Nitrergic neurotransmission within the lateral hypothalamus modulates cardiovascular responses to stress in rats
Gabriela A. Silva, Lilian L. Reis-Silva, Lucas Gomes-de-Souza, Lígia R. R. Tavares, Lucas Barretto-de-Souza, Vinícius Pelarin, Carlos C. Crestani
TL;DR
This study shows that nitric oxide signaling in the lateral hypothalamus of rats helps control heart rate and blood pressure during stress.
Contribution
The study identifies a specific role of neuronal nitric oxide synthase in modulating cardiovascular responses to stress in the lateral hypothalamus.
Findings
Inhibiting nNOS in the lateral hypothalamus enhanced heart rate responses to stress.
Nitric oxide signaling in the lateral hypothalamus modulates cardiovascular adjustments during acute stress.
The effect of nitrergic signaling appears to involve mechanisms independent of inducible NOS.
Abstract
The lateral hypothalamus (LH) is a key brain region involved in integrating cardiovascular and autonomic components of the stress response, yet the local neurochemical mechanisms governing these functions remain poorly understood. This study examined the contribution of nitrergic neurotransmission within the LH to cardiovascular adjustments induced by acute restraint stress in rats. To this end, animals received bilateral microinjections into the LH of either the nonselective nitric oxide synthase (NOS) inhibitor L-NAME, the selective neuronal NOS (nNOS) inhibitor Nω-propyl-L-arginine hydrochloride (NPLA), the selective inducible NOS (iNOS) inhibitor 1400W, or the nitric oxide (NO) donor MAHMA NONOate (NOC-9) prior to the restraint session. Intra-LH administration of L-NAME significantly attenuated both the pressor and tachycardic responses elicited by restraint. In contrast, the…
Genes, proteins, chemicals, diseases, species, mutations and cell lines named across the full text — each resolved to its canonical identifier and authoritative record.
Click any figure to enlarge with its caption.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7- —Universidade Estadual Paulista Júlio De Mesquita Filho
Peer Reviews
No public reviews on file for this paper yet. If you reviewed it on a platform where reviews are public (OpenReview, ICLR, NeurIPS, ICML), you can paste yours below so the community can read it here.
Videos
No videos yet. Explain this paper in a talk, walkthrough, or lecture? Add one.
Taxonomy
TopicsNitric Oxide and Endothelin Effects · Neuroscience of respiration and sleep · Neuroendocrine regulation and behavior
Introduction
Stress is associated with behavioral and physiological responses that are adaptive in the short term, facilitating maintenance of homeostasis, well-being and survival [21, 70]. Autonomic and cardiovascular responses to acute stress include sympathetic activation, elevations in arterial pressure and heart rate, redistribution of blood flow, and modulation of baroreflex function [21, 24]. Although evidence that these adjustments depend on the coordinated activation of brain circuits that integrate physiological and behavioral components of the stress response [16, 28, 66], the specific neurobiological substrates mediating cardiovascular/autonomic stress responses are not yet fully elucidated.
The lateral hypothalamus (LH) is part of the neural network that regulates autonomic and cardiovascular responses to stressful stimuli [16, 44, 66]. Accordingly, this hypothalamic region has been shown to be activated by emotional stressors, such as restraint stress [17, 22, 48]. Pharmacological studies provided evidence that LH plays an inhibitory role in restraint-evoked tachycardia via local glutamatergic signaling [25]; and this control is counterbalanced by local GABAergic neurotransmission [33, 34]. Moreover, expression of both pressor and tachycardiac responses during restraint seems to be mediated by activation of local corticotropin-releasing factor (CRF) receptors within the LH [11, 12]. However, the influence of local neuromodulators on the regulation of stress responses mediated by LH excitatory and inhibitory neurotransmissions remains unknown.
Nitric oxide (NO) is a gaseous signaling molecule synthesized from L-arginine by nitric oxide synthase (NOS) in the presence of NADPH and oxygen, yielding L-citrulline as a byproduct [2, 71]. Three isoforms of NOS have been identified, each with distinct localization and regulation: the neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) [2, 71]. NO is recognized as an atypical neurotransmitter in the brain playing a crucial modulatory role in controlling the release of various neurotransmitters, so influencing synaptic plasticity and brain function [31, 32, 56]. The nNOS isoform is Ca^2+^-dependent and consist in the predominant source of NO in the central nervous system (CNS) [31, 32, 38]. eNOS is also Ca^2^⁺-dependent, and NO released from this isoform expressed in endothelial cells and astrocytes may modulate synaptic activity in the brain [31, 32, 65]. iNOS is Ca^2^⁺-independent, and its activity is primarily regulated at the transcriptional level [53]. Although iNOS is typically not expressed under physiological conditions, constitutive expression has been reported in certain tissues, including the brain [7, 45, 52].
Nitric oxide (NO)-mediated signaling within the brain has been implicated in modulating both behavioral and physiological responses to stressors [8, 20, 30]. Regarding the cardiovascular responses, previous studies documented that systemic or intracerebroventricular treatment with NOS inhibitors affected cardiovascular changes evoked by stressful stimuli [3, 19, 78]. This control was described to be mediated mainly by nNOS-derived NO in specific brain sites, including the dorsal hippocampus [5, 47], medial prefrontal cortex (mPFC) [59], periaqueductal grey (PAG) [1], paraventricular nucleus of the hypothalamus (PVN) [18], and bed nucleus of the stria terminalis (BNST) [9, 10, 37].
Neurons capable of synthesizing NO were identified within the LH [23, 61, 75, 77], and these neurons were activated by aversive stimuli [43, 63]. These observations suggest that nitrergic signaling within the LH may contribute to the neural processing of stress-related responses. However, the involvement of local NO signaling in the control by LH of cardiovascular responses to emotional stress has not yet been described. Therefore, the present study aimed to elucidate the role of nitrergic mechanisms within the LH in modulating cardiovascular responses elicited by acute restraint stress in rats.
Experimental Procedures
Animals
Twenty-eight male Wistar rats (60 days old, weighing 240–260 g) were used in this study. The animals were obtained from the animal breeding facility of São Paulo State University (UNESP, Botucatu, São Paulo, Brazil) and transferred to the Animal Facility of the Laboratory of Pharmacology, School of Pharmaceutical Sciences of Araraquara (FCFAR–UNESP) at least seven days before the start of the experimental procedures. Rats were housed in groups of four per cage under controlled temperature (24 ± 2 °C) and a 12:12 h light/dark cycle (lights on at 7:00 a.m.), with food and water available ad libitum except during brief testing sessions. All experiments were conducted during the light phase. Experimental procedures were approved by the Ethical Committee for the Use of Animals of the School of Pharmaceutical Sciences/UNESP (approval # 15/2022) and complied with Brazilian and international guidelines for the care and use of laboratory animals.
Surgical procedures
Stereotaxic surgery
The animals were anesthetized with a mixture of ketamine (87 mg/kg) and xylazine (13 mg/kg) administered intraperitoneally (i.p.). After trichotomy, the head was positioned in a stereotaxic frame (Stoelting, Wood Dale, Illinois, USA), and local anesthesia with 2% lidocaine containing a vasoconstrictor was applied subcutaneously to minimize pain and bleeding. A midline scalp incision (~ 1.5 cm) was made, the skull was exposed, and the periosteum was carefully removed using 10% hydrogen peroxide. Stainless-steel guide cannulas (23G, 12 mm length) were bilaterally implanted into the LH through small burr holes drilled in the skull, according to the coordinates from the rat brain atlas of Paxinos and Watson [54]: anteroposterior, −1.8 mm from bregma; lateral, ± 2.3 mm from the midline; vertical, − 7.8 mm from the skull surface; with the incisor bar set at − 3.2 mm (Paxinos and Watson, 1997). The guide cannulas were fixed to the skull with dental acrylic resin, reinforced with a stainless-steel screw implanted into the skull bone. A 0.2-mm stainless-steel mandril was inserted into each cannula to prevent occlusion during the recovery period. At the end of surgery, animals received veterinary a poly-antibiotic containing streptomycins and penicillins (560 mg/ml/kg, i.m.) and the non-steroidal anti-inflammatory flunixin meglumine (0.5 mg/ml/kg, s.c.) as a prophylactic measure.
Femoral artery cannulation
One day before the cardiovascular recording session, rats were again anesthetized with a mixture of ketamine (87 mg/kg) and xylazine (13 mg/kg) administrated via i.p., and a polyethylene catheter was implanted into the femoral artery to record of cardiovascular parameters. The catheter was made of PE-10 tubing (4–5 cm) heat-welded to PE-50 tubing (12–13 cm; Clay Adams, Parsippany, USA), prefilled with heparinized saline (50 IU/mL; Hepamax-S®, Blausiegel, Cotia, SP, Brazil) and sealed with a metal pin. The catheter was tunneled subcutaneously, exteriorized on the animal’s dorsal region, and secured with surgical suture. At the end of surgery, animals received the nonsteroidal anti-inflammatory flunixin meglumine (0.5 mg/mL/kg, s.c.). Rats were housed individually during the postoperative recovery and trial.
Acute restraint stress
Acute restraint stress was performed by placing the rats into ventilated cylindrical plastic tubes (6.5 cm in diameter, 15 cm in length) containing ½-inch holes covering approximately 20% of the surface to allow airflow. Animals remained restrained for 30 min [10, 35, 36]. Each rat was subjected to a single restraint session to prevent habituation to the stressor [13, 14, 64].
Arterial pressure and heart rate recording
The arterial catheter implanted into femoral artery was connected to a pressure transducer (DPT100, Utah Medical Products Inc., USA) via a PE-50 tubing. Pulsatile arterial pressure (PAP) was amplified (Bridge Amp ML221, ADInstruments, Australia) and recorded using a computerized data acquisition system (PowerLab 4/30, ML866, ADInstruments, Australia) with LabChart Pro software (ADInstruments, Australia). Mean arterial pressure (MAP) and heart rate (HR) were derived from the PAP signal. Systolic (SAP) and diastolic arterial pressure (DAP) were also derived from PAP recordings, and the results for these parameters are presented in the Supplementary file.
Tail skin temperature recording
Activation of vasomotor sympathetic activity during aversive situations decreases cutaneous blood flow [15], which consequently reduces skin temperature [12, 18, 74]. In this context, the reduction in tail skin temperature was used as an indirect index of vasomotor sympathetic response in cutaneous vascular beds during restraint stress.
Tail skin temperature was recorded with a thermographic camera (IRI4010, InfraRed Integrated Systems Ltd, Northampton, UK). Thermal images were analyzed with specific software, and temperature variations were represented by differences in color intensity. For each image, temperature was measured at five points along the tail, and the mean value was calculated for each recording [12, 33].
Intra-brain microinjection procedure
Stainless steel needles (33 G; Small Parts, Miami Lakes, FL, USA) extending 1 mm longer than the guide cannulas were used for intra-LH microinjections. The needle was connected to a 2 μL microsyringe (7002 KH, Hamilton, Reno, NV, USA) via a polyethylene tube (PE-10). Microinjections into the LH were carried out over approximately 5 s, and the injection needle remained in place for an additional 60 s to prevent reflux. The injected volume was 100 nL per hemisphere [11, 12, 25, 33].
Drugs and solutions
The non-selective NOS inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME; Sigma-Aldrich, St. Louis, Missouri, USA); the selective nNOS inhibitor Nω-Propyl-L-Arginine hydrochloride (NPLA; Tocris, Ellisville, Missouri, USA); the selective iNOS inhibitor 1400 W (Cayman Chemical Company, Ann Arbor, Michigan, USA) and urethane (Sigma–Aldrich) were dissolved in saline (0.9% NaCl). MAHMA NONOate (NOC‐9) (Sigma‐Aldrich) was dissolved in 1 M Tris–HCl solution. NOC‐9 solution was prepared at pH 10 to prevent NO release before it reached the brain tissue. NOC‐9 is stable at an alkaline pH (> 10.0) and releases NO at physiological pH (7.4) [41]. The poly-antibiotic preparation (Pentabiotico®; Fort Dodge, Campinas, São Paulo, Brazil) and the non-steroidal anti-inflammatory flunixin meglumine (Banamine®, Schering-Plough, Cotia, São Paulo, Brazil) were used as supplied by the manufacturers.
Histological determination of intra-brain microinjection sites
At the end of each experiment, animals were anesthetized with urethane (250 mg/mL, 200 g body weight, i.p.), and 100 nL of 1% Evans Blue dye was bilaterally microinjected to verify the injection site. Brains were then removed, post-fixed in 10% formalin for at least 48 h, and sectioned into 40 μm-thick coronal slices using a cryostat (CM1900, Leica, Wetzlar, Germany). The location of the microinjection sites was assessed under a light microscope with reference to the rat brain atlas of Paxinos and Watson [54].
Immunohistochemistry
The animals were anesthetized with urethane (1.2 g/kg, i.p.) and perfused with saline phosphate (PBS) (1X pH 7.4) accompanied by 4% paraformaldehyde in solution with phosphate buffer (pH 7.4). Then, the brain was removed and post-fixed in paraformaldehyde for 2 h and transferred to 30% sucrose solution in PBS at 4 °C. Two days later, the brains were frozen in dry ice powder for 1 h, and then stored in a freezer at − 80 °C until processing. Before the immunohistochemistry procedures, the brains were sectioned in a cryostat (− 20 °C) (CM1900, Leica, Germany) with a thickness of 40 μm according to coordinates of Paxinos and Watson [54]. The slices containing the LH and dorsal hippocampus regions were washed 3 times (10 min each wash) in PBS and incubated in blocking solution (3% goat serum and 0.25% Triton X-100) dissolved in PBS for one hour at room temperature. After the blockage, the slices were incubated with anti-nNOS primary antibody (cat. #51973; dilution 1:1000; ABBKine, Atlanta, GA, USA) for 24 h at 4 °C. After the incubation, the slices were washed with PBS 3 times (10 min each wash) and incubated with biotinylated anti-rabbit secondary antibody (cat.# BA-1000; dilution 1:600; Vector Laboratories, Burlingame, CA, USA) in PBS-Tx (0.25% Triton X-100) and 3% goat serum for 2 h at room temperature. The slices were then washed with PBS 3 times (10 min each wash) and incubated for 1 h in avidin–biotin-peroxidase solution (cat.# PK-6100; Vectastain Elite ABC-HRP Kit, Peroxidase—Standard; Vector Laboratories, Burlingame, CA, USA), 0.5% Triton X-100 and PBS. The slices were then washed 3 times (10 min each wash) and incubated in 3,3′-diaminobenzidine – DAB (cat.# D5637, Sigma-Aldrich, St Louis, MO, USA) for seven min. Then, they were washed 4 times (5 min each wash), transferred to PBS solution and mounted on gelatinized slides. After drying, the slides were hydrated in distilled water and then gradient dehydrated by increasing ethanol titrations (30%, 60%, 90%, 95% and 100%) and xylol (LabSynth, São Paulo, Brazil). Finally, they were covered with Permount (Sigma-Aldrich, St. Louis, MA, USA) and coverslips. Immunostaining of nNOS was captured in a microscope coupled to a camera (Zeiss Axioskop 2). The LH and hippocampus were identified according to the atlas of rats brain of Paxinos and Watson [54].
Experimental design
A schematic representation of the experimental protocol used to assess the role of nitrergic neurotransmission within the LH on cardiovascular responses to restraint stress is presented in Fig. 1. Animals were randomly assigned to one of five groups: vehicle, L-NAME (non-selective NOS inhibitor), NPLA (selective nNOS inhibitor), 1400 W (selective iNOS inhibitor), or NOC-9 (NO donor). All animals underwent stereotaxic surgery for the implantation of bilateral guide cannulas targeting the LH and were allowed a five-day recovery period. One day before the trial, femoral artery cannulation was performed to enable measurement of blood pressure and heart rate. Animals were housed in their home cages in the experimental room overnight prior to testing, which was temperature-controlled (24 °C) and acoustically isolated.Fig. 1. Schematic representation of the experimental protocol used to evaluate the role of nitrergic neurotransmission within the lateral hypothalamus (LH) on cardiovascular responses to restraint stress. Top: Surgical procedures, including stereotaxic surgery and femoral artery cannulation. Bottom: Timeline illustrating the sequence of events on the experimental day
On the trial day, animals were connected to the cardiovascular recording system, and basal parameters were recorded for 10–20 min. Ten minutes prior to the onset of acute restraint stress, animals received bilateral microinjections into the LH of either saline (100nL, vehicle solution for L-NAME, NPLA and 1400 W preparation), NPLA (0.04 nmol/100nL), L-NAME (4 nmol/100nL), 1400 W (0.04 nmol/100 nL), Tris–HCl (200nL, vehicle solution for NOC-9 preparation) or NOC-9 (75 nmol/200nL). The doses of the drugs were chosen based on previous studies from our group [4, 9, 10, 18]. Cardiovascular parameters were continuously monitored throughout the 30-min restraint period and the subsequent 40-min recovery period. Tail skin temperature was recorded at 10, 5, and 0 min before restraint; at 5, 10, 20, and 30 min during restraint; and at 10, 20, 30, and 40 min during the recovery period. At the end of the recovery period, animals were anesthetized with urethane and euthanized by decapitation. Brains were rapidly removed, post-fixed, and the microinjection sites were examined.
Data and statistical analysis
Data were expressed as mean ± SEM. Basal values of MAP, HR and tail cutaneous temperature before and after treatment of the LH were compared using paired Student t test. Time-course curves of cardiovascular and tail skin temperature changes were analyzed using two-way ANOVA, with treatment as an independent factor and time as a repeated measure. The mean values of the time-course data during the restraint and recovery periods (representing the average response across each phase) were compared using two-way ANOVA, with the same factors applied as in the time-course analysis. Sidak post-hoc test was used following two-way ANOVA to assess specific differences between the experimental groups. Results with P < 0.05 were considered statistically significant.
Results
Figure 2 presents schematic illustrations, adapted from the rat brain atlas of Paxinos and Watson [54], showing the microinjection sites in the LH for all animals included in the study. A coronal photomicrograph from a representative animal illustrating microinjection sites in the LH is also provided in the Fig. 2.Fig. 2. Microinjection sites within the lateral hypothalamus (LH). (A) Photomicrograph of a coronal brain section from a representative rat showing bilateral microinjection sites within the LH. (B) Schematic representation based on the rat brain atlas of Paxinos and Watson (1997) showing the microinjection sites within the LH for animals that received saline (white circles), the non-selective nitric oxide synthase (NOS) inhibitor L-NAME (green circles), the neuronal NOS inhibitor NPLA (red circles) or the inducible NOS inhibitor 1400 W (blue circles)
Effect of non-selective NOS inhibition within the LH on the cardiovascular responses to acute restraint stress
Bilateral microinjection of the non-selective NOS inhibitor L-NAME (n = 8) into the LH did not alter baseline values of either MAP (t = 0.83, df = 7, P = 0.4312), HR (t = 1.3, df = 7, P = 0.2149), or tail skin temperature (t = 0.6, df = 7, P = 0.5750) (Table 1). L-NAME treatment also did not affect basal parameters of either SAP (t = 0.7, df = 7, P = 0.4935) or DAP (t = 0.6, df = 7, P = 0.5664) (Supplementary Table 1).Table 1. Basal parameters (i.e., pre-stress values) of mean arterial pressure (MAP), heart rate (HR), and tail skin temperature (TST) before and after treatment of the lateral hypothalamus (LH)GroupMAP (mmHg)HR (bpm)TST (^o^C)nbeforeafterbeforeafterbeforeafterVehicle (saline)791 ± 692 ± 5365 ± 24373 ± 2131.4 ± 0.830.5 ± 1.1L-NAME896 ± 496 ± 4373 ± 16389 ± 2232.0 ± 0.931.5 ± 1.0NPLA792 ± 396 ± 4358 ± 12369 ± 1431.1 ± 0.630.4 ± 0.81400W6102 ± 4106 ± 3333 ± 8356 ± 931.1 ± 0.531.2 ± 0.4Tris–HCl695 ± 494 ± 4343 ± 6333 ± 734.1 ± 1.932.9 ± 1.4NOC-96103 ± 3102 ± 2356 ± 10367 ± 2032.1 ± 0.530.9 ± 1.2Data were expressed as error ± standard error of the mean (SEM). Student t test
Microinjection of saline (vehicle solution of L-NAME, NPLA and 1400 W) (n = 7) did not affect MAP (t = 1.1, df = 6, P = 0.3071), HR (t = 0.9, df = 6, P = 0.3643), or tail skin temperature (t = 0.8, df = 6, P = 0.4609) baseline (Table 1). SAP (t = 0.99, df = 6, P = 0.3602) and DAP (t = 0.92, df = 6, P = 0.4391) were also not affected by saline microinjection (Supplementary Table 1).
Analysis of the time-course curves revealed that acute restraint stress produced significant increases in MAP (F_(4.463, 58.02)_ = 8, P < 0.0001) and HR (F_(4.263,55.42)_ = 4, P = 0.0095) accompanied by a decrease in tail skin temperature (F_(3.897,50.67)_ = 4, P = 0.0090) (Fig. 3). Two-way ANOVA further demonstrated that L-NAME microinjection into the LH significantly reduced MAP (F_(1,13)_ = 6, P = 0.0242) and HR (F_(1,13)_ = 9, P = 0.0097) responses, without affecting the restraint-evoked decrease in tail skin temperature (F_(1,13)_ = 0.008, P = 0.9298) (Fig. 3). Analysis also indicated a time x treatment interaction for MAP (F_(40,520)_ = 2, P = 0.0028), but not for HR (F_(40,520)_ = 0.9, P = 0.5230) or tail skin temperature (F_(11,143)_ = 0.8, P = 0.6716) (Fig. 3). Analysis of the time-course curves of change in both SAP (treatment factor: F_(1,13)_ = 6, P = 0.0324) and DAP (treatment factor: F_(1,13)_ = 6, P = 0.0264) also indicated reduced responses in animals treated with L-NAME (Supplementary Figure 1).Fig. 3. Effect of non-selective NOS inhibition within the LH on the cardiovascular responses to acute restraint stress. (Left) Time-course curves of changes in mean arterial pressure (ΔMAP), heart rate (ΔHR), and tail skin temperature (Δtail skin temperature) during the basal, restraint, and recovery periods in rats subjected to intra-HL microinjection of vehicle (saline, 100 nL; white circles; n = 7) or the non-selective NOS inhibitor L-NAME (4 nmol/100 nL; blue circles; n = 8). The shaded area represents the restraint period. # P < 0.05 over the entire restraint and recovery periods compared to the control group (vehicle), two-way ANOVA. (Right) Mean ΔMAP, ΔHR and Δ tail temperature during restraint and recovery periods in rats subjected to intra-HL microinjection of vehicle (saline, 100 nL; white circles; n = 7) or the non-selective NOS inhibitor L-NAME (4 nmol/100 nL; blue circles; n = 8). *P < 0.05, two-way ANOVA followed by Sidak’s post hoc test. Circles (left graphs) and bars (right graphs) represent mean ± SEM
Analysis of average responses during the restraint and recovery periods did not indicate effect of the time factor for MAP (F_(1,13)_ = 2, P = 0.1973), HR (F_(1,13)_ = 0.17, P = 0.6882), or tail skin temperature (F_(1,13)_ = 3, P = 0.1251) (Fig. 3). However, treatment significantly affected MAP (F_(1,13)_ = 7, P = 0.0212) and HR (F_(1,13)_ = 8, P = 0.0135) responses, without influencing the tail skin temperature drop (F_(1,13)_ = 0.4, P = 0.5336) (Fig. 3). No significant time x treatment interaction was found for either MAP (F_(1,13)_ = 2, P = 0.1692), HR (F_(1,13)_ = 0.003, P = 0.9544), or tail temperature (F_(1,13)_ = 0.002, P = 0.9620) (Fig. 3). Post-hoc analysis revealed that L-NAME treatment significantly reduced the pressor response during the restraint period (P = 0.012), while tachycardiac response was reduced during both restraint (P = 0.043) and recovery (P = 0.038) periods (Fig. 3). Analysis of SAP (treatment factor: F_(1,13)_ = 7, P = 0.0277) and DAP (treatment factor: F_(1,13)_ = 7, P = 0.0264) responses during the restraint and recovery periods also indicated an effect of L-NAME (Supplementary Figure 1). Post-hoc analysis revealed reduced values during the restraint period for both SAP (P = 0.0158) and DAP (P = 0.0151) (Supplementary Figure 1).
Effect of selective nNOS inhibition within the LH on the cardiovascular responses to acute restraint stress
Bilateral microinjection of the selective nNOS inhibitor NPLA (n = 7) into the LH did not alter baseline values of either MAP (t = 2.3, df = 6, P = 0.0603), HR (t = 1.1, df = 6, P = 0.2893), or tail skin temperature (t = 1.3, df = 6, P = 0.2561) (Table 1). NPLA into the LH also did not affect basal parameters of either SAP (t = 2.3, df = 6, P = 0.0652) or DAP (t = 2.3, df = 6, P = 0.0575) (Supplementary Table 1).
Analysis of the time-course curves revealed that acute restraint stress increased MAP (F_(4.691, 56.29)_ = 12, P < 0.0001) and HR (F_(3.791, 45.50)_ = 7, P = 0.0003), along with decrease in tail skin temperature (F_(3.766, 45.19)_ = 5, P = 0.0041) (Fig. 4). Two-way ANOVA further demonstrated that NPLA microinjection into the LH did not affect pressor (F_(1,12)_ = 2, P = 0.2102), tachycardic (F_(1,12)_ = 2, P = 0.2034) or decrease in tail skin temperature (F_(1,12)_ = 0.3, P = 0.5701) responses(Fig. 4). Nevertheless, a significant time x treatment interaction was observed for HR (F_(41,492)_ = 3, P < 0.0001), but not for MAP (F_(41,492)_ = 1, P = 0.1101) or tail skin temperature (F_(11,132)_ = 0.4, P = 0.9663) (Fig. 4). Post-hoc analysis revealed increased HR values at 8-, 14- and 16-min of the restraint session (P < 0.05), when compared with saline-treated animals (Fig. 4). Such as observed for MAP, analysis of the time-course curves also showed that NPLA treatment had no effect on either SAP (treatment factor: F_(1,12)_ = 1, P = 0.3346) or DAP (treatment factor: F_(1,12)_ = 1, P = 0.3239) responses (Supplementary Figure 2).Fig. 4. Effect of selective nNOS inhibition within the LH on the cardiovascular responses to acute restraint stress. (Left) Time-course curves of changes in mean arterial pressure (ΔMAP), heart rate (ΔHR), and tail skin temperature (Δtail skin temperature) during the basal, restraint, and recovery periods in rats subjected to intra-HL microinjection of vehicle (saline, 100 nL; white circles; n = 7) or the selective nNOS inhibitor NPLA (0.04 nmol/100nL, red circles, n = 7). The shaded area represents the restraint period. *P < 0.05, two-way ANOVA. (Right) Mean ΔMAP, ΔHR and Δ tail temperature during restraint and recovery periods in rats subjected to intra-HL microinjection of vehicle (saline, 100 nL; white circles; n = 7) or the selective nNOS inhibitor NPLA (0.04 nmol/100nL, red circles, n = 7). *P < 0.05, two-way ANOVA followed by Sidak’s post hoc test. Circles (left graphs) and bars (right graphs) represent mean ± SEM
Analysis of mean responses during the restraint and recovery periods indicated a main effect of time on MAP (F_(1,12)_ = 21, P = 0.0006) and HR (F_(1,12)_ = 6, P = 0.0284), but not on tail skin temperature (F_(1,12)_ = 0.4, P = 0.5583) (Fig. 4). Two-way ANOVA did not indicate an effect of treatment on MAP (F_(1,12)_ = 1.4, P = 0.2613), HR (F_(1,12)_ = 3, P = 0.1197) or tail skin temperature (F_(1,12)_ = 0.3, P = 0.6213) (Fig. 4). A time x treatment interaction was detected for HR (F_(1,12)_ = 5, P = 0.0493), but not for MAP (F_(1,12)_ = 1.5, P = 0.2411) or tail temperature (F_(1,12)_ = 0.01, P = 0.9912) (Fig. 4). Post-hoc analysis revealed enhanced HR increase during the restraint session in NPLA-treated animals when compared to rats subjected to saline microinjection into the LH (P = 0.0268) (Fig. 4). Analysis of SAP (treatment factor: F_(1,12)_ = 0.7, P = 0.3916) and DAP (treatment factor: F_(1,12)_ = 0.8, P = 0.3898) responses during the restraint and recovery periods did not indicate effect of NPLA treatment (Supplementary Figure 2).
Effect of selective iNOS inhibition within the LH on the cardiovascular responses to acute restraint stress
Bilateral microinjection of the selective iNOS inhibitor 1400 W (n = 6) into the LH did not alter baseline values of either MAP (t = 0.8, df = 5, P = 0.4455), HR (t = 1.7, df = 5, P = 0.1585), or tail skin temperature (t = 0.4, df = 5, P = 0.7316) (Table 1). 1400 W also did not affect basal parameters of either SAP (t = 1.7, df = 5, P = 0.1473) or DAP (t = 1.6, df = 5, P = 0.1587) (Supplementary Table 1).
Analysis of the time-course curves indicated that acute restraint stress increased MAP (F_(4.274,47.01)_ = 9, P < 0.0001) and HR (F_(3.916,43.08)_ = 4, P = 0.0136), while it decreased tail skin temperature (F_(3.301,36.31)_ = 5, P = 0.0054) (Fig. 5). Treatment of the LH with the iNOS inhibitor did not produce effects on MAP (F_(1,11)_ = 0.09, P = 0.7743), HR (F_(1,11)_ = 0.1, P = 0.7348) or tail skin temperature (F_(1,11)_ = 3, P = 0.0965) responses (Fig. 5). Moreover, analysis did not indicate a time x treatment interaction for MAP (F_(41,451)_ = 1, P = 0.3263), HR (F_(41,451)_ = 0.5, P = 0.9989) or tail skin temperature (F_(11,121)_ = 0.7, P = 0.7833) (Fig. 5). Such as observed for MAP, analysis of the time-course curves also showed that 1400 W had no effect on either SAP (treatment factor: F_(1,11)_ = 0.006, P = 0.9352) or DAP (treatment factor: F_(1,11)_ = 0.01, P = 0.9118) responses (Supplementary Figure 3).Fig. 5. Effect of selective iNOS inhibition within the LH on the cardiovascular responses to acute restraint stress. (Left) Time-course curves of changes in mean arterial pressure (ΔMAP), heart rate (ΔHR), and tail skin temperature (Δtail skin temperature) during the basal, restraint, and recovery periods in rats subjected to intra-HL microinjection of vehicle (saline, 100 nL; white circles; n = 7) or the selective iNOS inhibitor 1400 W (0.04 nmol/100nL,purple circles, n = 6). The shaded area represents the restraint period. Two-way ANOVA. (Right) Mean ΔMAP, ΔHR and Δ tail temperature during restraint and recovery periods in rats subjected to intra-HL microinjection of vehicle (saline, 100 nL; white circles; n = 7) or with the selective iNOS inhibitor 1400 W (0.04 nmol/100nL, purple circles, n = 6). Two-way ANOVA. Circles (left graphs) and bars (right graphs) represent mean ± SEM
Analysis of mean responses during the restraint and recovery periods revealed no effect of time on either MAP (F_(1,11)_ = 2, P = 0.1694), HR (F_(1,11)_ = 0.1, P = 0.7296), or tail skin temperature (F_(1,11)_ = 2, P = 0.1762) (Fig. 5). Similarly, no significant effect of treatment was indicated for MAP (F_(1,11)_ = 0.1, P = 0.7561), HR (F_(1,11)_ = 0.1, P = 0.7237), or tail skin temperature (F_(1,11)_ = 2, P = 0.1950) (Fig. 5). Analysis also did not indicate time x treatment interaction for MAP (F_(1,11)_ = 1, P = 0.2961), HR (F_(1,11)_ = 0.008, P = 0.9301), or tail skin temperature (F_(1,11)_ = 0.0001, P = 0.9999) (Fig. 5). Analysis of SAP (treatment factor: F_(1,11)_ = 0.001, P = 0.9682) and DAP (treatment factor: F_(1,11)_ = 0.004, P = 0.9468) responses during the restraint and recovery periods did not indicate effect of NPLA treatment (Supplementary Figure 3).
Effect of intra-LH microinjection of a NO donor on cardiovascular responses to acute restraint stress
Bilateral microinjection of the NO donor NOC-9 (n = 6) into the LH did not affect basal parameters of either MAP (t = 0.8, df = 5, P = 0.4436), HR (t = 0.39, df = 5, P = 0.7181), or tail skin temperature (t = 0.97, df = 5, P = 0.3881) (Table 1). Treatment with NOC-9 also did not affect baseline values of either SAP (t = 0.9, df = 5, P = 0.4015) or DAP (t = 1.4, df = 5, P = 0.2048) (Supplementary Table 1).
Microinjection of NOC-9 vehicle solution (i.e., TRISS-HCl) (n = 7) also did not affect MAP (t = 0.5, df = 5, P = 0.6264), HR (t = 0.97, df = 5, P = 0.3797), or tail skin temperature (t = 1.9, df = 5, P = 0.1198) baseline (Table 1). SAP (t = 0.5, df = 5, P = 0.6102) and DAP (t = 0.7, df = 5, P = 0.5015) were also not affected by TRISS-HCl treatment (Supplementary Table 1).
Analysis of the time-course curves showed that acute restraint stress increased MAP (F_(4.339,43.39)_ = 20, P < 0.0001) and HR (F(4.802,48.02) = 10, P < 0.0001), along with decreased tail skin temperature values (F_(3.305, 33.05)_ = 4, P = 0.0197) (Fig. 6). Treatment with the NO donor into LH decreased restraint-evoked tachycardia (F_(1,10)_ = 5.1, P = 0.04857), but without affecting MAP (F_(1,10)_ = 1.7, P = 0.2219) and tail skin temperature (F_(1,10)_ = 0.1, P = 0.7528) responses (Fig. 6). Moreover, analysis indicated time x treatment interaction for MAP (F_(40,400)_ = 1.6, P = 0.0227) and HR (F_(40,400)_ = 1.5, P = 0.0323), but not for tail skin temperature (F_(11,110)_ = 0.84, P = 0.5998) (Fig. 6). Such as observed for MAP, analysis of the time-course curves also showed that NOC-9 had no effect on either SAP (treatment factor: F_(1,10)_ = 1, P = 0.2638) or DAP (treatment factor: F_(1,10)_ = 1, P = 0.3065) responses (Supplementary Figure 4).Fig. 6. Effect of intra-LH microinjection of a NO donor on cardiovascular responses to acute restraint stress. (Left) Time-course curves of changes in mean arterial pressure (ΔMAP), heart rate (ΔHR), and tail skin temperature (Δtail skin temperature) during the basal, restraint, and recovery periods in rats subjected to intra-HL microinjection of vehicle (Tris–HCl, 200 nL; white circles; n = 7) or the NO donor NOC-9 (75 nmol/200nL, green circles, n = 6). The shaded area represents the restraint period. # P < 0.05 over the entire restraint and recovery periods compared to the control group (vehicle), two-way ANOVA. (Right) Mean ΔMAP, ΔHR and Δ tail temperature during restraint and recovery periods in rats subjected to intra-HL microinjection of vehicle (Tris–HCl, 200 nL; white circles; n = 7) or the NO donor NOC-9 (75 nmol/200nL, green circles, n = 6). *P < 0.05, two-way ANOVA followed by Sidak’s post hoc test. Circles (left graphs) and bars (right graphs) represent mean ± SEM
Analysis of mean responses during the restraint and recovery periods revealed effect of the time factor for both MAP (F_(1,10)_ = 33, P = 0.0002), HR (F_(1,10)_ = 7, P = 0.0242), and tail skin temperature (F_(1,10)_ = 15, P = 0.0029) (Fig. 6). Analysis indicated effect of the treatment factor for HR (F_(1,10)_ = 15, P = 0.0032), but not for MAP (F_(1,10)_ = 1.8, P = 0.2173) and tail skin temperature (F_(1,10)_ = 0.1, P = 0.8478) (Fig. 6). Analysis indicated time x treatment interaction for tail skin temperature (F_(1,10)_ = 10, P = 0.0112), but not for MAP (F_(1,10)_ = 0.2, P = 0.6609) and HR (F_(1,10)_ = 0.5, P = 0.4969) (Fig. 5) (Fig. 6). Post-hoc analysis revealed reduced HR values during restraint stress period in animals treated with NOC-9 (P = 0.0281) (Fig. 6). Analysis of SAP (treatment factor: F_(1,10)_ = 1, P = 0.2542) and DAP (treatment factor: F_(1,10)_ = 1, P = 0.2926) responses during the restraint and recovery periods did not indicate effect of NOC-9 (Supplementary Figure 4).
Expression of nNOS within the LH
Immunohistochemistry was performed to confirm the expression of nNOS within the LH. The dorsal hippocampus was included in the analysis as a positive control, as the presence of nNOS-synthesizing neurons in this region is well described [29, 40, 72, 76]. As shown in Fig. 7, numerous cells within the lateral hypothalamus (LH) were found to express nNOS. Notably, nNOS-synthesizing cells were observed in regions corresponding to the sites reached by the intra-brain microinjections of the pharmacological studies.Fig. 7. Immunohistochemical detection of neuronal nitric oxide synthase (nNOS) in the lateral hypothalamus (LH) and hippocampus. Representative sections show nNOS-immunoreactive neurons (brown/DAB staining) distributed throughout both regions. Images illustrate the presence of nitrergic cells within the LH in regions corresponding to the sites reached by the intra-brain microinjections of the pharmacological studies
Discussion
The present findings provide the first evidence that nitrergic neurotransmission within the lateral hypothalamus (LH) contributes to cardiovascular responses evoked by adverse environmental stimuli. Specifically, nNOS-derived NO within the LH appears to exert an inhibitory influence on restraint-evoked tachycardia, without affecting the pressor response or sympathetically-mediated cutaneous vasoconstriction. Conversely, non-selective NOS inhibition suggests that isoforms other than nNOS is involved in expression of both pressor and tachycardic responses, but the lack of effect of 1400 W indicates that this facilitatory control is unlikely to involve iNOS.
The cardiovascular effects of non-selective NOS inhibition in specific brain sites have largely been attributed to the suppression of nNOS activity [10, 60, 68], as this isoform is the most abundant and constitutes the primary source of NO in the brain under physiological conditions [42, 69]. Nevertheless, we identified different effects following local LH treatment with the non-selective NOS inhibitor L-NAME and the selective nNOS inhibitor NPLA. Indeed, while L-NAME decreased both pressor and tachycardiac responses to restraint, NPLA enhanced selectively the HR increase. This finding differs from prior observations in animals subjected to NOS inhibition in the BNST, where both L-NAME and NPLA produced comparable effects on restraint-induced cardiovascular changes [10]. Therefore, this opposite effect indicated that NOS isoform other than nNOS would be related to L-NAME effect in the LH. In this context, we investigated the effect of selective iNOS inhibition within the LH. Although iNOS expression is primarily induced under inflammatory conditions, constitutive expression has been reported in certain brain regions [7, 52], where its nitrergic signaling has been implicated in the modulation of stress-related responses [46]. However, we did not identify changes in restraint-evoked cardiovascular responses following iNOS inhibition within the LH. Based on this finding, one possibility is that L-NAME effects would be related to local eNOS inhibition. In this sense, it has been suggested that NO produced by eNOS in vascular endothelial cells near synapses may influence neuronal activity [31, 32]. However, we were unable to test this hypothesis pharmacologically, as a selective eNOS inhibitor is not currently available [2]. Therefore, future studies using genetic strategies to selectively downregulate eNOS expression in the LH will be essential to determine its role in the L-NAME–induced effects described here.
Although the different effects of L-NAME and NPLA treatments to suggest the involvement of NOS isoform other than nNOS within the LH in control of restraint-evoked cardiovascular responses, we observed that facilitation of nitrergic neurotransmission via local microinjection of the NO donor NOC-9 produced effects opposite to those seen following selective nNOS inhibition. This finding highlights a prominent role of nNOS in mediating the control of restraint-evoked cardiovascular responses by local nitrergic signaling. This idea is further reinforced by identification of nNOS-synthetizing cells within the LH (see Fig. 7), a finding consistent with previous reports of studies in rats [23, 77].
Such as observed in the present study following LH treatment with the selective nNOS inhibitor, previous studies demonstrated that non-selective synaptic inhibition within the LH via local treatment with CoCl_2_ enhanced the restraint-evoked tachycardia, without affecting the pressor effect [25]. The same study identified that this inhibitory control was mediated by local glutamatergic neurotransmission through activation of local NMDA receptors [25]. Conversely, activation of GABA_A_ receptors [33] and CRF_1_ and CRF_2_ receptors [12] has been shown to exert opposing effects, contributing to the expression of the tachycardic response to restraint stress. In this sense, the findings of the present study provide new evidence that activation of local nNOS and the subsequent release of NO are also components of the LH-mediated inhibitory control of the cardiac response to restraint stress. It is well established that nNOS is physically coupled to NMDA glutamate receptors via scaffold proteins such as PSD-95 and PSD-93, whereby NMDA receptor–mediated Ca^2^⁺ influx represents a prominent mechanism underlying nNOS activation in the brain [31, 32, 62]. In this context, the nNOS-mediated modulation of restraint-evoked tachycardia within the LH may represent a downstream mechanism of local NMDA receptor activation [25], constituting a component of an NMDA receptor/nNOS signaling pathway.
Previous evidence from studies using local non-selective synaptic inhibition and antagonism of glutamatergic, GABAergic, and CRF receptors within the LH indicated a selective modulation of restraint-evoked tachycardia, without affecting the pressor response [11, 12, 25, 33, 34]. In this context, results of the present study in animals subjected to local non-selective NOS inhibition consist of the first evidence that LH is also involved in control of arterial pressure response observed during restraint stress. This finding is supported by previous findings that LH is involved in expression of arterial pressure response observed during expression of fear conditioned [6, 39], thus providing further evidence that LH is also part of brain pathways regulating arterial pressure during aversive situations. Studies performed in unanesthetized non-stressed animals indicated that glutamatergic neurotransmission within the LH is linked with depressor responses [50, 51]. Conversely, pressor response was observed following microinjection of acetylcholine into the LH in unanesthetized non-stressed rats [55], suggesting that the control of the pressor response to restraint stress by local nitrergic mechanisms could involve an interaction with local cholinergic neurotransmission. In this sense, previous studies indicated that cardiovascular control by muscarinic cholinergic receptors is mediated by stimulation of NO release in the prefrontal cortex and hippocampus [26, 27, 67, 73]. Thus, the alteration in the restraint-evoked pressor response seen in L-NAME–treated animals may reflect a downstream pathway associated with muscarinic receptor activation. However, to the best of our knowledge, the involvement of LH cholinergic neurotransmission – as well as its interaction with local nitrergic neurotransmission—in the regulation of cardiovascular responses to stress has not yet been documented.
The LH has been described as part of the brain circuits that regulates the cardiovascular responses to stress by connecting limbic structures, such as the hippocampus, infralimbic cortex, central amygdala (CeA) and BNST, to effector structures in the brainstem and sympathetic neurons in the intermediolateral column [49, 66]. Indeed, previous findings indicated that the LH is a component of the neural pathway through which the BNST regulates cardiovascular responses to stress [34]. However, this pathway from BNST seemed to be mediated by GABAergic inputs to the LH [34]. As stated above, nNOS activation is likely mediated by local NMDA glutamate receptors. In this sense, projections arising from cerebral cortex sites such as mPFC, insula and hippocampus constitute relevant sources of glutamatergic inputs within the LH [57]. However, a role of these pathways in cardiovascular responses to stressful stimuli has never been directly assessed. Therefore, further studies are necessary to elucidate the pathway(s) related to the control by LH nitrergic neurotransmission of restraint-evoked cardiovascular responses documented in the present study.
Tachycardia responses during restraint stress seem to be resulted of the co-activation of both cardiac sympathetic and parasympathetic activities [21]. Indeed, it was previously reported that antagonism of muscarinic cholinergic receptors facilitated, while β_1_-adrenoceptor blockade decreased this response [58]. In this sense, it was reported that the inhibitory control of HR response to restraint stress by NMDA glutamate receptors within the LH was mediated by activation of faciliatory pathways to parasympathetic centers controlling the cardiac function [25]. As noted above, control by nNOS-derived NO may represent part of the downstream pathway of NMDA receptor activation. Accordingly, the parasympathetic-mediated regulation of restraint-evoked tachycardia previously described by LH glutamatergic neurotransmission may also involve the LH nitrergic neurotransmission observed in the present study [25]. However, considering that LH neurons projects directly, and indirectly via brainstem regions, to sympathetic neurons in the intermediolateral column [49, 66], the control of tachycardia to restraint by LH nitrergic neurotransmission may also be mediated by activation of inhibitory drive to sympathetic neurons controlling cardiac function. Conversely, the facilitatory control of pressor and tachycardiac responses evidenced following LH treatment with the non-selective NOS inhibitor L-NAME is probably related to by activation of facilitatory drive to sympathetic neurons and/or inhibitory influence on parasympathetic neurons.
In summary, present study provide evidence of a prominent role of local NO-mediated signaling within the LH in the regulation of cardiovascular responses to stress. Our results indicate that nNOS activation within the LH plays an inhibitory role in stress-evoked tachycardia, whereas other NOS isoforms seem to be involved in the expression of this response. Our results also provide new evidence that LH is involved in expression of pressor response to restraint stress, which seems to be mediated by local nitrergic neurotransmission via activation NOS isoform other than nNOS.
Supplementary Information
Below is the link to the electronic supplementary material.Supplementary file1 (PDF 436 KB)