Working Heart-brainstem Preparation Research Articles

Exploring Serotonergic mechanisms in Opioid Induced Respiratory Depression

Opioids profoundly depress breathing which can be fatal in the case of overdose. Despite growing knowledge of the pontine and medullary respiratory control circuits that are inhibited by opioids, the circuit and cellular mechanisms of opioid induced respiratory depression are not fully understood. Serotonin neurons located in the caudal medullary raphe modulate breathing and possess mu opioid receptors. Therefore, we hypothesized that serotonin-producing raphe neurons contribute to opioid induced respiratory depression. To test this, we employed to experimental approaches: 1) arterially perfused in situ working heart brainstem preparations and 2) ex vivo acute brain slice electrophysiology to identify and characterize serotonin neuron inputs onto brainstem respiratory neurons. To specifically activate serotonin neurons, we used ePet-Cre::Ai32 mice which Cre-dependently express channelrhodopsin-2, a light-activated cation channel, in serotonin neurons. At the circuit level, using the in situ preparation, we found that activation of caudal medullary raphe serotonin neurons increased respiratory rate by decreasing inspiratory and expiratory duration. Fentanyl application blocked the respiratory effects induced by serotonin neuron activation which were subsequently restored using naloxone. These results suggest serotonin neurons in the caudal medullary raphe are inhibited by opioids. To characterize the serotonin-mediated mechanisms at the cellular level, we made acute brain slices containing dorsal raphe neurons and lateral parabrachial neurons from ePet-Cre::Ai32 mice. We verified that optical stimulation resulted in action potentials and serotonin release from dorsal raphe serotonin neurons. Optically evoked serotonin release caused 5HT1A receptor-mediated outward currents in dorsal raphe serotonin neurons. To locate and characterize serotonin neuron inputs onto lateral parabrachial neurons, we performed whole-cell voltage clamp recordings from lateral parabrachial neurons and delivered paired light pulses (10 ms duration, 50 ms interval). We observed optically evoked inhibitory post-synaptic currents (IPSCs) that were blocked by the GABA-A receptor antagonist gabazine (1 μM). Optically evoked IPSCs had long latency (> 5 ms) and high jitter (> 1 ms), indicating stimulation of serotonin terminals resulted in polysynaptic GABA release onto lateral parabrachial neurons. Future work will examine the influence of opioids on this raphe-parabrachial circuit. Uncovering the role of serotonin neurons in opioid induced respiratory depression may reveal targets for restoring breathing in the presence of opioids or other conditions where life-threatening apneas occur. This work was supported by the National Institutes of Health (NIDA) R01DA047978-05 grant to ESL. 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.

Fiber optical imaging of astroglial calcium signaling in the respiratory network in the working heart brainstem preparation.

The neuronal activity in the respiratory network strongly depends on a variety of different neuromodulators. Given the essential role of astrocytes in stabilizing respiratory network activity generated by neurons in the preBötzinger complex (preBötC), our aim was to investigate astrocytic calcium signaling in the working heart brainstem preparation using fiber-optical imaging. By using transgenic mice that express GCaMP6s specifically in astrocytes, we successfully recorded astrocytic calcium signals in response to norepinephrine from individual astrocytes.

Angiotensin II increases respiratory rhythmic activity in the preBötzinger complex without inducing astroglial calcium signaling.

Angiotensin II (Ang II) is the primary modulator of the renin-angiotensin system and has been widely studied for its effect on the cardiovascular system. While a few studies have also indicated an involvement of Ang II in the regulation of breathing, very little is known in this regard and its effect on brainstem respiratory regions such as the preBötzinger complex (preBötC), the kernel for inspiratory rhythm generation, has not been investigated yet. This study reports that Ang II temporarily increases phrenic nerve activity in the working heart-brainstem preparation, indicating higher central respiratory drive. Previous studies have shown that the carotid body is involved in mediating this effect and we revealed that the preBötC also plays a part, using acute slices of the brainstem. It appears that Ang II is increasing the respiratory drive in an AT1R-dependent manner by optimizing the interaction of inhibitory and excitatory neurons of the preBötC. Thus, Ang II-mediated effects on the preBötC are potentially involved in dysregulating breathing in patients with acute lung injury.

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.

Comparison of the effects of fentanyls and other μ opioid receptor agonists on the electrical activity of respiratory muscles in the rat.

Introduction: Deaths due to overdose of fentanyls result primarily from depression of respiration. These potent opioids can also produce muscle rigidity in the diaphragm and the chest muscles, a phenomenon known as Wooden Chest Syndrome, which further limits ventilation. Methods: We have compared the depression of ventilation by fentanyl and morphine by directly measuring their ability to induce muscle rigidity using EMG recording from diaphragm and external and internal intercostal muscles, in the rat working heart-brainstem preparation. Results: At equipotent bradypnea-inducing concentrations fentanyl produced a greater increase in expiratory EMG amplitude than morphine in all three muscles examined. In order to understand whether this effect of fentanyl was a unique property of the phenylpiperidine chemical structure, or due to fentanyl's high agonist intrinsic efficacy or its lipophilicity, we compared a variety of agonists with different properties at concentrations that were equipotent at producing bradypnea. We compared carfentanil and alfentanil (phenylpiperidines with relatively high efficacy and high to medium lipophilicity, respectively), norbuprenorphine (orvinolmorphinan with high efficacy and lipophilicity) and levorphanol (morphinan with relatively low efficacy and high lipophilicity). Discussion: We observed that, agonists with higher intrinsic efficacy were more likely to increase expiratory EMG amplitude (i.e., produce chest rigidity) than agonists with lower efficacy. Whereas lipophilicity and chemical structure did not appear to correlate with the ability to induce chest rigidity.

Pro-opiomelanocortin neurons in the nucleus of the solitary tract mediate endorphinergic endogenous analgesia in mice.

The nucleus of the solitary tract (NTS) contains pro-opiomelanocortin (POMC) neurons that are 1 of the 2 major sources of β-endorphin in the brain. The functional role of these NTS POMC neurons in nociceptive and cardiorespiratory function is debated. We have shown that NTS POMC optogenetic activation produces bradycardia and transient apnoea in a working heart-brainstem preparation and chemogenetic activation with an engineered ion channel (PSAM) produced opioidergic analgesia in vivo. To better define the role of the NTS POMC neurons in behaving animals, we adopted in vivo optogenetics (ChrimsonR) and excitatory/inhibitory chemogenetic DREADD (hM3Dq/hM4Di) strategies in POMC-Cre mice. We show that optogenetic activation of NTS POMC neurons produces time-locked, graded, transient bradycardia and bradypnoea in anaesthetised mice that is naloxone sensitive (1 mg/kg, i.p.), suggesting a role of β-endorphin. Both optogenetic and chemogenetic activation of NTS POMC neurons produces sustained thermal analgesia in behaving mice that can be blocked by naloxone. It also produced analgesia in an inflammatory pain model (carrageenan) but not in a neuropathic pain model (tibial nerve transection). Inhibiting NTS POMC neurons does not produce any effect on basal nociception but inhibits stress-induced analgesia (unlike inhibition of arcuate POMC neurons). Activation of NTS POMC neuronal populations in conscious mice did not cause respiratory depression, anxiety, or locomotor deficit (in open field) or affective preference. These findings indicate that NTS POMC neurons play a key role in the generation of endorphinergic endogenous analgesia and can also regulate cardiorespiratory function.

Generation, breakdown and responses of ATP in the carotid body

Purinergic signaling involving adenosine triphosphate (ATP) acting on P2X2/3 receptors modulates physiological carotid body (CB) afferent discharge and chemoreflex activation however, the upregulation of P2X3 receptors on petrosal sensory neurons is partly responsible for aberrant CB tonicity and hyperreflexia, and the increase in blood pressure in the Spontaneously Hypertensive rat (SHR). The enhanced chemoreflex occurs prior to the onset of hypertension and elevated CB activity is thought to contribute to the underlying pathophysiology of the disease but the exact mechanisms remain unclear. We hypothesize that there is greater release of ATP and/or decreased ATP breakdown in the pre‐hypertensive SHR CB compared with that in the Wistar rat, and this evokes an increase in the chemoreflex response and subsequent activation of the sympathetic nervous system. We performed three studies. (i) Using digital droplet PCR (ddPCR), we measured mRNA expression levels of enzymes involved in the breakdown of ATP in CBs extracted from male 4‐week‐old prehypertensive SHR and Wistar rats; these included: ectonucleotide pyrophosphatase/phosphodiesterase 1‐3 (Enpp1‐3), ectonucleoside triphosphate diphosphohydrolase 2‐3 (Entpd2‐3), and ecto‐5’‐nucleotidase (Nt5e). (ii) We quantified ATP release from CBs in vitro during baseline conditions and during cyanide‐evoked ATP release using a colorimetric assay (213‐579‐1 ‐ Millipore), comparing between male 4‐week‐old pre‐hypertensive SHR and Wistar rats. (iii) In an in situ working heart‐brainstem preparation of 4‐week‐old Wistar rats, simultaneous recordings were made from phrenic (PN), hypoglossal (HN), recurrent laryngeal (RLN), abdominal (ABN), and thoracic sympathetic nerves (tSNA). The peripheral chemoreceptors were stimulated with either potassium cyanide (KCN 20‐100 μL, 0.04%) or ATP at varying concentrations (10‐100 µL, 50 µM ‐ 2 mM) delivered via the internal carotid artery. In whole CB extracts, ddPCR revealed increased Enpp2‐3 and Nt5e expression (p<0.05) in pre‐hypertensive SHRs relative to Wistar rats suggesting greater capacity to break down ATP in the pre‐hypertensive SHR CB. There were no differences in the expressions of Enpp1 or Entpd2‐3 between rat strains. However, the amount of ATP released from the CB in both baseline and during cyanide stimulation was two‐fold greater in the pre‐hypertensive SHR (P<0.05). Stimulation of the CB with KCN evoked hyperpnoea, bradycardia and sympathoexcitation at all doses. In contrast, CB injection of low dose ATP increased PN rate with little effect on tSNA, whereas higher doses induced apnea accompanied by repetitive burst discharge of post‐inspiratory activity in the RLN with tonic elevations in the discharge of both ABN and tSNA. In sum, despite the upregulation of several ATP degrading enzymes in the CBs of the SHR, CB release of ATP is higher in this species. We hypothesize that the increase in ATP release in the SHR CB drives the CB hyperexcitability and that the shift towards an upregulation of ATP degrading enzymes even prior to the development of hypertension is compensatory to oppose the increase in ATP signaling. The dose‐dependent effect of exogenous ATP at supraphysiological levels evoking greater CB‐mediated expiratory and sympathetic outflow in the Wistar rat mimics aspects of pathophysiological CB chemoreflex signaling in the SHR.

Uncovering Anti‐Arrhythmic Potential of Stellate Ganglion Purinergic Receptors

Cardiovascular disease affects over 30% of people worldwide and is one of the leading causes of death each year. Elevated sympathetic nerve activity is a common feature of cardiovascular disease, contributing to end‐organ damage, morbidity and mortality. Recent findings indicate that short‐circuiting sympathetic nerve overactivity by removal of the stellate ganglion can eradicate arrhythmias, emphasising the need for novel therapeutic targets to correct signalling non‐invasively.We have revealed upregulation of the P2X3 purinergic receptor in stellate ganglia of Spontaneously Hypertensive (SHR) compared to Wistar rats (16 week‐old), via RNASeq transcriptional profiling. We hypothesise that these purinergic receptors within cardiac stellate ganglia play a role in the excessive sympathetic drive to the heart in cardiovascular disease and can initiate cardiac arrhythmias. Thus, we have investigated the functional role of purinergic receptors in the stellate ganglion.Administration of ATP to acutely isolated post‐ganglionic sympathetic neurones from the stellate ganglia of Wistar rats (4‐6 week‐old) evokes a significant increase (Median; 0.21) in [Ca2+]i as measured by Fura‐2AM imaging (n=15, Wilcoxon matched‐pairs test; p<0.0001). This ATP‐induced calcium transient was inhibited by specific P2X3 receptor antagonism with either NF‐10 or AF‐130.Cardiac effects of stellate ganglion purinergic stimulation were investigated in the working heart‐brainstem preparation of Wistar rats (4‐6 week‐old). Increasing doses of ATP (50‐250 µg) delivered via microinjection (50‐250 µL) directly to the right stellate ganglion produced either a tachycardia (18 ± 6 bpm, n=6) or bradycardia (‐24 ± 12 bpm, n=5); these responses were typically found at distinct sites within the ganglion. Preliminary data from SHR suggest that higher doses of ATP (≥250µg) may be arrhythmogenic (n=3).P2X3 purinergic receptors are present in the sympathetic stellate ganglion and contribute to calcium ion flux and cardiac chronotropic regulation. Overexpression of P2X3 receptors is likely to contribute to excessive cardiac sympathetic activity and the development of hypertension and cardiac arrhythmias, making them a promising novel therapeutic target.

Advancing respiratory-cardiovascular physiology with the working heart-brainstem preparation over 25 years.

Twenty‐five years ago, a new physiological preparation called the working heart–brainstem preparation (WHBP) was introduced with the claim it would provide a new platform allowing studies not possible before in cardiovascular, neuroendocrine, autonomic and respiratory research. Herein, we review some of the progress made with the WHBP, some advantages and disadvantages along with potential future applications, and provide photographs and technical drawings of all the customised equipment used for the preparation. Using mice or rats, the WHBP is an in situ experimental model that is perfused via an extracorporeal circuit benefitting from unprecedented surgical access, mechanical stability of the brain for whole cell recording and an uncompromised use of pharmacological agents akin to in vitro approaches. The preparation has revealed novel mechanistic insights into, for example, the generation of distinct respiratory rhythms, the neurogenesis of sympathetic activity, coupling between respiration and the heart and circulation, hypothalamic and spinal control mechanisms, and peripheral and central chemoreceptor mechanisms. Insights have been gleaned into diseases such as hypertension, heart failure and sleep apnoea. Findings from the in situ preparation have been ratified in conscious in vivo animals and when tested have translated to humans. We conclude by discussing potential future applications of the WHBP including two‐photon imaging of peripheral and central nervous systems and adoption of pharmacogenetic tools that will improve our understanding of physiological mechanisms and reveal novel mechanisms that may guide new treatment strategies for cardiorespiratory diseases.

Modified working heart brainstem preparation for studying spike propagation through the dorsal root ganglion

The dorsal root ganglion (DRG) contains cell bodies of peripheral somatosensory nerves. These nerves generate and transmit to the CNS signals related to physical changes in the body's immediate environment or state of internal organs. DRG neurons are pseudounipolar with a short stem axon branching into peripheral and central (dorsal root; DR) aspects. Peripheral branches innervate the end organs (e.g. skin, muscle etc.), while DRs synapse in the spinal cord. On the way from peripheral nerve endings to synaptic terminals, spikes pass through branching points (t-junctions) in the ganglia.

Keep in touch: Nodal connectivity in the control of breathing and blood pressure.

Brainstem and suprapontine structures controlling cardiorespiratory function are reasonably well defined, but many subtleties of the intricate neural circuitry are yet to be revealed. Contemporary experimental approaches allow for elegant delineation of nodal connectivity underpinning integrative behaviours. The highly co-ordinated contemporaneous activation of respiratory motor and autonomic outputs at rest and in response to stress is crucial to survival. In this issue of Experimental Physiology, Malheiros-Lima et al. (2022) extend our understanding of pontomedullary communication in the control of breathing and blood pressure. Medullary catecholaminergic C1 neurons and ventrolateral pontine adrenergic A5 neurons are two key nodes in the brainstem network controlling cardiorespiratory function. Both are strongly implicated in chemoreflex control of respiratory and sympathetic outflows, but the nature of the interaction between the nodes is not fully appreciated. In a sophisticated experimental series, Malheiros-Lima et al. (2022) set out to establish: (1) whether C1 neurons projecting directly to A5 neurons are glutamatergic (excitatory); (2) whether retrograde ablation of C1 neurons via the A5 region reduces hypoxia-dependent activation of C1 neurons; and (3) whether ionotropic glutamatergic receptor blockade in A5 neurons decreases the cardiorespiratory effects of selective activation of C1 neurons. The experimental findings demonstrate a crucial link between C1 and A5 neurons in the cardiorespiratory control network. In adult male rats, lentiviral transfection of catecholaminergic (tyrosine hydroxylase-positive) neurons in the rostral ventrolateral medulla (C1 neurons) with a fluorescent protein provided confirmation that transduced C1 terminals, which are predominantly glutamatergic, make direct contact with ipsilateral A5 neurons of the pons, amongst other sites, such as the A6 locus coeruleus. Retrograde destruction of C1 neurons was achieved by microinjection of a selective poison unilaterally into the A5 region, which decreased ipsilateral C1 catecholaminergic neurons by 25% compared with the contralateral neuronal count. Exposure to hypoxia for 3 h elicited the expected increases in c-Fos expression in C1 neurons; however, after selective poisoning, the density of c-Fos-positive non-catecholaminergic C1 neurons was unchanged, whereas the total number of c-Fos-positive catecholaminergic neurons was reduced compared with the contralateral population. Bilateral injections of kynurenic acid into the A5 region abrogated, in part, the increase in blood pressure, splanchnic sympathetic nerve activity and phrenic nerve motor activity elicited by selective optogenetic activation of C1 catecholaminergic neurons (transfected with channelrhodopsin2 and activated by laser). The study reveals that medullary C1 neurons activated by hypoxia project directly to pontine A5 neurons. Moreover, glutamatergic receptor blockade in the A5 node suppressed sympathetic and respiratory responses to selective C1 neuronal activation. Although C1 neurons are traditionally considered catecholaminergic, possessing the enzymatic portfolio to produce various catecholamines, Malheiros-Lima et al. (2022) found that > 90% of the varicosities in the A5 region are glutamatergic. The physiological effects of selective pharmacological blockade of glutamate neurotransmission corroborate the neuroanatomical observations suggesting that the C1–A5 connection is mediated via glutamate and accounts for an appreciable amount of C1-dependent cardiorespiratory activation. A5 neurons might also have capacity for a direct independent contribution owing to their intrinsic chemosensitivity (Kanbar et al., 2011), which appears to be a ubiquitous feature of brainstem neural circuits. There is growing evidence for a contribution by C1 neurons to breathing (Malheiros et al., 2018), extending to the respiratory response to hypoxia (Malheiros et al., 2020). The present study extends this work, revealing a contribution by the C1–A5 pathway to respiratory motor drive, shown by retardation in phrenic motor amplitude in response to hypoxia after glutamatergic inhibition in the A5 region. The precise link between A5 neurons and other pontine and medullary sites implicated in the chemoreflex-dependent drive to breathe remains to be determined, but the lack of effect of A5 inhibition on hypoxia-dependent tachypnoea might exclude connectivity with rhythmogenic sites, such as the pre-Bötzinger complex. Confusingly, however, previous work reported that A5 lesions do not affect ventilatory responses to hypoxia in conscious rats nor the phrenic nerve response in the in situ working heart–brainstem preparation (Taxini et al., 2017). This suggests that anaesthesia affects the relevant circuitry behaviour and, as such, is a confounder in the present study. Thus, it appears that the C1–A5 pathway forms an intrinsic component of the chemoreflex circuitry mediating sympathoexcitation and the pressor response to stress, with a potential contribution to the accompanying respiratory excitation. Pontine adrenergic neurons project to most structures of the brain in a highly organized manner. Therefore, it might be that specific elements of the fight or flight response, beyond cardiorespiratory adjustments, are also manifest through the C1–A5 axis. We can anticipate continued elegant work from this research group striving to unravel these complex connections. Let's keep in touch! None declared.

Sympathetic Modulation By Glucagon Like Peptide 1 And Melanocortin 4 Receptors In The Carotid Body Of Wistar Rats

Diabetes is a chronic metabolic disorder that occurs when there is raised levels of glucose in the blood (hyperglycemia) caused by either insulin deficiency or an inadequate response to insulin. Globally, the clinical problem of diabetes and obesity is huge (8.5% of total population) and this is reflected in Aotearoa by 6% of the population. Diabetes increases the likelihood of stroke, heart attack, vascular and visual disorders. Such a clinical tsunami makes understanding mechanisms of glucose homeostasis more critical than ever. An area that remains unexplained are the mechanisms by which sympathetic nervous activity (SNA) is heightened in diabetes as this contributes substantially to blood hyperglycaemia. Given the influential role of the carotid body (CB) in regulating SNA together with its ability to sense blood glucose, insulin and adrenaline, we tested the hypothesis that two newly revealed receptors from transcriptomic sequencing - glucagon-like peptide-1 (GLP1R) and melanocortin-4 (MC4R) located within the CB play a significant role in controlling SNA. The working heart-brainstem preparation was used to evaluate functionally the cardiovascular (SNA, heart rate, arterial pressure) and respiratory (phrenic nerve) activity in Wistar rats. The specific agonists and antagonists of GLP1R (Exendin-4 and Exendin-3, respectively) and MC4R (THIQ and HS014, respectively) were injected into the internal carotid artery (ICA) which, via the carotid body artery, perfuses the CB. Data are expressed 10 min post injection. The CB was stimulated with low doses of sodium cyanide (i.a.). Immunocytochemistry detected GLP1 and MC4 receptors localised to distinct cell groups within the carotid body. Stimulation of GLP1R within the CB inhibited the chemoreflex evoked sympathetic response (p<0.05) whilst it was augmented powerfully by the MC4R agonist (p<0.05) compared to baseline. In contrast, heart rate, arterial pressure and phrenic activity were unaffected. GLP1Rs antagonist alone increased the chemoreflex evoked sympathetic response above basline (p<0.05) while antagonising MC4Rs was without effect. Injections of vehicle did not alter either the chemoreflex evoked or basal SNA. For the first time, stimulation of metabolism-linked receptors – GLP1 and MC4 within the CB modulate SNA differentially. Our data purport endogenous secretion of GLP-1 within the CB affecting sympathetic activity. Our study provides novel targets within the CB that might be pharmacologically probed to control metabolism via modulation of SNA; such data may provide the much needed guidance for controlling blood glucose in diabetes.

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.

Selective Peripheral Chemoreceptor Hyperreflexia Evoked after Sympathetic Stimulation

Previous studies have demonstrated that the integrity of the carotid bodies is essential for the development and maintenance of hypertension. In this condition, both increased tonicity and hyperreflexia are displayed by chemosensitive neurons in the petrosal ganglia of spontaneous hypertensive rats (SHR). However, what drives carotid body hyper‐excitability is not known. We hypothesised this was triggered by the sympathetic nerves innervating the carotid body, which originate from the superior cervical ganglion (SCG). The aim of our study was test if activation of the SCG could modulate carotid body activity. Experiments were carried out in 6 juvenile Wistar rats (60–80g) using the decerebrated in situ working heart‐brainstem preparation (WHBP). Simultaneous recordings of phrenic (PN) and thoracic sympathetic nerves (tSNA) were obtained using glass suction electrodes. Heart rate (HR) was derived from the electrocardiogram (ECG). Chemoreflex was activated using NaCN (50μL, 0.04%, i.a.) and the SCG was stimulated electrically (ES; 30Hz, 2 ms, 10V) using a Grass SD9 stimulator via a bipolar microelectrode. The chemoreflex was evoked before and immediately after ES of the SCG. Changes in sympathetic activity, perfusion pressure, phrenic and heart rate were analysed quantitatively. Data are displayed as mean ± SEM with a level of significance set at P&lt;0.05; differences were tested using paired student‐t test.The only component of the chemoreflex augmented by SCG stimulation was the evoked increase in sympathetic activity. In Wistars, the control response of 29.3 ± 3.2% from baseline was augmented to 46.04 ± 3.58% after ES (P=0.006). Within 10 min of the stimulation, the sympatho hyperreflexia had returned to control (20.24 ± 5.96%). SHR also presented sympatho hyperreflexia (43.0 ± 6.3 vs 62.9 ± 9.2 %, P= 0.015) but the change of sensitization was of similar magnitude between rat strains. The SCG induced carotid body hyperreflexia was prevented by application of lignocaine into the SCG. We conclude that stimulation of the efferent sympathetic nerves innervating the carotid body can increase the gain of the chemoreflex symathoexcitatory response. We are currently assessing whether chemoreflex hyperreflexia observed in neurogenic hypertension is mediated by sympathetic nerves innervating the carotid body.Support or Funding InformationHealth Research Council of New Zealand

Epigenetic silencing by early-life hypoxic stress programmes respiratory motor control.

Epigenetic silencing by early-life hypoxic stress programmes respiratory motor control.

Shift of leading pacemaker site during reflex vagal stimulation and altered electrical source-to-sink balance.

Vagal reflexes slow heart rate and can change where the heartbeat originates within the sinoatrial node (SAN). The mechanisms responsible for this process - termed leading pacemaker (LP) shift - have not been investigated fully. We used optical mapping to measure the effects of baroreflex, chemoreflex and carbachol on pacemaker entrainment and electrical conduction across the SAN. All methods of stimulation triggered shifts in LP site from the central SAN to one or two caudal pacemaker regions. These shifts were associated with reduced current generation capacity centrally and increased electrical load caudally. Previous studies suggest LP shift is a rate-dependent phenomenon whereby acetylcholine slows central pacemaker rate disproportionately, enabling caudal cells that are less acetylcholine sensitive to assume control. However, our findings indicate the LP region is defined by both pacemaker rate and capacity to drive activation. Shifts in LP site provide an important homeostatic mechanism for rapid switches in heart rate. Reflex vagal activity causes abrupt heart rate slowing with concomitant caudal shifts of the leading pacemaker (LP) site within the sinoatrial node (SAN). However, neither the mechanisms responsible nor their dynamics have been investigated fully. Therefore, the objective of this study was to elucidate the mechanisms driving cholinergic LP shift. Optical maps of right atrial activation were acquired in a rat working heart-brainstem preparation during baroreflex and chemoreflex stimulation or with carbachol. All methods of stimulation triggered shifts in LP site from the central SAN to caudal pacemaker regions, which were positive for HCN4 and received uniform cholinergic innervation. During baroreflex onset, the capacity of the central region to drive activation declined with a decrease in amplitude and gradient of optical action potentials (OAPs) in the surrounding myocardium. Accompanying this decline, there was altered entrainment in the caudal SAN as shown by decreased conduction velocity, OAP amplitude, gradient and activation time. Atropine abolished these responses. Chemoreflex stimulation produced similar effects but central capacity to drive activation was preserved before the LP shift. In contrast, carbachol produced a prolonged period of reduced capacity to drive and altered entrainment. Previous studies suggest LP shift is a rate-dependent phenomenon whereby acetylcholine slows central pacemaker rate disproportionately, enabling caudal cells that are less acetylcholine sensitive to assume control. Our findings indicate that cholinergic LP shifts are also determined by altered electrical source-to-sink balance in the SAN. We conclude that the LP region is defined by both rate and capacity to drive atrial activation.

Cardiovascular autonomic effects of transcutaneous auricular nerve stimulation via the tragus in the rat involve spinal cervical sensory afferent pathways

BackgroundElectrical stimulation on select areas of the external auricular dermatome influences the autonomic nervous system. It has been postulated that activation of the Auricular Branch of the Vagus Nerve (ABVN) mediates such autonomic changes. However, the underlying neural pathways mediating these effects are unknown and, further, our understanding of the anatomical distribution of the ABVN in the auricle has now been questioned. ObjectiveTo investigate the effects of electrical stimulation of the tragus on autonomic outputs in the rat and probe the underlying neural pathways. MethodsCentral neuronal projections from nerves innervating the external auricle were investigated by injections of the transganglionic tracer cholera toxin B chain (CTB) into the right tragus of Wistar rats. Physiological recordings of heart rate, perfusion pressure, respiratory rate and sympathetic nerve activity were made in an anaesthetic free Working Heart Brainstem Preparation (WHBP) of the rat and changes in response to electrical stimulation of the tragus analysed. ResultsNeuronal tracing from the tragus revealed that the densest CTB labelling was within laminae III-IV of the dorsal horn of the upper cervical spinal cord, ipsilateral to the injection sites. In the medulla oblongata, CTB labelled afferents were observed in the paratrigeminal nucleus, spinal trigeminal tract and cuneate nucleus. Surprisingly, only sparse labelling was observed in the vagal afferent termination site, the nucleus tractus solitarius. Recordings made from rats at night time revealed more robust sympathetic activity in comparison to day time rats, thus subsequent experiments were conducted in rats at night time. Electrical stimulation was delivered across the tragus for 5 min. Direct recording from the sympathetic chain revealed a central sympathoinhibition by up to 36% following tragus stimulation. Sympathoinhibition remained following sectioning of the cervical vagus nerve ipsilateral to the stimulation site, but was attenuated by sectioning of the upper cervical afferent nerve roots. ConclusionsInhibition of the sympathetic nervous system activity upon electrical stimulation of the tragus in the rat is mediated at least in part through sensory afferent projections to the upper cervical spinal cord. This challenges the notion that tragal stimulation is mediated by the auricular branch of the vagus nerve and suggests that alternative mechanisms may be involved.

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.

An arterially perfused brainstem preparation of guinea pig to study central mechanisms of airway defense

The perfused working heart brainstem preparation of rodents has become a widely used tool to study brainstem function. Here, we adapt this experimental technique for newborn guinea pigs (postnatal day 7–14) to develop a tool that enables investigation of airway defense mechanisms not observed in other rodents. The perfused guinea pig brainstem preparation generates a stable eupnea-like motor pattern recorded from the phrenic, recurrent laryngeal and intercostal nerves and basic cardio-respiratory reflexes, including the arterial chemoreceptor, the baroreceptor reflex. In addition a fictive laryngeal cough reflex can be reliably elicited after mechanical stimulation of the trachea. Single unit recordings within the ponto-medullary respiratory column show robust central respiratory neuronal activity. Additionally, as in other species ponto-medullary transection of the brainstem produces apneusis. The latter suggests that the preparation fully preserves ponto-medullary synaptic connectivity that is required for eupnea-like respiratory rhythm and pattern formation and the mediation of various cardio-respiratory reflexes. We conclude that this novel research tool provides an alternative to established rat and mouse preparations and may become a experimental tool for the investigation of central mechanisms that mediate laryngeal cough.