Respiratory-sympathetic Coupling Research Articles

Postinspiratory and preBötzinger complexes contribute to respiratory-sympathetic coupling in mice before and after chronic intermittent hypoxia.

The sympathetic nervous system modulates arterial blood pressure. Individuals with obstructive sleep apnea (OSA) experience numerous nightly hypoxic episodes and exhibit elevated sympathetic activity to the cardiovascular system leading to hypertension. This suggests that OSA disrupts normal respiratory-sympathetic coupling. This study investigates the role of the postinspiratory complex (PiCo) and preBötzinger complex (preBötC) in respiratory-sympathetic coupling under control conditions and following exposure to chronic intermittent hypoxia (CIH) for 21 days (5% O2-80 bouts/day). The surface of the ventral brainstem was exposed in urethane (1.5 g/kg) anesthetized, spontaneously breathing adult mice. Cholinergic (ChAT), glutamatergic (Vglut2), and neurons that co-express ChAT and Vglut2 at PiCo, as well as Dbx1 and Vglut2 neurons at preBötC, were optogenetically stimulated while recording activity from the diaphragm (DIA), vagus nerve (cVN), and cervical sympathetic nerve (cSN). Following CIH exposure, baseline cSN activity increased, breathing frequency increased, and expiratory time decreased. In control mice, stimulating PiCo specific cholinergic-glutamatergic neurons caused a sympathetic burst during all phases of the respiratory cycle, whereas optogenetic activation of cholinergic-glutamatergic PiCo neurons in CIH mice increased sympathetic activity only during postinspiration and late expiration. Stimulation of glutamatergic PiCo neurons increased cSN activity during the postinspiratory phase in control and CIH mice. Optogenetic stimulation of ChAT containing neurons in the PiCo area did not affect sympathetic activity under control or CIH conditions. Stimulating Dbx1 or Vglut2 neurons in preBötC evoked an inspiration and a concomitant cSN burst under control and CIH conditions. Taken together, these results suggest that PiCo and preBötC contribute to respiratory-sympathetic coupling, which is altered by CIH, and may contribute to the hypertension observed in patients with OSA.

Respiratory Modulation of Muscle Sympathetic Nerve Activity in Type 2 Diabetes Mellitus

Dysfunctional sympathetic neural regulation is present in patients with type 2 diabetes (T2D) with augmented muscle sympathetic nerve activity (MSNA) responses to handgrip exercise, cold pressor testing, and augmented sympathetic-blood pressure transduction identified. MSNA is modulated by respiration such that it is inhibited at higher lung volumes and increased at lower lung volumes. Herein we aimed to determine whether T2D affects the respiratory modulation of MSNA. Resting MSNA (microneurography) and respiration pattern (strain-gauge pneumobelt) were measured simultaneously in 20 patients with T2D (age 49.1±7.4 years, body mass index [BMI] 31.9±4.1 kg∙m−2, mean ± SD) and 13 age- and BMI-matched, healthy controls (46.3±9.4 years; 32.1±4.6 kg∙m−2). MSNA was stratified by the phase of respiration. Further comparisons were made between low lung volume (defined as 60-100% expiration + 0-60% inspiration) and high lung volume states (60-100% inspiration + 0-60% expiration). Resting MSNA burst frequency, burst incidence and total activity were similar between T2D and controls (p=0.953, p=0.624, and p=0.884, respectively). Both groups exhibited peak MSNA in late expiration to early inspiration, whilst inhibition of MSNA occurred during late inspiration to early expiration (respiratory phase p<0.001 for burst frequency, incidence and total activity), though no group x respiratory phase interaction was observed (p>0.05 for all MSNA measures). When MSNA responses were compared between lung volume states, percent reduction of MSNA burst incidence from low lung volume to high lung volume was less in T2D than controls (26.1±33.9 vs 50.1±25.6%, p=0.025). Furthermore, in T2D, there was a shift in the distribution of MSNA bursts with a greater proportion of bursts occurring at high lung volumes (i.e., burst count during high lung volume divided by total burst count) compared to controls (43.8±10.5 vs. 37.8±12.5% respectively, group x volume interaction p=0.04). Notably, the normal pattern of respiratory modulation of MSNA was absent in 5 T2D patients (non-modulators), whilst this did not occur in any control participants. Non-modulators had higher systolic blood pressure compared to modulators (138.7±16.6 vs. 123.2±10.6 mmHg respectively, p=0.024), with no differences in age, BMI, HbA1c, duration of T2D, or overall MSNA parameters (all p<0.05). These results suggest that whilst respiratory modulation of MSNA occurs in T2D, its effect appears blunted compared to healthy controls. Notably, the usual pattern of respiratory-sympathetic coupling is absent in some T2D patients, which may be associated with higher resting systolic blood pressure. MP is supported by the Greenlane Research and Educational Fund (21/01/4153). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Maternal high-fat diet changes breathing pattern and causes excessive sympathetic discharge in juvenile offspring rat.

Early life over-nutrition, as experienced in maternal obesity, is a risk factor for developing cardiorespiratory and metabolic diseases. Here we investigated the effects of high-fat diet (HFD) consumption on the breathing pattern and sympathetic discharge to blood vessels in juvenile offspring from dams fed with HFD (O-HFD). Adult female Holtzman rats were given a standard diet (SD) or HFD from 6 wk before gestation to weaning. At weaning (P21), the male offspring from SD dams (O-SD) and O-HFD received SD until the experimental day (P28-P45). Nerve recordings performed in decerebrated in situ preparations demonstrated that O-HFD animals presented abdominal expiratory hyperactivity under resting conditions and higher vasoconstrictor sympathetic activity levels. The latter was associated with blunted respiratory-related oscillations in sympathetic activity, especially in control animals. When exposed to elevated hypercapnia or hypoxia levels, the O-HFD animals mounted similar ventilatory and respiratory motor responses as the control animals. Hypercapnia and hypoxia exposure also increased sympathetic activity in both groups but did not reinstate the respiratory-sympathetic coupling in the O-HFD rats. In freely behaving conditions, O-HFD animals exhibited higher resting pulmonary ventilation and larger variability of arterial pressure levels than the O-SD animals due to augmented sympathetic modulation of blood vessel diameter. Maternal obesity modified the functioning of cardiorespiratory systems in offspring at a young age, inducing active expiration and sympathetic overactivity under resting conditions. These observations represent new evidence about pregnancy-related complications that lead to the development of respiratory distress and hypertension in children of obese mothers.NEW & NOTEWORTHY Maternal obesity is a risk factor for developing cardiorespiratory and metabolic diseases. This study highlights the changes on the breathing pattern and sympathetic discharge to blood vessels in juvenile offspring from dams fed with HFD. Maternal obesity modified the functioning of cardiorespiratory systems in offspring, inducing active expiration and sympathetic overactivity. These observations represent new evidence about pregnancy-related complications that lead to the development of respiratory distress and hypertension in children of obese mothers.

Evidences That Sympathetic Overactivity and Neurogenic Hypertension Correlate with Changes in the Respiratory Pattern in Rodent Models of Experimental Hypoxia.

The main question of this chapter is as follows: What is the contribution of changes in the sympathetic-respiratory coupling to the hypertension observed in some experimental models of hypoxia? Although there is evidence supporting the concept that sympathetic-respiratory coupling is increased in different models of experimental hypoxia [chronic intermittent hypoxia (CIH) and sustained hypoxia (SH)], it was also observed that in some strains of rats and in mice, these experimental models of hypoxia do not affect the sympathetic-respiratory coupling and the baseline arterial pressure. The data from studies performed in rats (different strains, male and female, and in the natural sleep cycle) and mice submitted to chronic CIH or SH are critically discussed. The main message from these studies performed in freely moving rodents and in the in situ working heart-brainstem preparation is that experimental hypoxia changes the respiratory pattern, which correlates with increased sympathetic activity and may explain the hypertension observed in male and female rats previously submitted to CIH or SH.

Augmented Respiratory-Sympathetic Coupling and Hemodynamic Response to Acute Mild Hypoxia in Female Rodents With Chronic Kidney Disease.

Carotid body feedback and hypoxia may serve to enhance respiratory–sympathetic nerve coupling (respSNA) and act as a driver of increased blood pressure. Using the Lewis polycystic kidney (LPK) rat model of chronic kidney disease, we examined respSNA in adult female rodents with CKD and their response to acute hypoxia or hypercapnia compared to Lewis control animals. Under urethane anesthesia, phrenic nerve activity, splanchnic sympathetic nerve activity (sSNA), and renal sympathetic nerve activity (rSNA) were recorded under baseline conditions and during mild hypoxic or hypercapnic challenges. At baseline, tonic SNA and blood pressure were greater in female LPK rats versus Lewis rats (all P < 0.05) and respSNA was at least two-fold larger [area under the curve (AUC), sSNA: 7.8 ± 1.1 vs. 3.4 ± 0.7 μV s, rSNA: 11.5 ± 3 vs. 4.8 ± 0.7 μV s, LPK vs. Lewis, both P < 0.05]. Mild hypoxia produced a larger pressure response in LPK [Δ mean arterial pressure (MAP) 30 ± 6 vs. 12 ± 6 mmHg] and augmented respSNA (ΔAUC, sSNA: 8.9 ± 3.4 vs. 2 ± 0.7 μV s, rSNA: 6.1 ± 1.2 vs. 3.1 ± 0.7 μV s, LPK vs. Lewis, all P ≤ 0.05). In contrast, central chemoreceptor stimulation produced comparable changes in blood pressure and respSNA (ΔMAP 13 ± 3 vs. 9 ± 5 mmHg; respSNA ΔAUC, sSNA: 2.5 ± 1 vs. 1.3 ± 0.7 μV s, rSNA: 4.2 ± 0.9 vs. 3.5 ± 1.4 μV s, LPK vs. Lewis, all P > 0.05). These results demonstrate that female rats with CKD exhibit heightened respSNA coupling at baseline that is further augmented by mild hypoxia, and not by hypercapnia. This mechanism may be a contributing driver of hypertension in this animal model of CKD.

Sex Differences in the Effect of Acute Intermittent Hypoxia on Respiratory‐Sympathetic Coupling in Humans

Background Sex-related differences in respiratory-sympathetic coupling have been described in preclinical models of intermittent hypoxia (IH) and have been implicated in the development of neurogenic hypertension. Based on sex disparities in the respiratory and blood pressure response to acute IH in humans as well as changes in respiratory-sympathetic coupling in clinical sleep apnea, we examined sex-related differences in respiratory modulation of muscle sympathetic nerve activity (MSNA) following acute IH in healthy humans. We hypothesized the magnitude of respiratory-sympathetic coupling would increase in both men and women following IH; however, the respiratory patterning of MSNA following IH would be sex-specific. Methods Heart rate, MSNA, and respiration were assessed in 21 healthy young men (30±1 yrs) and 10 healthy young women (28±1 yrs) during normoxic rest prior to and following IH. IH was achieved by alternating between a hypercapnic hypoxic (3% carbon dioxide, 5% oxygen) gas cylinder and room air (21% oxygen) to target 15 hypoxic events over 30-min. Respiratory modulation of MSNA was assessed by fitting polynomials to cross-correlation histograms constructed between sympathetic spikes and respiration. Results MSNA was elevated following IH in both men (34±2 to 41±3 bursts/100 heart beats) and women (30±4 to 35±5 bursts/100 heart beats) (p<0.01). When examining the normalized temporal profile of MSNA, both men and women exhibited changes in respiratory patterning of MSNA following IH; however, the pattern of MSNA differed by sex. Following IH, women exhibited more sympathetic activity during late expiration and less during post-inspiration (interaction of IH and time, p<0.01). In contrast, men exhibited greater sympathetic activity during post-inspiration following IH (interaction of IH and time, p<0.01). Conclusions Both men and women exhibit changes in respiratory patterning of MSNA following acute IH; however, this pattern differs by sex. These data support sex disparities in respiratory-sympathetic coupling and advance our understanding of sex-specific changes in sympathetic activation following IH which may have important implications for the development of neurogenic hypertension.

Optogenetic stimulation of Dbx1 neurons enhances the respiratory‐sympathetic coupling in in vivo CIH mice

Under resting conditions, breathing is generated by synchronized activity of a complex respiratory network located in the ventral respiratory column in the brainstem. The preBötzinger Complex (preBötC), critical for generating the inspiratory phase of breathing, contains glutamatergic neurons derived from progenitor cells that are characterized by the transcription factor “developing brain homeobox (Dbx1)”. These neurons are thought to be important for inspiratory rhythmogenesis. Excitatory and inhibitory synaptic inputs from the respiratory networks modulate the sympathetic outflow which is generated by presympathetic neurons from the rostroventrolateral medulla (RVLM). Changes in the respiratory-sympathetic coupling contribute to imbalances as demonstrated in chronic intermittent hypoxia (CIH). To investigate the contribution of Dbx1 neurons in the respiratory-sympathetic coupling, we used an optogenetic in vivo mouse preparation previously exposed to CIH for 10 -12 days (5% O2 – 80 bouts/day). The ventral surface of the brainstem in spontaneously breathing, anaesthetized (Urethane 1.5mg/kg) Dbx1-ERT2Cre/Ai32 adult mice was exposed and an optical fiber positioned bilaterally over the preBötC, while recording the hypoglossal (HN), cervical vagus (cVN) and cervical sympathetic nerves (cSN). Our preliminary data indicate that CIH exposure increased the respiratory frequency and mean sympathetic activity in the baseline conditions. Random light stimulation (200ms) of preBötC Dbx1 neurons (~ 9mV, 447nm) significantly promoted inspiratory activity, increasing the respiratory frequency and amplitude of HN and cSN in relation to baseline in both groups. During optogenetic stimulation of Dbx1 neurons, the respiratory-sympathetic coupling was enhanced in both groups, without significant difference in variability of cSN amplitude. This is possibly due to the increase in mean cSN activity under baseline conditions in CIH mice. Our data demonstrates that stimulation of preBötC Dbx1 neurons aids in regulation of autonomic control modulating the cSN activity, leading to increased sympathetic drive and hypertension in the CIH.

Changes in the autonomic and respiratory patterns of mice submitted to short‐term sustained hypoxia

Short-term sustained hypoxia (SH) in rats induces changes in sympathetic-respiratory coupling and sympathetic overactivity, which seems to be linked to hypertension. However, there is no previous experimental data about the effects of SH on the autonomic and respiratory pattern in mice. In the present study we tested the hypothesis that mice submitted to SH present autonomic and respiratory changes similarly to rats. To reach this goal we evaluated the effects of 24-hour SH exposure on cardiovascular, respiratory and autonomic parameters in baseline conditions and upon chemoreflex activation.C57BL/6J mice (7-8 weeks old, ~25g) were deeply anesthetized for femoral artery and jugular vein catheterization. Four days later, they were submitted to SH (24h, FiO2 0.1) or normoxia. At the end of SH protocol, arterial pressure, heart rate and ventilation were recorded in vivo. Peripheral chemoreflex was activated via KCN injection (0.16 mg/Kg, i.v.). In another experimental group, we performed in situ recordings of phrenic (PN), abdominal, (AbN), cervical vagus (cVN) and thoracic sympathetic (tSN) nerves activities of SH mice using the working heart-brainstem preparation. All experimental protocols were approved by the Institutional Ethics Committee (#140/2019 and #163/2019). In the in vivo approach, although SH mice (n=13) presented a significant decrease in baseline HR (510 ± 22 vs 633 ± 16 beats min-1; P = 0.002) compared with the controls (n=17), no differences were observed in mean arterial pressure levels (109 ± 2 vs 106 ± 2 mmHg; P=0.443). SH mice (n=14) also showed significantly increased respiratory frequency [(fR) 277 ± 8 vs 203 ± 6 breaths.min-1; P <0.0001], tidal volume [(VT) 14 ± 1 vs. 12 ± 1mL.Kg1; P = 0.0019)], and ventilation [(VE) 3927 ± 164 vs 2390 ± 104 mL.Kg-1.min-1; P < 0.0001)] compared with controls (n=17). The magnitudes of bradycardic responses to chemoreflex activation were greater in SH mice (n=8) than in controls (n=11; -329 ± 36 vs -210 ± 30 beats min-1, P=0.02). In the in situ approach, baseline PN discharge (PND) frequency was significantly reduced in SH mice (n=11) in relation to controls (n=26) (0.69 ± 0.06 vs 1.13 ± 0.10 Hz, P=0.0154). The incidence of Late-E bursts in AbN activity in SH mice (n=10) was significantly increased in relation to controls (n=20) (83.3 ± 7.7 vs 24.3 ± 8.0%, P<0.0001). The duration of expiration in SH mice (n=11) was longer than in controls (n=22) (1.13 ± 0.16 vs 0.67 ± 0.07s, P= 0.0002) due to an increase in the duration of post-inspiration (0.78 ± 0.12 vs 0.35 ± 0.04s, P= 0.0032). tSNA in the SH group (n=10) was significantly reduced during late-expiration (E2) compared with the control group (n=16) (19.0 ± 4.3 vs 39.7 ± 4.2%, P= 0.0203). The data are showing that 24-h SH protocol in mice produces: a) an increase in parasympathetic tone to the heart, which seems to be related to changes in their breathing pattern; and b) prolonged expiration and active expiration associated with a reduction in sympathetic activity during E2. This autonomic imbalance favoring the parasympathetic componente may explain why mice, different of rats, do not develop hypertension after SH.

Changes in the autonomic and respiratory patterns in mice submitted to short-term sustained hypoxia.

What is the central question of this study? Do mice submitted to sustained hypoxia present autonomic and respiratory changes similarly to rats? What is the main finding and its importance? Arterial pressure in the normal range, reduced baseline heart rate and tachypnoea were observed in behaving sustained hypoxia mice. Recordings in the in situ preparation of mice submitted to sustained hypoxia show an increase in cervical vagus nerve activity and a simultaneous reduction in thoracic sympathetic nerve activity correlated with changes in the respiratory cycle. Therefore, mice are an important model for studies on the modulation of sympathetic activity to the cardiovascular system and the vagus innervation of the upper airways due to changes in the respiratory network induced by sustained hypoxia. Short-term sustained hypoxia (SH) in rats induces sympathetic overactivity and hypertension due to changes in sympathetic-respiratory coupling. However, there are no consistent data about the effect of SH on mice due to the different protocols of hypoxia and difficulties associated with the handling of these rodents under different experimental conditions. In situ recordings of autonomic and respiratory nerves in SH mice have not been performed yet. Herein, we evaluated the effects of SH ( =0.1 for 24h) on baseline mean arterial pressure (MAP), heart rate (HR), respiratory frequency (fR ) and responses to chemoreflex activation in behaving SH mice. A characterization of changes in cervical vagus (cVN), thoracic sympathetic (tSN), phrenic (PN) and abdominal (AbN) nerves in SH mice using the in situ working heart-brainstem preparation was also performed. SH mice presented normal MAP, significant reduction in baseline HR, increase in baseline fR , as well as increase in the magnitude of bradycardic response to chemoreflex activation. In in situ preparations, SH mice presented a reduction in PN discharge frequency, and increases in the time of expiration and incidence of late-expiratory bursts in AbN activity. Nerve recordings also indicated a significant increase in cVN activity and a significant reduction in tSN activity during expiration in SH mice. These findings make SH mice an important experimental model for better understanding how changes in the respiratory network may impact on the modulation of vagal control to the upper airways, as well as in the sympathetic activity to the cardiovascular system.

The role of glycinergic inhibition in respiratory pattern formation and cardio-respiratory coupling in rats.

Cardio-respiratory coupling is reflected as respiratory sinus arrhythmia (RSA) and inspiratory-related bursting of sympathetic nerve activity. Inspiratory-related inhibitory and/or postinspiratory-related excitatory drive of cardiac vagal motoneurons (CVMs) can generate RSA. Since respiratory oscillations may depend on synaptic inhibition, we investigated the effects of blocking glycinergic neurotransmission (systemic and local application of the glycine receptor (GlyR) antagonist, strychnine) on the expression of the respiratory motor pattern, RSA and sympatho-respiratory coupling. We recorded heart-rate, phrenic, recurrent laryngeal and thoracic sympathetic nerve activities (PNA, RLNA, t-SNA) in a working-heart-brainstem preparation of rats, and show that systemic strychnine (50–200 ​nM) abolished RSA and triggered a shift of postinspiratory RLNA into inspiration, while t-SNA remained unchanged. Bilateral strychnine microinjection into the ventrolateral medullary area containing CVMs and laryngeal motoneurons (LMNs) of the nucleus ambiguus (NA/CVLM), the nucleus tractus solitarii, pre-Bötzinger Complex, Bötzinger Complex or Kölliker-Fuse nuclei revealed that only NA/CVLM strychnine microinjections mimicked the effects of systemic application. In all other target nuclei, except the Bötzinger Complex, GlyR-blockade attenuated the inspiratory-tachycardia of the RSA to a similar degree while evoking only a modest change in respiratory motor patterning, without changing the timing of postinspiratory-RLNA, or t-SNA. Thus, glycinergic inhibition at the motoneuronal level is involved in the generation of RSA and the separation of inspiratory and postinspiratory bursting of LMNs. Within the distributed ponto-medullary respiratory pre-motor network, local glycinergic inhibition contribute to the modulation of RSA tachycardia, respiratory frequency and phase duration but, surprisingly it had no major role in the mediation of respiratory-sympathetic coupling.

Erratum: Toor et al., "Neurons in the Intermediate Reticular Nucleus Coordinate Postinspiratory Activity, Swallowing, and Respiratory-Sympathetic Coupling in the Rat".

Erratum: Toor et al., "Neurons in the Intermediate Reticular Nucleus Coordinate Postinspiratory Activity, Swallowing, and Respiratory-Sympathetic Coupling in the Rat".

Optogenetic stimulation of Dbx1 neurons promote increase in sympathetic activity in vivo

Under resting conditions, breathing is characterized by inspiration and postinspiration. Located in the ventrolateral medulla, the preBötzinger Complex (pre‐BötC) is critical for generating the inspiratory phase of breathing. This microcircuit contains glutamatergic neurons derived from progenitor cells that are characterized by the transcription factor “developing brain homeobox (Dbx1)”, and that are thought to be important for inspiratory rhythmogenesis. Located rostromedial of the preBötC is the so called postinspiratory complex (PiCo), which seems to be critical for the generation of the postinspiratory phase of breathing. Important for generating the postinspiratory phase are glutamateric‐cholinergic PiCo neurons. At resting conditions, sympathetic and parasympathetic activity is modulated by excitatory and inhibitory synaptic inputs from these respiratory networks. It is thought that changes in the respiratory‐sympathetic coupling contribute to imbalances that lead to hypertension, as demonstrated in various animal models of chronic intermittent hypoxia. Here we combine optogenetic and electrophysiological approaches in vivo and in vitro in order to investigate the respiratory‐sympathetic coupling. Spontaneously breathing Dbx1‐ERT2Cre/Ai32 and ChAT‐Cre/Ai32 (ChR2) adult mice were anaesthetized (Urethane 1.5mg/kg), and the ventral surface of their brainstem exposed in order to place an optical fiber bilaterally over the pre‐BötC and/or PiCo, while recording the hypoglossal (HN), cervical vagus (cVN) and cervical sympathetic nerves (cSN). Our preliminary data indicate that continuous light stimulation (10s) of pre‐BötC Dbx1 neurons (~ 9mV, 447nm) significantly increased the respiratory frequency, the amplitude of HN, cSN and cVN in relation to baseline. During the optogenetic stimulation of Dbx1 neurons the respiratory‐sympathetic coupling was enhanced. Using the in vivo approach as well as in vitro horizontal slices we are currently also characterizing the role of PiCo in regulating this coupling under control conditions and following CIH. Our preliminary data suggest that PiCo activity is enhanced following CIH exposure, which also contributes to the regulation of respiratory‐sympathetic coupling. Unraveling how the respiratory microcircuits regulate autonomic control will be critical to understand how CIH leads to increased sympathetic drive and hypertension.Support or Funding InformationFAPESP (2016/23513‐1; 2019/11969‐2), and the NIH: P01 HL090554, R01 HL144801 and R01 HL126523

Early activation of ubiquitin-proteasome system at the diaphragm tissue occurs independently of left ventricular dysfunction in SHR rats.

Hypertensive status induces modifications in the respiratory profile. Previous studies have indicated that hypertensive rats show increased respiratory-sympathetic coupling compared to normotensive rats. However, these effects and especially the mechanisms underlying such effects are not well known. Thus, we evaluated the influence of high blood pressure and autonomic dysfunction on a ventilatory pattern associated with lung injury and on the ubiquitin-proteasome system of the diaphragm muscle. Autonomic cardiovascular modulation (systolic BP variance and low-frequency band and pulse interval variance) and arterial blood gases patterns (pH, pO2, HCO3, SpO2), can be changed by hypertension, as well exacerbated chemoreflex pressor response. We observed that the diaphragm muscle of SHR showed increase in type I cross-sectional fiber (16%) and reduction in type II cross-sectional fiber area (41%), increased activity of the ubiquitin-proteasome system and lipid peroxidation, with no differences between groups in the analysis of ubiquitinated proteins and misfolded proteins. Our results showed that hypertension induced functional compensatory/adverse alterations associated with diaphragm fiber type changes and protein degradation as well as changed autonomic control of circulation. In conclusion, we believe there is an adaptation in ventilatory pattern in regarding to prevent the development of fatigue and muscle weakness and improve ventilatory endurance. Impact statement It was well known that hypertension can be driven by increased sympathetic activity and has been documented as a central link between autonomic dysfunction and alterations in the respiratory pattern. Our study demonstrated the impact of hypertension in ventilatory mechanics and their relationship with diaphragm muscle protein degradation. These findings may assist us in future alternative treatments to prevent diaphragm fatigue and weakness in hypertensive patients.

Neurons in the Intermediate Reticular Nucleus Coordinate Postinspiratory Activity, Swallowing, and Respiratory-Sympathetic Coupling in the Rat.

Breathing results from sequential recruitment of muscles in the expiratory, inspiratory, and postinspiratory (post-I) phases of the respiratory cycle. Here we investigate whether neurons in the medullary intermediate reticular nucleus (IRt) are components of a central pattern generator (CPG) that generates post-I activity in laryngeal adductors and vasomotor sympathetic nerves and interacts with other members of the central respiratory network to terminate inspiration. We first identified the region of the (male) rat IRt that contains the highest density of lightly cholinergic neurons, many of which are glutamatergic, which aligns well with the putative postinspiratory complex in the mouse (Anderson et al., 2016). Acute bilateral inhibition of this region reduced the amplitudes of post-I vagal and sympathetic nerve activities. However, although associated with reduced expiratory duration and increased respiratory frequency, IRt inhibition did not affect inspiratory duration or abolish the recruitment of post-I activity during acute hypoxemia as predicted. Rather than representing an independent CPG for post-I activity, we hypothesized that IRt neurons may instead function as a relay that distributes post-I activity generated elsewhere, and wondered whether they could be a site of integration for para-respiratory CPGs that drive the same outputs. Consistent with this idea, IRt inhibition blocked rhythmic motor and autonomic components of fictive swallow but not swallow-related apnea. Our data support a role for IRt neurons in the transmission of post-I and swallowing activity to motor and sympathetic outputs, but suggest that other mechanisms also contribute to the generation of post-I activity.SIGNIFICANCE STATEMENT Interactions between multiple coupled oscillators underlie a three-part respiratory cycle composed from inspiratory, postinspiratory (post-I), and late-expiratory phases. Central post-I activity terminates inspiration and activates laryngeal motoneurons. We investigate whether neurons in the intermediate reticular nucleus (IRt) form the central pattern generator (CPG) responsible for post-I activity. We confirm that IRt activity contributes to post-I motor and autonomic outputs, and find that IRt neurons are necessary for activation of the same outputs during swallow, but that they are not required for termination of inspiration or recruitment of post-I activity during hypoxemia. We conclude that this population may not represent a distinct CPG, but instead may function as a premotor relay that integrates activity generated by diverse respiratory and nonrespiratory CPGs.

Cardiovascular and respiratory profiles during the sleep-wake cycle of rats previously submitted to chronic intermittent hypoxia.

What is the central question of this study? Chronic intermittent hypoxia (CIH) causes increased arterial pressure (AP), sympathetic overactivity and changes in expiratory modulation of sympathetic activity. However, changes in the short-term sleep-wake cycle pattern after CIH and their potential impact on cardiorespiratory parameters have not been reported previously. What is the main finding and its importance? Exposure to CIH for 10 days elevates AP in wakefulness and sleep but does not cause major changes in short-term sleep-wake cycle pattern. A higher incidence of muscular expiratory activity was observed in rats exposed to CIH only during wakefulness, indicating that active expiration is not required for the increase in AP in rats submitted to CIH. Chronic intermittent hypoxia (CIH) increases arterial pressure (AP) and changes sympathetic-respiratory coupling. However, the alterations in the sleep-wake cycle after CIH and their potential impact on cardiorespiratory parameters remain unknown. Here, we evaluated whether CIH-exposed rats present changes in their short-term sleep-wake cycle pattern and in cardiorespiratory parameters. Male Wistar rats (∼250g) were divided into CIH and control groups. The CIH rats were exposed to 8hday-1 of cycles of normoxia (fraction of inspired O2 =0.208, 5min) followed by hypoxia (fraction of inspired O2 =0.06, 30-40 s) for 10 days. One day after CIH, electrocorticographic activity, cervical EMG, AP and heart rate were recorded for 3h. Plethysmographic recordings were collected for 2h. A subgroup of control and CIH rats also had the diaphragm and oblique abdominal muscle activities recorded. Chronic intermittent hypoxia did not alter the time for sleep onset, total time awake, durations of rapid eye movement (REM) and non-REM (NREM) sleep and number of REM episodes in the 3h recordings. However, a significant increase in the duration of REM episodes was observed. The AP and heart rate were increased in all phases of the cycle in rats exposed to CIH. Respiratory frequency and ventilation were similar between groups in all phases, but tidal volume was increased during NREM and REM sleep in rats exposed to CIH. An increase in the incidence of active expiration during wakefulness was observed in rats exposed to CIH. The data show that CIH-related hypertension is not caused by changes in the sleep-wake cycle and suggest that active expiration is not required for the increase in AP in freely moving rats exposed to CIH.

Pre- and post-inspiratory neurons change their firing properties in female rats exposed to chronic intermittent hypoxia

Obstructive sleep apnea patients face episodes of chronic intermittent hypoxia (CIH), which has been suggested as a causative factor for increased sympathetic activity (SNA) and hypertension. Female rats exposed to CIH develop hypertension and exhibit changes in respiratory–sympathetic coupling, marked by an increase in the inspiratory modulation of SNA. We tested the hypothesis that enhanced inspiratory-modulation of SNA is dependent on carotid bodies (CBs) and are associated with changes in respiratory network activity. For this, in CIH-female rats we evaluated the effect of CBs ablation on respiratory–sympathetic coupling, recorded from respiratory neurons in the working heart–brainstem preparation and from NTS neurons in brainstem slices. CIH-female rats had an increase in peripheral chemoreflex response and in spontaneous excitatory neurotransmission in NTS. CBs ablation prevents the increase in inspiratory modulation of SNA in CIH-female rats. Pre-inspiratory/inspiratory (Pre-I/I) neurons of CIH-female rats have a reduced firing frequency. Post-inspiratory neurons are active for a longer period during expiration in CIH-female rats. Further, using the computational model of a brainstem respiratory–sympathetic network, we demonstrate that a reduction in Pre-I/I neuron firing frequency simulates the enhanced inspiratory SNA modulation in CIH-female rats. We conclude that changes in respiratory–sympathetic coupling in CIH-female rats is dependent on CBs and it is associated with changes in firing properties of specific respiratory neurons types.

Sympathoexcitation following intermittent hypoxia in rat is mediated by circulating angiotensin II acting at the carotid body and subfornical organ

In anaesthetized rats, acute intermittent hypoxia increases sympathetic nerve activity, sympathetic peripheral chemoreflex sensitivity and central sympathetic-respiratory coupling. Renin-angiotensin system inhibition prevents the sympathetic effects of intermittent hypoxia, with intermittent injections of angiotensin II into the systemic circulation replicating these effects. Bilateral carotid body denervation reduces the sympathetic effects of acute intermittent hypoxia and eliminates the increases in chemoreflex sensitivity and sympathetic-respiratory coupling. Pharmacological inhibition of the subfornical organ also reduces the sympathetic effects of acute intermittent hypoxia, although it has no effect on the increases in chemoreflex sensitivity and central sympathetic-respiratory coupling. Combining both interventions eliminates the sympathetic effects of both intermittent hypoxia and angiotensin II. Circulating angiotensin II (AngII) is vital for arterial pressure elevation following intermittent hypoxia in rats, although its importance in the induction of sympathetic changes is unclear. We tested the contribution of the renin-angiotensin system to the effects of acute intermittent hypoxia (AIH) in anaesthetized and ventilated rats. There was a 33.7±2.9% increase in sympathetic nerve activity (SNA), while sympathetic chemoreflex sensitivity and central sympathetic-respiratory coupling increased by one-fold following AIH. The sympathetic effects of AIH were prevented by blocking angiotensin type 1 receptors with systemic losartan. Intermittent systemic injections of AngII (Int.AngII) elicited similar sympathetic responses to AIH. To identify the neural pathways responsible for the effects of AIH and Int.AngII, we performed bilateral carotid body denervation, which reduced the increase in SNA by 56% and 45%, respectively. Conversely, pharmacological inhibition of the subfornical organ (SFO), an established target of circulating AngII, reduced the increase in SNA following AIH and Int.AngII by 65% and 59%, respectively, although it did not prevent the sensitization of the sympathetic peripheral chemoreflex, nor the increase in central sympathetic-respiratory coupling. Combined carotid body denervation and inhibition of the SFO eliminated the enhancement of SNA following AIH and Int.AngII. Repeated systemic injections of phenylephrine caused an elevation in SNA similar to AIH, and this effect was prevented by a renin inhibitor, aliskiren. Our findings show that the sympathetic effects of AIH are the result of RAS-mediated activations of the carotid bodies and the SFO.

OFFSPRING OF OBESE DAMS PRESENT CHANGES IN RESPIRATORY AND SYMPATHETIC ACTIVITIES

Obesity in dams is associated with the development of several pathologies in the offspring, including cardiovascular and hormonal dysfunctions. In this study, we investigated the pattern of respiratory and sympathetic activities in the offspring of dams treated with a high‐fat diet (HFD − 45% fat). Adult female Holtzman rats (250 g) were fed with standard diet (SD) or HFD during 6 weeks before, during gestation and lactation. After weaning, male offspring of SD dams (O‐SD) or HFD dams (O‐HFD) were fed with SD. At P28–32, O‐SD and O‐HFD rats were surgically prepared to obtain the arterially‐perfused in situ preparation to examine the thoracic sympathetic, phrenic, hypoglossal and abdominal activities under basal conditions and during peripheral chemoreceptors activation (KCN −0.05%). O‐HFD rats exhibited augmented baseline phrenic inspiratory (0.80 ± 0.04 vs O‐SD: 0.57 ± 0.07 s; n=6; p &lt; 0.05) and hypoglossal pre‐inspiratory times (0.60 ± 0.11 vs O‐SD: 0.27 ± 0.08 s, n=4; p &lt; 0.05) compared to O‐SD. Additional expiratory bursts were observed in baseline abdominal activity of O‐HFD group, indicating the presence of active expiration (8.5 ± 2.5 vs O‐SD: 1.3 ± 0.6 events/min, n = 4 p &lt; 0.05). Moreover, O‐HF rats showed weakened baseline respiratory‐related modulation of sympathetic activity compared to O‐HFD group. In response to stimulation of peripheral chemoreceptors, O‐HFD group showed greater abdominal reflex responses (212 ± 20 vs O‐SD: 134 ± 21 %; n = 4; p&lt;0.05), but no significant differences were noted in the others recorded nerves comparing to O‐SD. The results indicate that male offspring of HFD‐fed dams exhibit changes in baseline respiratory pattern associated with the presence of active expiration, and blunted respiratory‐sympathetic coupling. Part of these changes persist during peripheral chemoreceptor stimulation.Support or Funding InformationFAPESP (2016/23513‐1; 2013/17251‐6) and CNPq (425586/2016‐2).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Changes in the inspiratory pattern contribute to modulate the sympathetic activity in sino-aortic denervated rats.

What is the central question of this study? Sino-aortic denervated (SAD) rats present normal levels of sympathetic activity and mean arterial pressure. However, neural mechanisms regulating the sympathetic activity in the absence of arterial baroreceptors remain unclear. Considering that respiration modulates the sympathetic activity, we hypothesize that changes in the respiratory network contribute to keep the sympathetic outflow in the normal range after removal of arterial baroreceptors. What is the main finding and its importance? Despite longer inspiration observed in SAD rats, the respiratory-sympathetic coupling is working within a normal range of variation. These findings suggest that in the absence of arterial baroreceptors the respiratory modulation of sympathetic activity is maintained within the normal range. The activity of presympathetic neurons is under respiratory modulation, and changes in the central respiratory network may impact on the baseline sympathetic activity and mean arterial pressure. It is well known that after removal of baroreceptor afferents [sino-aortic denervation (SAD)], rats present an unexpected normal level of mean arterial pressure. We hypothesized that changes in the respiratory pattern and in the respiratory modulation of the sympathetic activity contribute to keep the sympathetic outflow within a normal range of variation in the absence of arterial baroreceptors in rats. To study these mechanisms, we recorded perfusion pressure and the activities of phrenic and thoracic sympathetic nerves in male juvenile rats using the working heart-brainstem preparation. The time of inspiration significantly increased in SAD rats, and this change was not dependent on the carotid bodies or on the vagal afferents. However, no changes were observed in the perfusion pressure or in the baseline thoracic sympathetic nerves in all phases of the respiratory cycle in SAD rats. Our data show that despite longer inspiratory activity, the baseline sympathetic activity is maintained at a normal level in SAD rats. These findings indicate that the respiratory-sympathetic coupling is normal after SAD and suggest that the respiratory modulation of sympathetic activity is maintained within the normal range after the removal of arterial baroreceptors.

Mapping and Analysis of the Connectome of Sympathetic Premotor Neurons in the Rostral Ventrolateral Medulla of the Rat Using a Volumetric Brain Atlas.

Spinally projecting neurons in the rostral ventrolateral medulla (RVLM) play a critical role in the generation of vasomotor sympathetic tone and are thought to receive convergent input from neurons at every level of the neuraxis; the factors that determine their ongoing activity remain unresolved. In this study we use a genetically restricted viral tracing strategy to definitively map their spatially diffuse connectome. We infected bulbospinal RVLM neurons with a recombinant rabies variant that drives reporter expression in monosynaptically connected input neurons and mapped their distribution using an MRI-based volumetric atlas and a novel image alignment and visualization tool that efficiently translates the positions of neurons captured in conventional photomicrographs to Cartesian coordinates. We identified prominent inputs from well-established neurohumoral and viscero-sympathetic sensory actuators, medullary autonomic and respiratory subnuclei, and supramedullary autonomic nuclei. The majority of inputs lay within the brainstem (88–94%), and included putative respiratory neurons in the pre-Bötzinger Complex and post-inspiratory complex that are therefore likely to underlie respiratory-sympathetic coupling. We also discovered a substantial and previously unrecognized input from the region immediately ventral to nucleus prepositus hypoglossi. In contrast, RVLM sympathetic premotor neurons were only sparsely innervated by suprapontine structures including the paraventricular nucleus, lateral hypothalamus, periaqueductal gray, and superior colliculus, and we found almost no evidence of direct inputs from the cortex or amygdala. Our approach can be used to quantify, standardize and share complete neuroanatomical datasets, and therefore provides researchers with a platform for presentation, analysis and independent reanalysis of connectomic data.