Respiratory Reflexes Research Articles

Practice Guidelines for Intravenous Conscious Sedation in Dentistry (Second Edition, 2017).

Practice Guidelines for Intravenous Conscious Sedation in Dentistry (Second Edition, 2017).

Sex-Specific Consequences of Neonatal Stress on Cardio-Respiratory Inhibition Following Laryngeal Stimulation in Rat Pups.

The presence of liquid near the larynx of immature mammals triggers prolonged apneas with significant O2 desaturations and bradycardias. When excessive, this reflex (the laryngeal chemoreflex; LCR) can be fatal. Our understanding of the origins of abnormal LCR are limited; however, perinatal stress and male sex are risk factors for cardio-respiratory failure in infants. Because exposure to stress during early life has deleterious and sex-specific consequences on brain development it is plausible that respiratory reflexes are vulnerable to neuroendocrine dysfunction. To address this issue, we tested the hypothesis that neonatal maternal separation (NMS) is sufficient to exacerbate LCR-induced cardio-respiratory inhibition in anesthetized rat pups. Stressed pups were separated from their mother 3 h/d from postnatal days 3 to 12. At P14–P15, pups were instrumented to monitor breathing, O2 saturation (Spo2), and heart rate. The LCR was activated by water injections near the larynx (10 µl). LCR-induced apneas were longer in stressed pups than controls; O2 desaturations and bradycardias were more profound, especially in males. NMS increased the frequency and amplitude of spontaneous EPSCs (sEPSCs) in the dorsal motor nucleus of the vagus (DMNV) of males but not females. The positive relationship between corticosterone and testosterone observed in stressed pups (males only) suggests that disruption of neuroendocrine function by stress is key to sex-based differences in abnormal LCR. Because testosterone application onto medullary slices augments EPSC amplitude only in males, we propose that testosterone-mediated enhancement of synaptic connectivity within the DMNV contributes to the male bias in cardio-respiratory inhibition following LCR activation in stressed pups.

Mechanistic link between diesel exhaust particles and respiratory reflexes

BackgroundDiesel exhaust particles (DEPs) are a major component of particulate matter in Europe's largest cities, and epidemiologic evidence links exposure with respiratory symptoms and asthma exacerbations. Respiratory reflexes are responsible for symptoms and are regulated by vagal afferent nerves, which innervate the airway. It is not known how DEP exposure activates airway afferents to elicit symptoms, such as cough and bronchospasm.ObjectiveWe sought to identify the mechanisms involved in activation of airway sensory afferents by DEPs.MethodsIn this study we use in vitro and in vivo electrophysiologic techniques, including a unique model that assesses depolarization (a marker of sensory nerve activation) of human vagus.ResultsWe demonstrate a direct interaction between DEP and airway C-fiber afferents. In anesthetized guinea pigs intratracheal administration of DEPs activated airway C-fibers. The organic extract (DEP-OE) and not the cleaned particles evoked depolarization of guinea pig and human vagus, and this was inhibited by a transient receptor potential ankyrin-1 antagonist and the antioxidant N-acetyl cysteine. Polycyclic aromatic hydrocarbons, major constituents of DEPs, were implicated in this process through activation of the aryl hydrocarbon receptor and subsequent mitochondrial reactive oxygen species production, which is known to activate transient receptor potential ankyrin-1 on nociceptive C-fibers.ConclusionsThis study provides the first mechanistic insights into how exposure to urban air pollution leads to activation of guinea pig and human sensory nerves, which are responsible for respiratory symptoms. Mechanistic information will enable the development of appropriate therapeutic interventions and mitigation strategies for those susceptible subjects who are most at risk.

Evolution of the hypoxia-sensitive cells involved in amniote respiratory reflexes.

The evolutionary origins of the hypoxia-sensitive cells that trigger amniote respiratory reflexes - carotid body glomus cells, and 'pulmonary neuroendocrine cells' (PNECs) - are obscure. Homology has been proposed between glomus cells, which are neural crest-derived, and the hypoxia-sensitive 'neuroepithelial cells' (NECs) of fish gills, whose embryonic origin is unknown. NECs have also been likened to PNECs, which differentiate in situ within lung airway epithelia. Using genetic lineage-tracing and neural crest-deficient mutants in zebrafish, and physical fate-mapping in frog and lamprey, we find that NECs are not neural crest-derived, but endoderm-derived, like PNECs, whose endodermal origin we confirm. We discover neural crest-derived catecholaminergic cells associated with zebrafish pharyngeal arch blood vessels, and propose a new model for amniote hypoxia-sensitive cell evolution: endoderm-derived NECs were retained as PNECs, while the carotid body evolved via the aggregation of neural crest-derived catecholaminergic (chromaffin) cells already associated with blood vessels in anamniote pharyngeal arches.

A Cellular Mechanism of Leptin Mediated Responses in Mouse NTS Neurons

The nucleus of the solitary tract (NTS) in the medulla is the site of first synapse for sensory afferents of the vagal and glosspharyngeal nerves. A dense termination of sensory input reflects a key function of the NTS as an integrative center for a number of vitally important homeostatic reflexes such as cardiovascular, respiratory and visceral reflexes. Little is known concerning the cellular rules of information integration in the NTS, especially how the metabolic state influences respiratory output. Leptin is an anorexigenic cytokine acting through activation of specific G‐protein‐coupled receptors (GPCRs). Leptin receptors are expressed in the NTS, but the mechanism of action following their activation remains unclear. Moreover, the precise identity of the cells expressing the leptin receptor in the NTS is also elusive.Our study has two main goals: 1‐ to uncover the expression of individual genes that identify neuronal subpopulations of cells expressing the leptin receptor; and 2‐ to identify the ion channel(s) mediating the action of leptin in these different cellular populations. To pursue these goals we combine genetic tagging of leptin receptor expressing neurons, electrophysiological recordings and single cell RT‐PCR to test the expression of candidate channels and neuropeptides. Our data show that NTS leptin receptor expressing neurons belong to two distinct populations characterized by different firing patterns (bursting vs. regular firing). Further, we found that leptin receptor activation leads to membrane depolarization that is mediated by a cationic conductance. This mechanism appears to be the same in both the cell populations. The PCR data suggest that the conductance activated by leptin is, at least in part, mediated by NALCN channels.Support or Funding InformationThis study was supported by NIH grant HL122921

Impact of high concentrations of sevoflurane on laryngeal reflex responses.

Exaggerated defensive upper airway reflexes, particularly laryngospasm, may cause hypoxemic damage, especially in children. General clinical experience suggests that laryngeal reflex responses are more common under light levels of anesthesia, and previous clinical studies have shown an inverse correlation between laryngeal responsiveness and depth of hypnosis. However, this seems to be less obvious in children anesthetized with sevoflurane. The aim of this study was to assess the impact of high concentrations of sevoflurane on laryngeal and respiratory reflex responses in spontaneously breathing children. Accordingly, we tested the hypothesis that laryngeal and respiratory reflex responses were completely suppressed in spontaneously breathing children when anesthetized with sevoflurane 4.7% (=MACED95Intubation ) as compared with sevoflurane 2.5% (=1MAC). In this prospective observational study, we tested the hypothesis that the incidence of laryngospasm evoked by laryngeal stimulation is diminished under high concentrations of sevoflurane. Following Ethics approval, trial registration, and informed consent, 40 children (3-7years) scheduled for elective surgery participated in the trial. All children received sevoflurane 2.5% (1 MAC) and 4.7% (ED95Intubation ) in random order with 5-min equilibration between the states. Under both conditions, distilled water was sprayed under bronchoscopic view onto the larynx. Potential laryngeal and respiratory reflex responses were assessed offline by a blinded reviewer. Laryngospasm (episodes lasting >10s) occurred in 12/38 (32%) of the patients anesthetized with sevoflurane 2.5%, vs 7/38 (18%) in those anesthetized with sevoflurane 4.7% (difference: OR 3.5; 95% CI [0.72-16.84], P=0.18). All other reflex responses (coughing, expiration reflexes, and spasmodic panting) were infrequent and were similar among the examined concentrations. Against our hypothesis, laryngospasm could still be observed in 18% of children under the higher concentration of sevoflurane (4.7%, ED95Intubation ).

Vagal Control of Breathing Pattern after Midcervical Contusion in Rats.

The present study was designed to establish a midcervical contusion model that can simulate long-term respiratory deficits, and investigate the breathing pattern during vagal-mediated respiratory reflexes following midcervical contusion. Moderate and severe (impactor height: 6.25 or 12.5 mm) contusion was induced at midline C3-4 spinal cord in adult Sprague-Dawley rats. The ventilatory behaviors of unanesthetized were evaluated by whole body plethysmography at 1 day and 1, 2, 4, and 8 weeks post-injury. The tidal volume was decreased and frequency was increased in contused animals compared with uninjured animals at the acute injury state. At 8 weeks post-injury, respiratory frequency was similar between groups; however, contused animals had lower tidal volume. The pulmonary chemoreflex induced by intrajugular capsaicin (1.5 μg/kg) injection and the Hering-Breuer inflation reflex induced by increasing positive end-expired pressure (9 cm H2O) were evoked in anesthetized animals at 3 days, or 2 or 8 weeks post-surgery. The result showed that capsaicin induced a significant prolongation of the expiratory duration in uninjured animals; however, this response was greatly attenuated in contused animals at 3 days post-injury. Increased positive end-expired pressure also caused an increase in the expiratory duration in uninjured and moderately contused animals; however, severely contused animals exhibited an attenuated response. At 2 and 8 weeks post-injury, both the pulmonary chemoreflex and the Hering-Breuer inflation reflex were similar between uninjured and contused animals. These data suggested that midcervical contusion can cause a long-term respiratory impairment and a transiently attenuation of vagal-mediated respiratory reflexes.

Comparison of Physiological Parameters and Anaesthesia Specific Observations during Isoflurane, Ketamine-Xylazine or Medetomidine-Midazolam-Fentanyl Anaesthesia in Male Guinea Pigs.

Guinea pigs (GPs) are difficult to anaesthetize successfully, the choices for anaesthesia are limited and physiological parameters are likely to be influenced substantially under anaesthesia. We implanted blood pressure radio-telemetry devices into 16 male GPs and subjected them to anaesthesia with ketamine-xylazine (KX), medetomidine-midazolam-fentanyl (MMF) or isoflurane (Iso, plus atropine premedication) in a randomized order with a 7 day interval between anaesthesias. Each anaesthesia lasted 40min, after which Iso was discontinued, MMF was fully antagonized with atipamezole-flumazenil-naloxone and KX was partially antagonized with atipamezole. Hemodynamics were recorded continuously for at least 240min after induction and the GPs were monitored for respiratory rate, reflex responses and specific observations until regaining of their righting reflex (RR). Blood for glucose testing was taken from the ear at 7.5, 20 and 40min during anaesthesia. Recovery time was short with MMF and Iso but long for KX. MMF induced only a transient blood pressure drop after antagonization, whereas Iso caused a marked hypotension during maintenance and KX led to moderate hypotension after antagonization. MMF and Iso produced tolerable heart rate changes, but KX led to long term post-anaesthetic bradycardia. Hypothermia occurred with all anaesthesias, but the GPs returned to normothermia the fastest under MMF, followed shortly by Iso. KX, however, caused a profound and prolonged hypothermia. The respiration was depressed with all anaesthesias, substantially with MMF (-41%) and KX (-52%) and severe during Iso maintenance (-71%). Blood glucose with MMF and KX increased throughout the anaesthesia, but the values remained within reference values with all anaesthetics. The reflex responses character and strength varied between the anaesthetics. In conclusion, MMF is the anaesthetic of choice and Iso may be used for short, non-painful procedures. We advise against the use of KX in GPs.

Neuropeptide Y in the brain of Euphlyctis cyanophlyctis tadpoles responds to hypoxic stress

Neuropeptide Y (NPY) has emerged as a novel peptide to antagonize various physiological consequences of stress within a mammalian brain. Hypoxia induced neuropeptide Y release in mammalian systems is well established. However, the possible role of NPY in regulating the effects of oxygen variation in lower vertebrates has not been investigated. We have studied the distribution and neuro-anatomical expression of NPY in the brain of Euphlyctis cyanophlyctis tadpoles, exposed to normal and reduced oxygen levels using immunohistochemistry. Animals exposed to hypoxia (<2mg/ml) exhibited a significant amplification of NPY-immunoreactivity throughout the brain. Increased NPY-ir perikarya appeared in all the sub-divisions of pallium, septum and preoptic area of telencephalon; suprachiasmatic nucleus, central and lateral thalamus, infundibulum and habenular regions of diencephalon; and nucleus cerebella and medulla of rhombencephalon. Most of these regions form the stress and anxiety regulating centers of a vertebrate brain and some of the parallel regions also respond to respiratory reflexes in mammals. Hence, our results suggest NPY induced modulation of hypoxia in Euphlyctis cyanophlyctis tadpoles.

Developmental cardiorespiratory physiology of the air-breathing tropical gar, Atractosteus tropicus.

The physiological transition to aerial breathing in larval air-breathing fishes is poorly understood. We investigated gill ventilation frequency (fG), heart rate (fH), and air breathing frequency (fAB) as a function of development, activity, hypoxia, and temperature in embryos/larvae from day (D) 2.5 to D30 posthatch of the tropical gar, Atractosteus tropicus, an obligate air breather. Gill ventilation at 28°C began at approximately D2, peaking at ∼75 beats/min on D5, before declining to ∼55 beats/min at D30. Heart beat began ∼36-48 h postfertilization and ∼1 day before hatching. fH peaked between D3 and D10 at ∼140 beats/min, remaining at this level through D30. Air breathing started very early at D2.5 to D3.5 at 1-2 breaths/h, increasing to ∼30 breaths/h at D15 and D30. Forced activity at all stages resulted in a rapid but brief increase in both fG and fH, (but not fAB), indicating that even in these early larval stages, reflex control existed over both ventilation and circulation prior to its increasing importance in older fishes. Acute progressive hypoxia increased fG in D2.5-D10 larvae, but decreased fG in older larvae (≥D15), possibly to prevent branchial O2 loss into surrounding water. Temperature sensitivity of fG and fH measured at 20°C, 25°C, 28°C and 38°C was largely independent of development, with a Q10 between 20°C and 38°C of ∼2.4 and ∼1.5 for fG and fH, respectively. The rapid onset of air breathing, coupled with both respiratory and cardiovascular reflexes as early as D2.5, indicates that larval A. tropicus develops "in the fast lane."

Time Domains of the Hypoxic Ventilatory Response and Their Molecular Basis.

Ventilatory responses to hypoxia vary widely depending on the pattern and length of hypoxic exposure. Acute, prolonged, or intermittent hypoxic episodes can increase or decrease breathing for seconds to years, both during the hypoxic stimulus, and also after its removal. These myriad effects are the result of a complicated web of molecular interactions that underlie plasticity in the respiratory control reflex circuits and ultimately control the physiology of breathing in hypoxia. Since the time domains of the physiological hypoxic ventilatory response (HVR) were identified, considerable research effort has gone toward elucidating the underlying molecular mechanisms that mediate these varied responses. This research has begun to describe complicated and plastic interactions in the relay circuits between the peripheral chemoreceptors and the ventilatory control circuits within the central nervous system. Intriguingly, many of these molecular pathways seem to share key components between the different time domains, suggesting that varied physiological HVRs are the result of specific modifications to overlapping pathways. This review highlights what has been discovered regarding the cell and molecular level control of the time domains of the HVR, and highlights key areas where further research is required. Understanding the molecular control of ventilation in hypoxia has important implications for basic physiology and is emerging as an important component of several clinical fields. © 2016 American Physiological Society. Compr Physiol 6:1345-1385, 2016.

Purinergic and Cholinergic Drugs Mediate Hyperventilation in Zebrafish: Evidence from a Novel Chemical Screen.

A rapid test to identify drugs that affect autonomic responses to hypoxia holds therapeutic and ecologic value. The zebrafish (Danio rerio) is a convenient animal model for investigating peripheral O2 chemoreceptors and respiratory reflexes in vertebrates; however, the neurotransmitters and receptors involved in this process are not adequately defined. The goals of the present study were to demonstrate purinergic and cholinergic control of the hyperventilatory response to hypoxia in zebrafish, and to develop a procedure for screening of neurochemicals that affect respiration. Zebrafish larvae were screened in multi-well plates for sensitivity to the cholinergic receptor agonist, nicotine, and antagonist, atropine; and to the purinergic receptor antagonists, suramin and A-317491. Nicotine increased ventilation frequency (fV) maximally at 100 μM (EC50 = 24.5 μM). Hypoxia elevated fV from 93.8 to 145.3 breaths min-1. Atropine reduced the hypoxic response only at 100 μM. Suramin and A-317491 maximally reduced fV at 50 μM (EC50 = 30.4 and 10.8 μM) and abolished the hyperventilatory response to hypoxia. Purinergic P2X3 receptors were identified in neurons and O2-chemosensory neuroepithelial cells of the gills using immunohistochemistry and confocal microscopy. These studies suggest a role for purinergic and nicotinic receptors in O2 sensing in fish and implicate ATP and acetylcholine in excitatory neurotransmission, as in the mammalian carotid body. We demonstrate a rapid approach for screening neuroactive chemicals in zebrafish with implications for respiratory medicine and carotid body disease in humans; as well as for preservation of aquatic ecosystems.

Orexin and Central Modulation of Cardiovascular and Respiratory Function.

Orexin makes an important contribution to the regulation of cardiorespiratory function. When injected centrally under anesthesia, orexin increases blood pressure, heart rate, sympathetic nerve activity, and the amplitude and frequency of respiration. This is consistent with the location of orexin neurons in the hypothalamus and the distribution of orexin terminals at all levels of the central autonomic and respiratory network. These cardiorespiratory responses are components of arousal and are necessary to allow the expression of motivated behaviors. Thus, orexin contributes to the cardiorespiratory response to acute stressors, especially those of a psychogenic nature. Consequently, upregulation of orexin signaling, whether it is spontaneous or environmentally induced, can increase blood pressure and lead to hypertension, as is the case for the spontaneously hypertensive rat and the hypertensive BPH/2J Schlager mouse. Blockade of orexin receptors will reduce blood pressure in these animals, which could be a new pharmacological approach for the treatment of some forms of hypertension. Orexin can also magnify the respiratory reflex to hypercapnia in order to maintain respiratory homeostasis, and this may be in part why it is upregulated during obstructive sleep apnea. In this pathological condition, blockade of orexin receptors would make the apnea worse. To summarize, orexin is an important modulator of cardiorespiratory function. Acting on orexin signaling may help in the treatment of some cardiovascular and respiratory disorders.

The Role of the Paratrigeminal Nucleus in Vagal Afferent Evoked Respiratory Reflexes: A Neuroanatomical and Functional Study in Guinea Pigs.

The respiratory tree receives sensory innervation from the jugular and nodose vagal sensory ganglia. Neurons of these ganglia are derived from embryologically distinct origins and as such demonstrate differing molecular, neurochemical and physiological phenotypes. Furthermore, whereas nodose afferent neurons project to the nucleus of the solitary tract (nTS), recent neuroanatomical studies in rats suggest that jugular neurons have their central terminations in the paratrigeminal nucleus (Pa5). In the present study we confirm that guinea pigs demonstrate a comparable distinction between the brainstem terminations of nodose and jugular ganglia afferents. Thus, microinjection of fluorescently conjugated cholera toxin B (CT-B) neural tracers into the caudal nTS and Pa5 resulted in highly specific retrograde labeling of neurons in the nodose and jugular ganglia, respectively. Whereas, nodose neurons more often expressed 160 KD neurofilament proteins and the alpha3 subunit of Na+/K+ ATPase, significantly more jugular neurons expressed the neuropeptides substance P (SP) and, especially, Calcitonin Gene-Related Peptide (CGRP). Indeed, terminal fibers in the Pa5 compared to the nTS were characterized by their significantly greater expression of CGRP, further supporting the notion that jugular afferents project to trigeminal-related brainstem regions. Electrical stimulation of the guinea pig larynx following selective surgical denervation of the nodose afferent innervation to the larynx (leaving intact the jugular innervation) resulted in stimulus dependent respiratory slowing and eventual apnea. This jugular ganglia neuron mediated response was unaffected by bilateral microinjections of the GABAA agonist muscimol into the nTS, but was abolished by muscimol injected into the Pa5. Taken together these data confirm that jugular and nodose vagal ganglia afferent neurons innervate distinct central circuits and support the notion that multiple peripheral and central pathways mediate sensory responses associated with airway irritations.

Localization of TRPV1 and P2X3 in unmyelinated and myelinated vagal afferents in the rat

The vagus nerve is dominated by afferent fibers that convey sensory information from the viscera to the brain. Most vagal afferents are unmyelinated, slow-conducting C-fibers, while a smaller portion are myelinated, fast-conducting A-fibers. Vagal afferents terminate in the nucleus tractus solitarius (NTS) in the dorsal brainstem and regulate autonomic and respiratory reflexes, as well as ascending pathways throughout the brain. Vagal afferents form glutamatergic excitatory synapses with postsynaptic NTS neurons that are modulated by a variety of channels. The organization of vagal afferents with regard to fiber type and channels is not well understood. In the present study, we used tract tracing methods to identify distinct populations of vagal afferents to determine if key channels are selectively localized to specific groups of afferent fibers. Vagal afferents were labeled with isolectin B4 (IB4) or cholera toxin B (CTb) to detect unmyelinated and myelinated afferents, respectively. We find that TRPV1 channels are preferentially found in unmyelinated vagal afferents identified with IB4, with almost half of all IB4 fibers showing co-localization with TRPV1. These results agree with prior electrophysiological findings. In contrast, we found that the ATP-sensitive channel P2X3 is found in a subset of both myelinated and unmyelinated vagal afferent fibers. Specifically, 18% of IB4 and 23% of CTb afferents contained P2X3. The majority of CTb-ir vagal afferents contained neither channel. Since neither channel was found in all vagal afferents, there are likely further degrees of heterogeneity in the modulation of vagal afferent sensory input to the NTS beyond fiber type.

Purinergic signalling mediates bidirectional crosstalk between chemoreceptor typeI and glial-like typeII cells of the rat carotid body.

Carotid body chemoreceptors are organized in clusters containing receptor type I and contiguous glial-like type II cells. While type I cells depolarize and release ATP during chemostimulation, the role of type II cells which express purinergic P2Y2 receptors (P2Y2Rs) and ATP-permeable pannexin-1 (Panx-1) channels, is unclear. Here, we show that in isolated rat chemoreceptor clusters, type I cell depolarization induced by hypoxia, hypercapnia, or high K(+) caused delayed intracellular Ca(2+) elevations (Δ[Ca(2+)]i) in nearby type II cells that were inhibited by the P2Y2R blocker suramin, or by the nucleoside hydrolase apyrase. Likewise, stimulation of P2Y2Rs on type II cells caused a delayed, secondary Δ[Ca(2+)]i in nearby type I cells that was inhibited by blockers of Panx-1 channels, adenosine A2A receptors and 5'-ectonucleotidase. We propose that reciprocal crosstalk between type I and type II cells contributes to sensory processing in the carotid body via purinergic signalling pathways. The mammalian carotid body (CB) is excited by blood-borne stimuli including hypoxia and acid hypercapnia, leading to respiratory and cardiovascular reflex responses. This chemosensory organ consists of innervated clusters of receptor type I cells, ensheathed by processes of adjacent glial-like type II cells. ATP is a major excitatory neurotransmitter released from type I cells and type II cells express purinergic P2Y2 receptors (P2Y2Rs), the activation of which leads to the opening of ATP-permeable, pannexin-1 (Panx-1) channels. While these properties support crosstalk between type I and type II cells during chemotransduction, direct evidence is lacking. To address this, we first exposed isolated rat chemoreceptor clusters to acute hypoxia, isohydric hypercapnia, or the depolarizing stimulus high K(+), and monitored intracellular [Ca(2+)] using Fura-2. As expected, these stimuli induced intracellular [Ca(2+)] elevations (Δ[Ca(2+)]i) in type I cells. Interestingly, however, there was often a delayed, secondary Δ[Ca(2+)]i in nearby type II cells that was reversibly inhibited by the P2Y2R antagonist suramin, or by the nucleoside hydrolase apyrase. By contrast, type II cell stimulation with the P2Y2R agonist uridine-5'-triphosphate (100 μm) often led to a delayed, secondary Δ[Ca(2+)]i response in nearby type I cells that was reversibly inhibited by the Panx-1 blocker carbenoxolone (5 μm). This Δ[Ca(2+)]i response was also strongly inhibited by blockers of either the adenosine A2A receptor (SCH 58261) or of the 5'-ectonucleotidase (AOPCP), suggesting it was due to adenosine arising from breakdown of ATP released through Panx-1 channels. Collectively, these data strongly suggest that purinergic signalling mechanisms mediate crosstalk between CB chemoreceptor and glial cells during chemotransduction.

Dorsal and ventral aspects of the most caudal medullary reticular formation have differential roles in modulation and formation of the respiratory motor pattern in rat.

The respiratory pattern generator of mammals is anatomically organized in lateral respiratory columns (LRCs) within the brainstem. LRC compartments serve specific functions in respiratory pattern and rhythm generation. While the caudal medullary reticular formation (cMRF) has respiratory functions reportedly related to the mediation of expulsive respiratory reflexes, it remains unclear whether neurons of the cMRF functionally belong to the LRC. In the present study we specifically investigated the respiratory functions of the cMRF. Tract tracing shows that the cMRF has substantial connectivity with key compartments of the LRC, particularly the parafacial respiratory group and the Kölliker-Fuse nuclei. These neurons have a loose topography and are located in the ventral and dorsal cMRF. Systematic mapping of the cMRF with glutamate stimulation revealed potent respiratory modulation of the respiratory motor pattern from both dorsal and ventral injection sites. Pharmacological inhibition of the cMRF with the GABA-receptor agonist isoguvacine produced significant and robust changes to the baseline respiratory motor pattern (decreased laryngeal post-inspiratory and abdominal expiratory motor activity, delayed inspiratory off-switch and increased respiratory frequency) after dorsal cMRF injection, while ventral injections had no effect. The present data indicate that the ventral cMRF is not an integral part of the respiratory pattern generator and merely serves as a relay for sensory and/or higher command-related modulation of respiration. On the contrary, the dorsal aspect of the cMRF clearly has a functional role in respiratory pattern formation. These findings revive the largely abandoned concept of a dorsal respiratory group that contributes to the generation of the respiratory motor pattern.

Neuro-glial communication in NTS after hypoxia

Humans ascending to high altitudes are submitted to sustained hypoxia (SH), which activates the peripheral chemoreflex with several autonomic and respiratory responses. In this study we analyzed initially the effect of short-term SH (24 hours, FIO210%) on the processing of cardiovascular and respiratory reflexes using an in situ preparation of rats. SH increased both the sympatho-inhibitory and bradycardiac components of baroreflex and the sympathetic and respiratory responses of peripheral chemoreflex. The electrophysiological properties and synaptic transmission in the nucleus tractus solitarius (NTS) neurons, the first synaptic station of afferents of baro- and chemoreflexes, were evaluated using brainstem slices and whole-cell patch clamp. The 2nd-order NTS neurons were identified by previous application of fluorescent tracer onto carotid body for chemoreceptor afferents or onto aortic depressor nerve for baroreceptor afferents. SH increased the intrinsic excitability of NTS neurons. Delayed excitation, caused by A-type potassium current (IKA), was observed in most of NTS neurons from control rats. The IKA amplitude was higher in identified 2nd-order NTS neurons from control than in SH rats. SH also blunted the astrocytic inhibition of IKA in NTS neurons and increased the synaptic transmission in response to afferent fibers stimulation. The frequency of spontaneous excitatory currents was also increased in neurons from SH rats, indicating that SH increased the neurotransmission by pre-synaptic mechanisms. We conclude that short-term SH changed the glia-neuron interaction, increasing the excitability and excitatory transmission of NTS neurons, which may contribute to the observed increase in the reflex sensitivity of baro- and chemoreflex in in situ preparation. Financial support: FAPESP and CNPq.

Sex-specific response to hypoxia in a reduced brainstem preparation from Xenopus laevis

Respiratory reflexes and tolerance to hypoxia show significant sexual dimorphism. However, the data supporting this notion originates exclusively from mammals. To determine whether this concept is limited to this group of vertebrates, we examined the sex-specific response to acute hypoxia in an adult reduced brainstem preparation from Xenopus laevis. Within the first 5min of exposure to hypoxic aCSF (98% N2/2% CO2), recordings of respiratory-related activity show a stronger increase in fictive breathing frequency in males than females. This initial response was followed by a decrease in respiratory-related activity; this depression occurred 6min sooner in males than females. These results represent new evidences of sexual dimorphism in respiratory control in amphibians and provide potential insight in understanding the homology with other groups of vertebrates, including mammals.

Development of a Clinically Applicable Protocol for Assessment of Hypoxic Response Through Measurement of the Endogenous Gasotransmitter Hydrogen Sulfide in Human Plasma.

Gasotransmitters are endogenously made, biologically active gases with unique physiological properties. In addition to participation in the hypoxic respiratory reflex of the carotid body, the gasotransmitter hydrogen sulfide (H(2)S) is thought to play a role in more localized vasodilatory hypoxic tissue responses. This pilot project describes a methodology suitable to the clinical environment that allows for H(2)S gas capture in human plasma utilizing the fluorescent trapping agent dansyl azide. Under an IRB-approved pilot project, 10 healthy male volunteers were spontaneously ventilated on room air, hypoxic (15% oxygen, 85% nitrogen), and hyperoxic (100%) gas mixtures through a nonrebreather system. Venous whole-blood samples were collected at both internal jugular and antecubital sites following 7 minutes of exposure to the tested oxygen environments. Resultant plasma aliquots were treated with dansyl azide and submitted to fluorescence reading (excitation 340 nm, emission 517 nm). Compiled mean data from volunteer plasma samples demonstrated statistically significant findings (P<0.05) in measurement of increased fluorescent intensity between those samples collected under mildly hypoxic conditions compared with normoxic and hyperoxic samples submitted to the same laboratory criteria. To study the role of H(2)S as a marker of hypoxic response in humans, a reliable, robust, and safe protocol amenable to standard hospital laboratory procedures is needed. Through modification to methodologies described in the biochemistry literature, this pilot project demonstrates the feasibility of utilizing a fluorescent H2S gas trapping agent for assessment of hypoxic response in humans within the confines of a typical clinical collection and analysis environment.