDREADD rats markedly attenuated the minute phrenic response to hypoxia because of a decrease in phrenic amplitude. These blunted phrenic nerve activity responses were due to inhibition of PVN terminals specifically in the nTS since they were not observed in GiDREADD rats in which C21 was microinjected outside of the nTS. Furthermore, diminished phrenic activation to hypoxia was not due to nonspecific effects of C21 to diminish nTS neuronal excitability because cardiorespiratory responses to glutamate were similar or enhanced after C21, and C21 had no effect on phrenic responses to hypoxia in GFP rats. Coupled with our previous report that hypoxia activates nTS-projecting PVN neurons (42) and current data showing reduced hypoxia-induced nTS neuronal activation after PVN inhibition, these data indicate that, although respiratory responses remain, activation of a descending PVN-nTS pathway is critical for shaping ventilatory responses to hypoxia. Therefore, the blunted phrenic responses observed during PVN terminal inhibition are likely due to elimination of excitatory inputs from the PVN to chemosensitive nTS neurons.
Interestingly, general inhibition of PVN neurons in conscious rats blunted both the tidal volume and breathing frequency response to hypoxia, whereas selective silencing of PVN synaptic inputs to the nTS in anesthetized animals attenuated the increase in phrenic amplitude only. Hypoxia evokes glutamate release from chemoafferents, induces robust activation of nTS neurons, and produces intensity-dependent increases in both respiratory rate and tidal volume (36). The nTS is required for appropriate hypoxic ventilatory responses, but the extent to which the nTS shapes respiratory frequency and tidal volume responses is less clear. Some studies report that nTS inhibition or lesions reduce tidal volume but do not alter respiratory frequency responses to hypoxia (36, 51), whereas other reports indicate effects on both frequency and amplitude responses to peripheral chemoreceptor stimulation (8, 9). The reasons for the discrepancy in hypoxic ventilatory responses between plethysmography and microinjection experiments in the current study are not clear. One possibility is the effects of anesthesia or the specific preparation. Anesthesia alters glutamatergic and GABAergic neurotransmission in cardiorespiratory nuclei, including the PVN and nTS (22, 23), and blunts cardiorespiratory reflexes (37), and vagotomy in the anesthetized rats eliminates afferent inputs from the lungs (34). In addition, the Phr Freq response to hypoxia in the microinjection studies is substantially less than the increase observed in plethysmography experiments. Another possibility is that the PVN influences the tidal volume response to hypoxia via a specific projection to the nTS, whereas its effects to modulate the frequency of ventilation are mediated through projections to other brain regions, such as the pre-Bötzinger complex, retrotrapezoid nucleus, or raphe nuclei (31, 56). Regardless, the data in the present study support the concept that a descending PVN-nTS pathway plays an important role in shaping hypoxic ventilatory responses. PVN projections to the nTS contribute to activation of chemosensitive nTS neurons and augment hypoxic ventilatory responses by enhancing phrenic amplitude. Additional studies are required to verify the mechanism of PVN effects on breathing frequency.
The current studies suggest that the PVN-nTS pathway enhances nTS neuronal and ventilatory responses to hypoxia, but the mechanisms within the nTS are not known. The PVN contains numerous neuropeptides, and many of these neuropeptides are synthesized in PVN neurons that project to the nTS (6, 50). Our previous studies (42) showed that a large majority of nTS-projecting PVN neurons that are activated by hypoxia are immunoreactive for corticotropin-releasing hormones (CRH), and the CRH receptor CRFR2 is present in the nTS. Activation of CRH projections to the nTS increases discharge frequency in nTS neurons and arterial pressure, and nTS CRFR2s contribute to hypertension following intermittent hypoxia (54, 55). Thus, CRH in the nTS may contribute to the effects of the PVN on responses to acute hypoxia. In the nTS, CRFR2s are presynaptic on oxytocin-IR fibers and are closely associated with hypoxia-activated nTS neurons (42). PVN oxytocinergic projections to the nTS facilitate excitation of nTS neurons (5, 40). Thus, it is possible that the PVN-nTS projection exerts its effects on chemoreflex responses via CRH and/or oxytocin signaling in the nTS. In addition, CRH and oxytocin PVN neurons are predominantly glutamatergic (13), and the excitatory effects of these PVN projections to the nTS could be mediated by glutamate or an interaction between glutamate and neuropeptide signaling (40). Further studies are required to determine the mechanisms by which nTS-projecting PVN neurons influence cardiorespiratory chemoreflex responses and the specific projections (e.g., retrotrapezoid nucleus, VLM, raphe nuclei) from the nTS that may contribute to these effects.
In contrast to effects on phrenic nerve activity, selective inhibition of GiDREADD PVN terminals in the nTS produced a small augmentation of the sympathoexcitatory and tachycardic responses to hypoxia. There are several possibilities that could account for these results. PVN stimulation has been shown to produce both sympathoexcitatory and pressor responses and also depressor responses (16, 24, 54). Furthermore, reports indicate that activation of the PVN may either inhibit or activate nTS neurons, including barosensitive neurons (16, 24). Our cFos data suggest that the PVN contributes to hypoxia-induced activation of chemosensitive nTS neurons, and this likely contributes to the PVN-induced enhancement of ventilation and phrenic nerve activity during hypoxia. However, the PVN-nTS projection may also excite barosensitive sympathoinhibitory caudal ventrolateral medulla-projecting nTS neurons during hypoxia. If so, inhibition of PVN terminals in the nTS could result in augmented sympathoexcitation during hypoxia. In addition, GABA mechanisms in the nTS influence KCN-evoked chemoreflex function (7), and this could possibly play a role. For example, it is possible that activation of the PVN-nTS pathway during hypoxia directly or indirectly (via inhibitory GABAergic or glycinergic interneurons) inhibits RVLM-projecting nTS neurons, which are critical for the sympathoexcitation in response to hypoxia (29). Regardless of the mechanism, it appears that PVN inputs to the nTS dampen sympathoexcitatory and tachycardic responses to peripheral chemoreflex stimulation.
The nTS is important for the respiratory modulation of sympathetic nerve activity (11). Similar to previous studies (14), baseline sympathetic activity was coupled to the respiratory cycle. The majority of sSNA occurred during expiration, and the increase in sSNA during hypoxia was also primarily associated with expiration. Our data show that PVN terminal inhibition in the nTS slightly increased baseline sympathetic activity primarily during early expiration, suggesting a tonic input from the PVN to the nTS has a minor effect on basal sympathetic activity and its coupling to respiration. In contrast, the enhanced sympathoexcitation during PVN terminal inhibition in the nTS was not associated with altered cardiorespiratory coupling because of hypoxia. Thus, although the nTS plays a role in coupling, the change in respiratory and sympathetic coupling during hypoxia does not appear to require a projection from the PVN to the nTS.
Perspectives and Significance
The findings from the present study, in combination with previous work, strengthen the concept that the PVN, likely via a reciprocal nTS-PVN pathway, plays an important role in shaping cardiorespiratory responses to chemoreflex stimulation. Hypoxia activates a population of predominantly catecholaminergic nTS neurons that project to the PVN (25), and this projection contributes to hypoxia-evoked cardiorespiratory responses. In addition, we propose that these projections from the nTS in turn directly or indirectly activate PVN neurons that project back to the nTS (42). These nTS-projecting PVN neurons are neuropeptidergic and may corelease glutamate. The current data show that activation of the PVN, possibly via a descending PVN-nTS pathway, facilitates excitation of chemosensitive nTS neurons and enhances chemoreflex respiratory responses during acute hypoxia. It also may serve as a mechanism that limits activation of the sympathetic nervous system during hypoxia. It is possible that function of this pathway is enhanced in response to sustained hypoxic exposure or following intermittent hypoxia. If so, it could play an important role in adaptation to altitude and may also contribute to augmented chemoreflex function after acute intermittent hypoxia and in pathophysiological states, such as obstructive sleep apnea.
GRANTS
This work was supported by Grants R01-HL-98602 (to C. M. Heesch, D. D. Kline, and E. M. Hasser) and F31-HL-140858 (to B. C. Ruyle).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
B.C.R. and E.M.H. conceived and designed research; B.C.R. and D.M. performed experiments; B.C.R. and D.M. analyzed data; B.C.R., D.M., C.M.H., D.D.K., and E.M.H. interpreted results of experiments; B.C.R. and D.M. prepared figures; B.C.R. drafted manuscript; B.C.R., D.M., C.M.H., D.D.K., and E.M.H. edited and revised manuscript; B.C.R., D.M., C.M.H., D.D.K., and E.M.H. approved final version of manuscript.
ACKNOWLEDGMENTS
We thank Sarah A. Friskey for outstanding technical expertise. We also thank Allie Feinberg and Colbren Trogstad-Isaacson for excellent assistance with immunohistochemical procedures.
REFERENCES
- 1. . Caudal nuclei of the rat nucleus of the solitary tract differentially innervate respiratory compartments within the ventrolateral medulla. Neuroscience 190: 207–227, 2011. doi:10.1016/j.neuroscience.2011.06.005.
Crossref | PubMed | Web of Science | Google Scholar - 2. . Nucleus tractus solitarius—gateway to neural circulatory control. Annu Rev Physiol 56: 93–116, 1994. doi:10.1146/annurev.ph.56.030194.000521.
Crossref | PubMed | Web of Science | Google Scholar - 3. . Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc Natl Acad Sci USA 104: 5163–5168, 2007. doi:10.1073/pnas.0700293104.
Crossref | PubMed | Web of Science | Google Scholar - 4. . Hypothalamic PVN contributes to acute intermittent hypoxia-induced sympathetic but not phrenic long-term facilitation. J Appl Physiol (1985) 124: 1233–1243, 2018. doi:10.1152/japplphysiol.00743.2017.
Link | Web of Science | Google Scholar - 5. . Oxytocin innervation of caudal brainstem nuclei activated by cholecystokinin. Brain Res 993: 30–41, 2003. doi:10.1016/j.brainres.2003.08.036.
Crossref | PubMed | Web of Science | Google Scholar - 6. . Intra- and extrahypothalamic vasopressin and oxytocin pathways in the rat. Cell Tissue Res 186: 423–433, 1978. doi:10.1007/BF00224932.
Crossref | PubMed | Web of Science | Google Scholar - 7. . Activation of GABAA but not GABAB receptors in the NTSblocked bradycardia of chemoreflex in awake rats. Am J Physiol Heart Circ Physiol 276: H1902–H1910, 1999. doi:10.1152/ajpheart.1999.276.6.H1902.
Link | Web of Science | Google Scholar - 8. . Domoic acid lesions in nucleus of the solitary tract: time-dependent recovery of hypoxic ventilatory response and peripheral afferent axonal plasticity. J Neurosci 22: 3215–3226, 2002. doi:10.1523/JNEUROSCI.22-08-03215.2002.
Crossref | PubMed | Web of Science | Google Scholar - 9. . A midline area in the nucleus commissuralis of NTS mediates the phrenic nerve responses to carotid chemoreceptor stimulation. Brain Res 662: 127–133, 1994. doi:10.1016/0006-8993(94)90804-4.
Crossref | PubMed | Web of Science | Google Scholar - 10. . Acute hypoxia activates neuroendocrine, but not presympathetic, neurons in the paraventricular nucleus of the hypothalamus: differential role of nitric oxide. Am J Physiol Regul Integr Comp Physiol 312: R982–R995, 2017. doi:10.1152/ajpregu.00543.2016.
Link | Web of Science | Google Scholar - 11. . Glutamatergic antagonism in the NTS decreases post-inspiratory drive and changes phrenic and sympathetic coupling during chemoreflex activation. J Neurophysiol 103: 2095–2106, 2010. doi:10.1152/jn.00802.2009.
Link | Web of Science | Google Scholar - 12. . Intermittent activation of peripheral chemoreceptors in awake rats induces Fos expression in rostral ventrolateral medulla-projecting neurons in the paraventricular nucleus of the hypothalamus. Neuroscience 157: 463–472, 2008. doi:10.1016/j.neuroscience.2008.08.070.
Crossref | PubMed | Web of Science | Google Scholar - 13. . Neuroanatomical evidence for reciprocal regulation of the corticotrophin-releasing factor and oxytocin systems in the hypothalamus and the bed nucleus of the stria terminalis of the rat: implications for balancing stress and affect. Psychoneuroendocrinology 36: 1312–1326, 2011. doi:10.1016/j.psyneuen.2011.03.003.
Crossref | PubMed | Web of Science | Google Scholar - 14. . Entrainment pattern between sympathetic and phrenic nerve activities in the Sprague-Dawley rat: hypoxia-evoked sympathetic activity during expiration. Am J Physiol Regul Integr Comp Physiol 286: R1121–R1128, 2004. doi:10.1152/ajpregu.00485.2003.
Link | Web of Science | Google Scholar - 15. . Acute intermittent hypoxia increases both phrenic and sympathetic nerve activities in the rat. Exp Physiol 92: 87–97, 2007. doi:10.1113/expphysiol.2006.035758.
Crossref | PubMed | Web of Science | Google Scholar - 16. . Stimulation of the paraventricular nucleus modulates firing of neurons in the nucleus of the solitary tract. Am J Physiol Regul Integr Comp Physiol 277: R403–R411, 1999. doi:10.1152/ajpregu.1999.277.2.R403.
Link | Web of Science | Google Scholar - 17. . Cardiorespiratory components of defense reaction elicited from paraventricular nucleus. Physiol Behav 61: 325–330, 1997. doi:10.1016/S0031-9384(96)00410-6.
Crossref | PubMed | Web of Science | Google Scholar - 18. . Paraventricular hypothalamic nucleus: axonal projections to the brainstem. J Comp Neurol 518: 1460–1499, 2010. doi:10.1002/cne.22283.
Crossref | PubMed | Web of Science | Google Scholar - 19. . The sympathetic control of blood pressure. Nat Rev Neurosci 7: 335–346, 2006. doi:10.1038/nrn1902.
Crossref | PubMed | Web of Science | Google Scholar - 20. . Working model of the sympathetic chemoreflex in rats. Clin Exp Hypertens 17: 167–179, 1995. doi:10.3109/10641969509087063.
Crossref | PubMed | Web of Science | Google Scholar - 21. . Oxytocin neuron activation prevents hypertension that occurs with chronic intermittent hypoxia/hypercapnia in rats. Am J Physiol Heart Circ Physiol 310: H1549–H1557, 2016. doi:10.1152/ajpheart.00808.2015.
Link | Web of Science | Google Scholar - 22. . Increase in sympathetic outflow by paraventricular nucleus stimulation in awake rats. Am J Physiol Regul Integr Comp Physiol 256: R1325–R1330, 1989. doi:10.1152/ajpregu.1989.256.6.R1325.
Link | Web of Science | Google Scholar - 23. . Inhibition of renal sympathetic nerve activity by electrical stimulation of the hypothalamic paraventricular nucleus in anesthetized rats. J Auton Nerv Syst 21: 83–86, 1987. doi:10.1016/0165-1838(87)90094-4.
Crossref | PubMed | Google Scholar - 24. . Cardiovascular function of a glutamatergic projection from the hypothalamic paraventricular nucleus to the nucleus tractus solitarius in the rat. Neuroscience 153: 605–617, 2008. doi:10.1016/j.neuroscience.2008.02.076.
Crossref | PubMed | Web of Science | Google Scholar - 25. . Hypoxia activates nucleus tractus solitarii neurons projecting to the paraventricular nucleus of the hypothalamus. Am J Physiol Regul Integr Comp Physiol 302: R1219–R1232, 2012. doi:10.1152/ajpregu.00028.2012.
Link | Web of Science | Google Scholar - 26. . Acute systemic hypoxia activates hypothalamic paraventricular nucleus-projecting catecholaminergic neurons in the caudal ventrolateral medulla. Am J Physiol Regul Integr Comp Physiol 305: R1112–R1123, 2013. doi:10.1152/ajpregu.00280.2013.
Link | Web of Science | Google Scholar - 27. . Catecholaminergic neurons projecting to the paraventricular nucleus of the hypothalamus are essential for cardiorespiratory adjustments to hypoxia. Am J Physiol Regul Integr Comp Physiol 309: R721–R731, 2015. doi:10.1152/ajpregu.00540.2014.
Link | Web of Science | Google Scholar - 28. . Sensory afferent and hypoxia-mediated activation of nucleus tractus solitarius neurons that project to the rostral ventrolateral medulla. Neuroscience 167: 510–527, 2010. doi:10.1016/j.neuroscience.2010.02.012.
Crossref | PubMed | Web of Science | Google Scholar - 29. . NTS neurons with carotid chemoreceptor inputs arborize in the rostral ventrolateral medulla. Am J Physiol Regul Integr Comp Physiol 270: R1273–R1278, 1996. doi:10.1152/ajpregu.1996.270.6.R1273.
Link | Web of Science | Google Scholar - 30. . DREADDs: novel tools for drug discovery and development. Drug Discov Today 19: 469–473, 2014. doi:10.1016/j.drudis.2013.10.018.
Crossref | PubMed | Web of Science | Google Scholar - 31. . Stimulation of the hypothalamic paraventricular nucleus modulates cardiorespiratory responses via oxytocinergic innervation of neurons in pre-Botzinger complex. J Appl Physiol (1985) 102: 189–199, 2007. doi:10.1152/japplphysiol.00522.2006.
Link | Web of Science | Google Scholar - 32. . Clozapine N-oxide administration produces behavioral effects in long-evans rats: implications for designing DREADD experiments. eNeuro 3:
ENEURO.0219-16.2016 , 2016. doi:10.1523/ENEURO.0219-16.2016.
Crossref | PubMed | Google Scholar - 33. . Modulation of the sympathetic response to acute hypoxia by the caudal ventrolateral medulla in rats. J Physiol 587: 461–475, 2009. doi:10.1113/jphysiol.2008.161760.
Crossref | PubMed | Web of Science | Google Scholar - 34. . Analysis of cardiovascular responses evoked following changes in peripheral chemoreceptor activity in the rat. J Physiol 394: 393–414, 1987. doi:10.1113/jphysiol.1987.sp016877.
Crossref | PubMed | Web of Science | Google Scholar - 35. . Excitatory amino acid transporters tonically restrain nTS synaptic and neuronal activity to modulate cardiorespiratory function. J Neurophysiol 115: 1691–1702, 2016. doi:10.1152/jn.01054.2015.
Link | Web of Science | Google Scholar - 36. . In vivo release of glutamate in nucleus tractus solitarii of the rat during hypoxia. J Physiol 478: 55–66, 1994. doi:10.1113/jphysiol.1994.sp020229.
Crossref | PubMed | Web of Science | Google Scholar - 37. . The effect of halothane on phrenic and chemoreceptor responses to hypoxia in anesthetized kittens. Anesth Analg 83: 329–335, 1996. doi:10.1213/00000539-199608000-00022.
Crossref | PubMed | Web of Science | Google Scholar - 38. . Involvement of the paraventricular nucleus of the hypothalamus in the pressor response to chemoreflex activation in awake rats. Brain Res 895: 167–172, 2001. doi:10.1016/S0006-8993(01)02067-4.
Crossref | PubMed | Web of Science | Google Scholar - 39. . The Rat Brain in Stereotaxic Coordinates (6th ed.). London: Academic, 2007.
Google Scholar - 40. . Oxytocin enhances cranial visceral afferent synaptic transmission to the solitary tract nucleus. J Neurosci 28: 11731–11740, 2008. doi:10.1523/JNEUROSCI.3419-08.2008.
Crossref | PubMed | Web of Science | Google Scholar - 41. . Differential role of the paraventricular nucleus of the hypothalamus in modulating the sympathoexcitatory component of peripheral and central chemoreflexes. Am J Physiol Regul Integr Comp Physiol 289: R789–R797, 2005. doi:10.1152/ajpregu.00222.2005.
Link | Web of Science | Google Scholar - 42. . Hypoxia activates a neuropeptidergic pathway from the paraventricular nucleus of the hypothalamus to the nucleus tractus solitarii. Am J Physiol Regul Integr Comp Physiol 315: R1167–R1182, 2018. doi:10.1152/ajpregu.00244.2018.
Link | Web of Science | Google Scholar - 43. . Immunohistochemical identification of neurons in the paraventricular nucleus of the hypothalamus that project to the medulla or to the spinal cord in the rat. J Comp Neurol 205: 260–272, 1982. doi:10.1002/cne.902050306.
Crossref | PubMed | Web of Science | Google Scholar - 44. . Cardiorespiratory and metabolic responses to injection of bicuculline into the hypothalamic paraventricular nucleus (PVN) of conscious rats. Brain Res 895: 33–40, 2001. doi:10.1016/S0006-8993(01)02011-X.
Crossref | PubMed | Web of Science | Google Scholar - 45. . Integration in the PVN: another piece of the puzzle. Am J Physiol Regul Integr Comp Physiol 289: R653–R655, 2005. doi:10.1152/ajpregu.00386.2005.
Link | Web of Science | Google Scholar - 46. . Neurons in the hypothalamic paraventricular nucleus send collaterals to the spinal cord and to the rostral ventrolateral medulla in the rat. Brain Res 801: 239–243, 1998. doi:10.1016/S0006-8993(98)00587-3.
Crossref | PubMed | Web of Science | Google Scholar - 47. . Acute hypoxia activates hypothalamic paraventricular nucleus-projecting catecholaminergic neurons in the C1 region. Exp Neurol 285: 1–11, 2016. doi:10.1016/j.expneurol.2016.08.016.
Crossref | PubMed | Web of Science | Google Scholar - 48. . Role of ventrolateral medulla catecholamine cells in hypothalamic neuroendocrine cell responses to systemic hypoxia. J Neurosci 15: 7979–7988, 1995. doi:10.1523/JNEUROSCI.15-12-07979.1995.
Crossref | PubMed | Web of Science | Google Scholar - 49. . Chemogenetic synaptic silencing of neural circuits localizes a hypothalamus→midbrain pathway for feeding behavior. Neuron 82: 797–808, 2014. doi:10.1016/j.neuron.2014.04.008.
Crossref | PubMed | Web of Science | Google Scholar - 50. . Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunohistochemical study. Neuroendocrinology 36: 165–186, 1983. doi:10.1159/000123454.
Crossref | PubMed | Web of Science | Google Scholar - 51. . Role of GABA within the nucleus tractus solitarii in the hypoxic ventilatory decline of awake rats. Am J Physiol Regul Integr Comp Physiol 281: R1411–R1419, 2001. doi:10.1152/ajpregu.2001.281.5.R1411.
Link | Web of Science | Google Scholar - 52. . DREADD agonist 21 is an effective agonist for muscarinic-based DREADDs in vitro and in vivo. ACS Pharmacol Transl Sci 1: 61–72, 2018. doi:10.1021/acsptsci.8b00012.
Crossref | PubMed | Google Scholar - 53. . Behavioral effect of chemogenetic inhibition is directly related to receptor transduction levels in rhesus monkeys. J Neurosci 38: 7969–7975, 2018. doi:10.1523/JNEUROSCI.1422-18.2018.
Crossref | PubMed | Web of Science | Google Scholar - 54. . Corticotropin-releasing hormone projections from the paraventricular nucleus of the hypothalamus to the nucleus of the solitary tract increase blood pressure. J Neurophysiol 121: 602–608, 2019. doi:10.1152/jn.00623.2018.
Link | Web of Science | Google Scholar - 55. . CRHR2 (corticotropin-releasing hormone receptor 2) in the nucleus of the solitary tract contributes to intermittent hypoxia-induced hypertension. Hypertension 72: 994–1001, 2018. doi:10.1161/HYPERTENSIONAHA.118.11497.
Crossref | PubMed | Web of Science | Google Scholar - 56. . The paraventricular nucleus of the hypothalamus influences respiratory timing and activity in the rat. Neurosci Lett 232: 63–66, 1997. doi:10.1016/S0304-3940(97)00579-X.
Crossref | PubMed | Web of Science | Google Scholar - 57. . Silencing synapses with DREADDs. Neuron 82: 723–725, 2014. doi:10.1016/j.neuron.2014.05.002.
Crossref | PubMed | Web of Science | Google Scholar
