Melanin-concentrating hormone regulates the hypercapnic chemoreflex by acting in the lateral hypothalamic area.

NEW FINDINGS: What is the central question of this study? Melanin-concentrating hormone (MCH) suppresses the hypercapnic chemoreflex: what is the mechanism by which this effect is produced? What is the main finding and its importance? MCH acting in the lateral hypothalamic area but not in the locus coeruleus in rats, in the light period, attenuates the hypercapnic chemoreflex. The data provide new insight into the role of MCH in the modulation of the hypercapnic ventilatory response. ABSTRACT: Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide involved in a broad range of homeostatic functions including regulation of the hypercapnic chemoreflex. We evaluated whether MCH modulates the hypercapnic ventilatory response by acting in the lateral hypothalamic area (LHA) and/or in the locus coeruleus (LC). Here, we measured pulmonary ventilation ( V ̇ E ${\dot V_{\rm{E}}}$ ), body temperature, electroencephalogram (EEG) and electromyogram (EMG) of unanaesthetized adult male Wistar rats before and after microinjection of MCH (0.4 mM) or MCH receptor 1 (MCH1-R) antagonist (SNAP-94847; 63 mM) into the LHA and LC, in room air and 7% CO2 conditions during wakefulness and sleep in the dark and light periods. MCH intra-LHA caused a decreased CO2 ventilatory response during wakefulness and sleep in the light period, while SNAP-94847 intra-LHA increased this response, during wakefulness in the light period. In the LC, MCH or the MCH1-R antagonist caused no change in the hypercapnic ventilatory response. Our results suggest that MCH, in the LHA, exerts an inhibitory modulation of the hypercapnic ventilatory response during the light-inactive period in rats.

Lesion of Serotonergic Afferents to the Retrotrapezoid Nucleus Impairs the Tachypneic Response to Hypercapnia in Unanesthetized Animals.

Hypercapnia promotes an increase in pulmonary ventilation due to the stimulation of brainstem chemosensory cells that are connected to the respiratory network. Among these cells are the raphe serotonergic neurons which widely send projections to distinct central respiratory compartments. Nevertheless, the physiological role of specific raphe serotonergic projections to other chemosensitive sites on the emergence of hypercapnia ventilatory response in vivo still remains to be elucidated. Here we investigated whether the ventilatory response to hypercapnia requires serotonergic inputs to the chemosensitive cells of the retrotrapezoid nucleus (RTN) in the ventrolateral medulla. To test this, pulmonary ventilation was evaluated under baseline conditions and during hypercapnia (7% CO2) in unanesthetized juvenile Holtzman rats (60-90 g) that received bilateral microinjections of either vehicle (control) or anti-SERT-SAP (0.1 mM, 10 pmol/100 nl) toxin in the RTN to retrogradely destroy serotonergic afferents to this region. Fifteen days after microinjections, baseline ventilation was not different between anti-SERT-SAP (n = 8) and control animals (n = 9). In contrast, the ablation of RTN-projecting serotonergic neurons markedly attenuated the hypercapnia-induced increase in respiratory frequency which was correlated with reduced numbers of serotonergic neurons in the raphe obscurus and magnus, but not in the raphe pallidus. The increase in tidal volume during hypercapnia was not significantly affected by anti-SERT-SAP microinjections in the RTN. Our data indicate that serotoninergic neurons that send projections to the RTN region are required for the processing of ventilatory reflex response during exposure to high CO2 in unanesthetized conditions.

Differential modulation of active expiration during hypercapnia by the medullary raphe in unanesthetized rats.

Active expiration represents an important mechanism to improve ventilation in conditions of augmented ventilatory demand, such as hypercapnia. While a rostral ventromedullary region, the parafacial respiratory group (pFRG), has been identified as a conditional expiratory oscillator, little is known about how central chemosensitive sites contribute to modulate active expiration under hypercapnia. In this study, we investigated the influence of the medullary raphe in the emergence of phasic expiratory abdominal activity during hypercapnia in unanesthetized adult male rats, in a state-dependent manner. To do so, reverse microdialysis of muscimol (GABAA receptor agonist, 1 mM) or 8-OH-DPAT (5-HT1A agonist, 1 mM) was applied in the MR during sleep and wakefulness periods, both in normocapnic (room air) and hypercapnic conditions (7% CO2). Electromyography (EMG) of diaphragm and abdominal muscles was performed to measure inspiratory and expiratory motor outputs. We found that active expiration did not occur in room air exposure during wakefulness or sleep. However, hypercapnia did recruit active expiration, and differential effects were observed with the drug dialyses in the medullary raphe. Muscimol increased the diaphragm inspiratory motor output and also increased the amplitude and frequency of abdominal expiratory rhythmic activity during hypercapnia in wakefulness periods. On the other hand, the microdialysis of 8-OH-DPAT attenuated hypercapnia-induced active expiration in a state-dependent manner. Our data suggest that the medullary raphe can either inhibit or potentiate respiratory motor activity during hypercapnia, and the balance of these inhibitory or excitatory outputs may determine the expression of active expiration.

Respiratory control of acid-base status in lungfish.

The acid-base status is a tightly regulated physiological process, resulting from a balance of ions in the organism relevant to acid-base. The efficiency of the regulatory systems importantly determines the compensatory pH changes for a given disturb. Vertebrates minimize (or compensate) an acid-base disturb by general processes, which include ion transfer and/or PCO2 changes. Acid-base adjustment in fish is predominantly achieved by branchial exchange of acid-base relevant ions with correlated change in plasma HCO3- levels. Conversely, land vertebrates change blood PCO2 through ventilatory process and hence respiratory control of acid-base regulation plays an important role as a compensatory mechanism. Lungfishes (Dipnoi) have central position on vertebrate's evolution being considered as the sister group to the tetrapods. With an aquatic life mode, lungfish share similarities of respiratory function with tetrapods. This article reviews evidence showing that lungfish's respiratory system regulates acid-base status, like terrestrial ectothermic vertebrates. In the South American lungfish, Lepidosiren paradoxa, the presence of central CO2/pH chemoreceptors was unequivocally described. Also, the blood PCO2 and acid-base status are typical of a terrestrial vertebrate. These aspects are discussed under different environmental conditions that require respiratory acid-base adjustments, such as, exposure to hypercarbia, hypoxia, high temperature and aestivation. Interesting questions regarding the location and cell phenotype of CO2/pH central and peripheral chemoreceptors remain an open field to be explored in lungfish.

ATP in the lateral hypothalamus/perifornical area enhances the CO2 chemoreflex control of breathing.

NEW FINDINGS: What is the central question of this study? ATP is known to modulate the chemosensitivity of some brain areas. However, whether the ATP contributes specifically to the mechanism of chemoreception in the lateral hypothalamus/perifornical area (LH/PFA) remains to be determined. What is the main finding and its importance? ATP, acting on the LH/PFA, enhances the hypercapnic ventilatory response in rats during wakefulness, in the dark period. Our results highlight the importance of ATP as a modulator of central chemoreception and provide new insight regarding the mechanisms involved in LH/PFA chemosensitivity and the sleep-wake differences in the CO2 /H+ -dependent drive to breathe. ABSTRACT: The lateral hypothalamus/perifornical area (LH/PFA) is a central chemoreceptor site, which acts in an arousal state-dependent manner. It has been shown that purinergic signalling through ATP influences the CO2 /H+ responsiveness of other chemosensitive regions, but it is unknown whether ATP is also involved in the mechanisms that underlie LH/PFA chemoreception. Here, we studied the effects of microdialysis of a P2X-receptor agonist [α,ß-methylene ATP (α,ß-meATP), 10 mm] and a non-selective P2-receptor antagonist [pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), 1 mm] into the LH/PFA of conscious rats on ventilation in room air and in 7% CO2 . In the dark (active) phase, but not in the light, microdialysis of α,ß-meATP caused an augmented hypercapnic ventilatory response during wakefulness, but not during non-REM sleep (P < 0.001). PPADS caused no change in CO2 ventilatory responses in either the dark period or the light period. Our data suggest that ATP in LH/PFA contributes to the hypercapnic ventilatory response in conscious rats during wakefulness in the dark phase of the diurnal cycle.

Correlation between neuroanatomical and functional respiratory changes observed in an experimental model of Parkinson's disease.

NEW FINDINGS: What is the central question of this study? What is the relationship between neuroanatomical and functional respiratory changes in an experimental model of Parkinson's disease? What is the main finding and its importance? Sixty days after induction of Parkinson's disease in a rat model, there are decreases in baseline breathing and in the number of neurons, density of the neurokinin-1 receptor and density of astrocytes in the ventrolateral respiratory region. These results provide the first evidence that neuroanatomical changes occur before functional respiratory deficits in a Parkinson's disease model and that there is a positive correlation between those sets of changes. The neuroanatomical changes impair respiratory activity and are presumably a major cause of the respiratory problems observed in Parkinson's disease. ABSTRACT: We showed previously that 60 days after the induction of Parkinson's disease (PD) in a rat model, there are decreases in baseline breathing and in the number of phox2b-expressing neurons of the retrotrapezoid nucleus (RTN) and nucleus of the solitary tract (NTS), as well as a reduction in the density of the neurokinin-1 receptor (NK1r) in the pre-Bötzinger complex (preBötC) and rostral ventrolateral respiratory group (rVRG). Here, our aim was to evaluate the correlation between neuroanatomical and functional respiratory changes in an experimental model of PD. Male Wistar rats with bilateral injections of 6-hydroxydopamine (6-OHDA, 24 µg µl-1 ) or vehicle into the striatum had respiratory parameters assessed by whole-body plethysmography 1 day before and 30, 40 or 60 days after the ablation. From the 30th day after the ablation, we observed a reduction in the number of phox2b neurons in the RTN and NTS and a reduction in the density of astrocytes in the rVRG. At 40 days after the ablation, we observed decreases in the density of NK1r in the preBötC and rVRG and of astrocytes in the RTN region. At 60 days, we observed a reduction in the density of astrocytes in the NTS and preBötC regions. The functional data showed changes in the resting and hypercapnia-induced respiratory rates and tidal volume from days 40-60 after injury. Our data suggest that the neuroanatomical changes impair respiratory activity and are presumably a major cause of the respiratory problems observed in PD.

The Onset of the Fetal Respiratory Rhythm: An Emergent Property Triggered by Chemosensory Drive?

The mechanisms responsible for the onset of respiratory activity during fetal life are unknown. The onset of respiratory rhythm may be a consequence of the genetic program of each of the constituents of the respiratory network, so they start to interact and generate respiratory cycles when reaching a certain degree of maturation. Alternatively, generation of cycles might require the contribution of recently formed sensory inputs that will trigger oscillatory activity in the nascent respiratory neural network. If this hypothesis is true, then sensory input to the respiratory generator must be already formed and become functional before the onset of fetal respiration. In this review, we evaluate the timing of the onset of the respiratory rhythm in comparison to the appearance of receptors, neurotransmitter machinery, and afferent projections provided by two central chemoreceptive nuclei, the raphe and locus coeruleus nuclei.

Role of Astrocytes in Central Respiratory Chemoreception.

Astrocytes perform various homeostatic functions in the nervous system beyond that of a supportive or metabolic role for neurons. A growing body of evidence indicates that astrocytes are crucial for central respiratory chemoreception. This review presents a classical overview of respiratory central chemoreception and the new evidence for astrocytes as brainstem sensors in the respiratory response to hypercapnia. We review properties of astrocytes for chemosensory function and for modulation of the respiratory network. We propose that astrocytes not only mediate between CO2/H+ levels and motor responses, but they also allow for two emergent functions: (1) Amplifying the responses of intrinsic chemosensitive neurons through feedforward signaling via gliotransmitters and; (2) Recruiting non-intrinsically chemosensitive cells thanks to volume spreading of signals (calcium waves and gliotransmitters) to regions distant from the CO2/H+ sensitive domains. Thus, astrocytes may both increase the intensity of the neuron responses at the chemosensitive sites and recruit of a greater number of respiratory neurons to participate in the response to hypercapnia.

Blunted Hypercapnic Respiratory Drive Response in Subjects With Late-Onset Pompe Disease.

BACKGROUND: Patients with late-onset Pompe disease develop progressive hypercapnic respiratory failure that can be disproportionate to the respiratory muscle compromise and/or thoracic restriction. Although recent studies have reported the presence of a blunted hypercapnic respiratory response in some subjects with neuromuscular disorders and chronic hypercapnia, no study has evaluated the integrity of the respiratory drive in subjects with late-onset Pompe disease. Thus, we endeavor to determine the CO2 rebreathing response in subjects with late-onset Pompe disease. METHODS: Respiratory muscle strength was assessed by measuring the maximum inspiratory pressure, and the maximum expiratory pressure. The maximum inspiratory pressure reflects the strength of the diaphragm and other inspiratory muscles, whereas the maximum expiratory pressure reflects the strength of the abdominal muscles and other expiratory muscles. We studied the hypercapnic drive response (measured as the ratio of the change in airway-occlusion pressure 0.1 s after the start of inspiration and end-tidal PCO2 in 13 subjects with late-onset Pompe disease and 51 healthy controls. RESULTS: Overall inspiratory muscle strength was within normal limits or slightly diminished in the late-onset Pompe disease group. Five subjects (38.5%) were chronically hypercapnic, and 9 (69.2%) had an increased breath-holding time. Compared with controls, the change in airway-occlusion pressure 0.1 s/change in end-tidal CO2 pressure slope (hypercapnic respiratory drive) was lower in the late-onset Pompe disease group (median 0.050 [interquartile range 0.027-0.118] vs 0.183 [0.153-0.233], P < .001). Nine subjects (69.2%) had a blunted change in airway-occlusion pressure 0.1 s/change in end-tidal carbon dioxide pressure slope. CONCLUSIONS: Subjects with late-onset Pompe disease had an impaired hypercapnic respiratory drive response. The clinical impact of this phenomenon in this subject subset deserves further investigation.

The Alteration of Neonatal Raphe Neurons by Prenatal-Perinatal Nicotine. Meaning for Sudden Infant Death Syndrome.

Nicotine may link maternal cigarette smoking with respiratory dysfunctions in sudden infant death syndrome (SIDS). Prenatal-perinatal nicotine exposure blunts ventilatory responses to hypercapnia and reduces central respiratory chemoreception in mouse neonates at Postnatal Days 0 (P0) to P3. This suggests that raphe neurons, which are altered in SIDS and contribute to central respiratory chemoreception, may be affected by nicotine. We therefore investigated whether prenatal-perinatal nicotine exposure affects the activity, electrical properties, and chemosensitivity of raphe obscurus (ROb) neurons in mouse neonates. Osmotic minipumps, implanted subcutaneously in 5- to 7-day-pregnant CF1 mice, delivered nicotine bitartrate (60 mg kg(-1) d(-1)) or saline (control) for up to 28 days. In neonates, ventilation was recorded by head-out plethysmography, c-Fos (neuronal activity marker), or serotonin autoreceptors (5HT1AR) were immunodetected using light microscopy, and patch-clamp recordings were made from raphe neurons in brainstem slices under normocarbia and hypercarbia. Prenatal-perinatal nicotine exposure decreased the hypercarbia-induced ventilatory responses at P1-P5, reduced both the number of c-Fos-positive ROb neurons during eucapnic normoxia at P1-P3 and their hypercapnia-induced recruitment at P3, increased 5HT1AR immunolabeling of ROb neurons at P3-P5, and reduced the spontaneous firing frequency of ROb neurons at P3 without affecting their CO2 sensitivity or their passive and active electrical properties. These findings reveal that prenatal-perinatal nicotine reduces the activity of neonatal ROb neurons, likely as a consequence of increased expression of 5HT1ARs. This hypoactivity may change the functional state of the respiratory neural network leading to breathing vulnerability and chemosensory failure as seen in SIDS.

Activation of the brain melanocortin system is required for leptin-induced modulation of chemorespiratory function.

UNLABELLED: Melanocortin receptors (MC3/4R) mediate most of the metabolic and cardiovascular actions of leptin. AIM: Here, we tested if MC4R also contributes to leptin's effects on respiratory function. METHODS: After control measurements, male Holtzman rats received daily microinjections of leptin, SHU9119 (MC3/4R antagonist) or SHU9119 combined with leptin infused into the brain lateral ventricle for 7 days. On the 6th day of treatment, tidal volume (VT ), respiratory frequency (fR ) and pulmonary ventilation (VE ) were measured by whole-body plethysmography during normocapnia or hypercapnia (7% CO2 ). Baseline mean arterial pressure (MAP), heart rate (HR) and metabolic rate were also measured. VE , VT and fR were also measured in mice with leptin receptor deletion in the entire central nervous system (LepR/Nestin-cre) or only in proopiomelanocortin neurones (LepR/POMC-cre) and in MC4R knockout (MC4R(-/-) ) and wild-type mice. RESULTS: Leptin (5 µg day(-1) ) reduced body weight (~17%) and increased ventilatory response to hypercapnia, whereas SHU9119 (0.6 nmol day(-1) ) increased body weight (~18%) and reduced ventilatory responses compared with control-PBS group (Lep: 2119 ± 90 mL min(-1)  kg(-1) and SHU9119: 997 ± 67 mL min(-1)  kg(-1) , vs. PBS: 1379 ± 91 mL min(-1)  kg(-1) ). MAP increased after leptin treatment (130 ± 2 mmHg) compared to PBS (106 ± 3 mmHg) or SHU9119 alone (109 ± 3 mmHg). SHU9119 prevented the effects of leptin on body weight, MAP (102 ± 3 mmHg) and ventilatory response to hypercapnia (1391 ± 137 mL min(-1)  kg(-1) ). The ventilatory response to hypercapnia was attenuated in the LepR/Nestin-cre, LepR/POMC-cre and MC4R(-/-) mice. CONCLUSION: These results suggest that central MC4R mediate the effects of leptin on respiratory response to hypercapnia.

Central hydrogen sulphide mediates ventilatory responses to hypercapnia in adult conscious rats.

AIM: Hydrogen sulphide (H2S) is endogenously produced and plays an important role as a modulator of neuronal functions; however, its modulatory role in the central CO2 chemoreception is unknown. The aim of the present study was to assess the role of endogenously produced H2S in the ventilatory response to hypercapnia in adult conscious rats. METHODS: Cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) inhibitors (aminooxyacetate: AOA and propargylglycine: PAG respectively) and a H2S donor (sodium sulphide: Na2S) were microinjected into the fourth ventricle (4V). Ventilation (V̇(E)), oxygen consumption (V̇O2) and body temperature were recorded before (room air) and during a 30-min CO2 exposure (hypercapnia, 7% CO2). Endogenous H2S levels were measured in the nucleus tractus solitarius (NTS). RESULTS: Microinjection of Na2S (H2S donor), AOA (CBS inhibitor) or PAG (CSE inhibitor) did not affect baseline of the measured variables compared to control group (vehicle). In all experimental groups, hypercapnia elicited an increase in V̇(E). However, AOA microinjection, but not PAG, attenuated the ventilatory response to hypercapnia (P < 0.05), whereas Na2S elicited a slight, not significant, enhancement. Moreover, endogenous H2S levels were found higher in the NTS after hypercapnia (P < 0.05) compared to room air (normoxia) condition. CONCLUSION: There are a few reports on the role of gaseous transmitters in the control of breathing. Importantly, the present data suggest that endogenous H2S via the CBS-H2S pathway mediates the ventilatory response to hypercapnia playing an excitatory role.

Leptin into the ventrolateral medulla facilitates chemorespiratory response in leptin-deficient (ob/ob) mice.

AIM: Leptin, an adipocyte-derived hormone, is suggested to participate in the central control of breathing. We hypothesized that leptin may facilitate ventilatory responses to chemoreflex activation by acting on respiratory nuclei of the ventrolateral medulla. The baseline ventilation and the ventilatory responses to CO2 were evaluated before and after daily injections of leptin into the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) for 3 days in obese leptin-deficient (ob/ob) mice. METHODS: Male ob/ob mice (40-45 g, n = 7 per group) received daily microinjections of vehicle or leptin (1 µg per 100 nL) for 3 days into the RTN/pFRG. Respiratory responses to CO2 were measured by whole-body plethysmography. RESULTS: Unilateral microinjection of leptin into the RTN/pFRG in ob/ob mice increased baseline ventilation (VE ) from 1447 ± 96 to 2405 ± 174 mL min(-1) kg(-1) by increasing tidal volume (VT ) from 6.4 ± 0.4 to 9.1 ± 0.8 mL kg(-1) (P < 0.05). Leptin also enhanced ventilatory responses to 7% CO2 (Δ = 2172 ± 218 mL min(-1) kg(-1) , vs. control: Δ = 1255 ± 105 mL min(-1) kg(-1) ), which was also due to increased VT (Δ = 4.71 ± 0.51 mL kg(-1) , vs. control: Δ = 2.27 ± 0.20 mL kg(-1) ), without changes in respiratory frequency. Leptin treatment into the RTN/pFRG or into the surrounding areas decreased food intake (83 and 70%, respectively), without significantly changing body weight. CONCLUSION: The present results suggest that leptin acting in the respiratory nuclei of the ventrolateral medulla improves baseline VE and VT and facilitates respiratory responses to hypercapnia in ob/ob mice.