Caudal Ventral Respiratory Group Research Articles

Axonal projection of the medullary expiratory neurons in the feline thoracic spinal cord

Expiratory neurons in the caudal ventral respiratory group extend descending axons to the lumbar and sacral spinal cord, and they possess axon collaterals, the distribution of which has been well-documented. Likewise, these expiratory neurons extend axons to the thoracic spinal cord and innervate thoracic expiratory motoneurons. These axons also give rise to collaterals, and their distribution may influence the strength of synaptic connectivity between the axons and the thoracic expiratory motoneurons. We investigated the distribution of axon collaterals in the thoracic spinal cord using a microstimulation technique. This study was performed on cats; one cat was used to make an anatomical atlas and six were used in the experiment. Extracellular spikes of expiratory neurons were recorded in artificially ventilated cats. The thoracic spinal gray matter was microstimulated from dorsal to ventral sites at 100-μm intervals using a glass-insulated tungsten microelectrode with a current of 150–250 μA. The stimulation tracks were made at 1 mm intervals along the spinal cord in segments Th9 to Th13, and the effective stimulating sites of antidromic activation in axon collaterals were systematically mapped. The effective stimulating sites in the contralateral thoracic spinal cord with expiratory neurons in the caudal ventral respiratory group (cVRG) occupied 14.4% of the total length of the thoracic spinal cord examined. The mean percentage of effective stimulating tracks per unit was 18.6 ± 4.4%. The distribution of axon collaterals of expiratory neurons in the feline thoracic spinal cord indeed resembled that reported in the upper lumbar spinal cord. We propose that a single medullary expiratory neuron exerts excitatory effects across multiple segments of the thoracic spinal cord via its collaterals.

Efferent network of Neuromedin B neurons in the retrotrapezoid nucleus in mice

In the parafacial region of the medulla, the peptide Neuromedin B (NMB) is expressed in retrotrapezoid nucleus (RTN) neurons that are proposed to be central respiratory chemoreceptors. The efferent network of RTN Nmb neurons has been established in rats, but has not been examined in mice in detail. To map the downstream connections of RTN Nmb neurons we performed genetically-targeted tracing using novel Nmb-Cre knock-in mice. Adult Nmb-Cre mice were injected in the RTN unilaterally with a Cre-dependent AAV producing expression of red fluorescent protein (AAV-Cre ON -mScarlet). Expression of Nmb was identified by fluorescent in situ hybridization (FISH). Injections of AAV-Cre ON -mScarlet in the RTN (N=3) transduced 58 ± 11 % of Nmb-expressing neurons with a selectivity for Nmb neurons of 98 ± 1 %. Intensely labelled axons and terminals were observed in regions overlapping with the medullary and pontine respiratory network. We observe an intra-RTN network of fibers with close appositions between RTN Nmb neurons, as well as moderate projections to areas dorso-medial to the facial nucleus and medially adjacent to the locus coeruleus. There was a modest contralateral projection to the medullary respiratory network, but none to the pontine respiratory network. No labelled fibers were found rostral of the parabrachial nucleus. Anterograde tracing of RTN Nmb neurons was complemented using retrograde transport of Cre-recombinase (AAVrg-Cre) in R26-loxSTOPlox-L10-GFP mice (eGFP-reporter) mice combined with labelling of transcript for Nmb by FISH. Small volumes (3-6 nL) of AAVrg-Cre were unilaterally injected in the Pre-Bötzinger complex (PreBöt), lateral parabrachial nucleus (LPB), caudal ventral respiratory group (cVRG), and the nucleus of the solitary tract (NTS). We determined the proportion of total RTN Nmb neurons that were retro-labelled from each target (PreBöt- 75 ± 3%, n=3; LPB- 65 ± 3%, n=4; cVRG- 54 ± 6%, n=3; NTS- 38 ± 3%, n=3). This result suggests that RTN neurons have a high degree of collateralization within the medullary and pontine respiratory network. Injections of AAVrg-Cre in the PreBöt labelled 16 ± 11% of RTN Nmb neurons contralateral to the injection site. Collectively, these neuroanatomical observations show that the efferent network of RTN Nmb neurons broadly innervate and selectively the central respiratory network in alignment with prior descriptions in rat. Furthermore, we provide evidence for novel connectivity between RTN Nmb neurons. This anatomy supports the idea that RTN neurons generate a coordinated widespread excitation of the respiratory rhythm and pattern generating network in order to promote alveolar ventilation. Funding from NIH-HLBI #HL148004 This is the full abstract presented at the American Physiology Summit 2023 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.

Sneezing reflex is mediated by a peptidergic pathway from nose to brainstem

Sneezing is a vital respiratory reflex frequently associated with allergic rhinitis and viral respiratory infections. However, its neural circuit remains largely unknown. A sneeze-evoking region was discovered in both cat and human brainstems, corresponding anatomically to the central recipient zone of nasal sensory neurons. Therefore, we hypothesized that a neuronal population postsynaptic to nasal sensory neurons mediates sneezing in this region. By screening major presynaptic neurotransmitters/neuropeptides released by nasal sensory neurons, we found that neuromedin B (NMB) peptide is essential for signaling sneezing. Ablation of NMB-sensitive postsynaptic neurons in the sneeze-evoking region or deficiency in NMB receptor abolished the sneezing reflex. Remarkably, NMB-sensitive neurons further project to the caudal ventral respiratory group (cVRG). Chemical activation of NMB-sensitive neurons elicits action potentials in cVRG neurons and leads to sneezing behavior. Our study delineates a peptidergic pathway mediating sneezing, providing molecular insights into the sneezing reflex arc.

Effect of Positive Allosteric Modulation of Dopamine D2 Receptors on Respiration in Mouse Models of Rett Syndrome

Rett syndrome (RTT) is an autism spectrum disorder caused by loss-of-function mutations in the methyl-CPG-binding protein 2 (Mecp2) gene. Frequent apneas and respiratory disturbances are prevalent in Rett syndrome, and also occur in rodent models of the disorder, including Mecp2Bird and Mecp2R168X mice. Prior research has demonstrated that sarizotan, a serotonin 5-HT1a and dopamine D2-like receptor agonist, reduces the incidence of apneas and irregular breathing in mouse models of RTT. However, the contribution of D2 receptors in correcting these respiratory disturbances remains untested. Given that D2 receptors inhibit expiratory neurons in the parabrachial complex and the caudal ventral respiratory group, we hypothesized that compounds targeting the D2 receptor would correct apnea and irregular breathing in mouse models of RTT by enhancing dopamine responses in expiratory neurons. PAOPA, the dopamine D2 receptor positive allosteric modulator, and quinpirole, a dopamine D2 receptor agonist, were used in conjunction with whole-body plethysmography to evaluate whether activation of D2 receptors is sufficient to restore a normal respiratory phenotype in Mecp2Bird and Mecp2R168X mice. There were no significant differences in apnea incidence or irregularity score between saline and PAOPA treated RTT mice. PAOPA also had no effect on the ventilatory response to hypercapnia (7 % CO2). These results suggest that dopamine D2 receptor activity may not be an ideal candidate in the correction of respiratory abnormalities in Rett syndrome.

Differential Contribution of the Retrotrapezoid Nucleus and C1 Neurons to Active Expiration and Arousal in Rats.

Collectively, the retrotrapezoid nucleus (RTN) and adjacent C1 neurons regulate breathing, circulation and the state of vigilance, but previous methods to manipulate the activity of these neurons have been insufficiently selective to parse out their relative roles. We hypothesize that RTN and C1 neurons regulate distinct aspects of breathing (e.g., frequency, amplitude, active expiration, sighing) and differ in their ability to produce arousal from sleep. Here we use optogenetics and a combination of viral vectors in adult male and female Th-Cre rats to transduce selectively RTN (Phox2b+/Nmb+) or C1 neurons (Phox2b+/Th+) with Channelrhodopsin-2. RTN photostimulation modestly increased the probability of arousal. RTN stimulation robustly increased breathing frequency and amplitude; it also triggered strong active expiration but not sighs. Consistent with these responses, RTN innervates the entire pontomedullary respiratory network, including expiratory premotor neurons in the caudal ventral respiratory group, but RTN has very limited projections to brainstem regions that regulate arousal (locus ceruleus, CGRP+ parabrachial neurons). C1 neuron stimulation produced robust arousals and similar increases in breathing frequency and amplitude compared with RTN stimulation, but sighs were elicited and active expiration was absent. Unlike RTN, C1 neurons innervate the locus ceruleus, CGRP+ processes within the parabrachial complex, and lack projections to caudal ventral respiratory group. In sum, stimulating C1 or RTN activates breathing robustly, but only RTN neuron stimulation produces active expiration, consistent with their role as central respiratory chemoreceptors. Conversely, C1 stimulation strongly stimulates ascending arousal systems and sighs, consistent with their postulated role in acute stress responses.SIGNIFICANCE STATEMENT The C1 neurons and the retrotrapezoid nucleus (RTN) reside in the rostral ventrolateral medulla. Both regulate breathing and the cardiovascular system but in ways that are unclear because of technical limitations (anesthesia, nonselective neuronal actuators). Using optogenetics in unanesthetized rats, we found that selective stimulation of either RTN or C1 neurons activates breathing. However, only RTN triggers active expiration, presumably because RTN, unlike C1, has direct excitatory projections to abdominal premotor neurons. The arousal potential of the C1 neurons is far greater than that of the RTN, however, consistent with C1's projections to brainstem wake-promoting structures. In short, C1 neurons orchestrate cardiorespiratory and arousal responses to somatic stresses, whereas RTN selectively controls lung ventilation and arterial Pco2 stability.

Essential Role of the cVRG in the Generation of Both the Expiratory and Inspiratory Components of the Cough Reflex.

As stated by Korpáš and Tomori (1979), cough is the most important airway protective reflex which provides airway defensive responses to nociceptive stimuli. They recognized that active expiratory efforts, due to the activation of caudal ventral respiratory group (cVRG) expiratory premotoneurons, are the prominent component of coughs. Here, we discuss data suggesting that neurons located in the cVRG have an essential role in the generation of both the inspiratory and expiratory components of the cough reflex. Some lines of evidence indicate that cVRG expiratory neurons, when strongly activated, may subserve the alternation of inspiratory and expiratory cough bursts, possibly owing to the presence of axon collaterals. Of note, experimental findings such as blockade or impairment of glutamatergic transmission to the cVRG neurons lead to the view that neurons located in the cVRG are crucial for the production of the complete cough motor pattern. The involvement of bulbospinal expiratory neurons seems unlikely since their activation affects differentially expiratory and inspiratory muscles, while their blockade does not affect baseline inspiratory activity. Thus, other types of cVRG neurons with their medullary projections should have a role and possibly contribute to the fine tuning of the intensity of inspiratory and expiratory efforts.

Serotonergic projections to the ventral respiratory column from raphe nuclei in rats

The ventral respiratory column (VRC) generates rhythmical respiration and is divided into four compartments: the Bötzinger complex (BC), pre-Bötzinger complex (PBC), rostral ventral respiratory group (rVRG), and caudal ventral respiratory group (cVRG). Serotonergic nerve fibers are densely distributed in the rostral to caudal VRC and serotonin would be one of the important modulators for the respiratory control in the VRC. In the present study, to elucidate detailed distribution of serotonergic neurons in raphe nuclei projecting to the various rostrocaudal levels of VRC, we performed combination of retrograde tracing technique by cholera toxin B subunit (CTB) with immunohistochemistry for tryptophan hydroxylase 2 (TPH2). The double-immunoreactive neurons with CTB and TPH2 were distributed in the both rostral and caudal raphe nuclei, i.e. dorsal raphe nucleus, raphe magnus nucleus, gigantocellular reticular nucleus alpha and ventral parts, lateral paragigantocellular nucleus, parapyramidal area, raphe obscurus nucleus, and raphe pallidus nucleus. The distributions of double-immunoreactive neurons were similar among injection groups of BC, PBC, anterior rVRG, and posterior rVRG/cVRG. In conclusion, serotonergic neurons in both rostral and caudal raphe nuclei projected throughout the VRC and these serotonergic projections may contribute to respiratory responses to various environmental and vital changes.

Expression of the transcription factor FOXP2 in brainstem respiratory circuits of adult rat is restricted to upper-airway pre-motor areas

Expression of the transcription factor FOXP2 is linked to brain circuits that control motor function and speech. Investigation of FOXP2 protein expression in respiratory areas of the ponto-medullary brainstem of adult rat revealed distinct rostro-caudal expression gradients. A high density of FOXP2 immunoreactive nuclei was observed within the rostral pontine Kölliker-Fuse nucleus, compared to low densities in caudal pontine and rostral medullary respiratory nuclei, including the: (i) noradrenergic A5 and parafacial respiratory groups; (ii) Bötzinger and pre-Bötzinger complex and; (iii) rostral ventral respiratory group. Moderate densities of FOXP2 immunoreactive nuclei were observed in the caudal ventral respiratory group and the nucleus retroambiguus, with significant density levels found in the caudal half of the dorsal respiratory group and the hypoglossal pre-motor area lateral around calamus scriptorius. FOXP2 immunoreactivity was absent in all cranial nerve motor nuclei. We conclude that FOXP2 expression in respiratory brainstem areas selectively delineates laryngeal and hypoglossal pre-motor neuron populations essential for the generation of sound and voice.

Supraspinal respiratory plasticity following acute cervical spinal cord injury

Impaired breathing is a devastating result of high cervical spinal cord injuries (SCI) due to partial or full denervation of phrenic motoneurons, which innervate the diaphragm – a primary muscle of respiration. Consequently, people with cervical level injuries often become dependent on assisted ventilation and are susceptible to secondary complications. However, there is mounting evidence for limited spontaneous recovery of respiratory function following injury, demonstrating the neuroplastic potential of respiratory networks. Although many studies have shown such plasticity at the level of the spinal cord, much less is known about the changes occurring at supraspinal levels post-SCI. The goal of this study was to determine functional reorganization of respiratory neurons in the medulla acutely (>4h) following high cervical SCI. Experiments were conducted in decerebrate, unanesthetized, vagus intact and artificially ventilated rats. In this preparation, spontaneous recovery of ipsilateral phrenic nerve activity was observed within 4 to 6h following an incomplete, C2 hemisection (C2Hx). Electrophysiological mapping of the ventrolateral medulla showed a reorganization of inspiratory and expiratory sites ipsilateral to injury. These changes included i) decreased respiratory activity within the caudal ventral respiratory group (cVRG; location of bulbospinal expiratory neurons); ii) increased proportion of expiratory phase activity within the rostral ventral respiratory group (rVRG; location of inspiratory bulbo-spinal neurons); iii) increased respiratory activity within ventral reticular nuclei, including lateral reticular (LRN) and paragigantocellular (LPGi) nuclei. We conclude that disruption of descending and ascending connections between the medulla and spinal cord leads to immediate functional reorganization within the supraspinal respiratory network, including neurons within the ventral respiratory column and adjacent reticular nuclei.

In vivo Studies of Brainstem Neurons with Respiratory Rhythm Activity in a Rat Model of Rett Syndrome

People with Rett Syndrome (RTT), a neurodevelopmental disorder caused by mutations of the MECP2 gene, show breathing abnormalities that are attributable to the high incidence of sudden death. To understand the underlying mechanisms for the breathing disorders, direct in vivo recordings from brainstem neurons with respiratory rhythm are needed, which is a challenge in mouse RTT models. The newly available rat model may allow such a study. Therefore, we performed electrophysiological recording from individual neurons of the medullary ventral respiratory group (VRG) in decerebrate Mecp2‐null and wild‐type (WT) rats. In both groups of rats, neurons with expiratory rhythm were recorded from the rostral and caudal VRG, and cells with inspiratory rhythm were found in between. Firing frequency of the inspiratory neurons was significantly lower in null rats than in WT rats, while expiratory neurons in both rostral and caudal VRG did not show such a difference. Firing durations of both expiratory and inspiratory neurons were elongated in null rats compared with the WT. The ratio of inspiratory to expiratory firing duration (Ti/Te) was lower in null rats. A large number of cells showed E‐I phase‐spanning in null rats. The null rats had a frequent breathing pattern of apneusis followed by apnea, during which inspiratory neurons showed increased firing frequency and caudal expiratory neurons showed decreased firing frequency, whereas no significant change was observed in rostral expiratory neurons. Although this breathing pattern of apneusis followed by apnea was also seen in WT rats, the firing frequency of either respiratory neurons did not seem to change. These findings suggest that Mecp2 disruption appears to cause a large scale of changes in firing activity and pattern of medullary respiratory neurons consistent with the broad range of breathing abnormalities seen in people with RTT and animal models.Support or Funding InformationThis work was supported by NIH grant R01‐NS‐073875 and International Rett Syndrome Foundation.

Neuroanatomical and physiological evidence that the retrotrapezoid nucleus/parafacial region regulates expiration in adult rats

The rostroventrolateral medulla contains two functional neuronal populations: (1) the parafacial respiratory group (pFRG) neurons and (2) the chemosensitive retrotrapezoid nucleus (RTN) neurons. Using anatomical and physiological techniques, we investigated the role of the RTN/pFRG in CO2-induced active expiration (AE) in urethane-anesthetized rats. Anterograde tracing using biotinylated dextran amine (BDA) revealed dense neuronal projections emanating from the RTN/pFRG to the caudal ventral respiratory group (cVRG), 60% of which contained vesicular glutamate transporter-2. The minority (16%) of the RTN projections to the cVRG emanated from Phox2b positive neurons. Hypercapnia (10% CO2) increased DiaEMG and elicited AbdEMG activity. Bilateral injections of muscimol (2mM) into the RTN/pFRG reduced the activation of DiaEMG (23±4%) and abolished AE-induced by chemoreflex stimulation. Taken together, these results support the presence of direct excitatory projections from RTN/pFRG neurons to cVRG expiratory premotor neurons, playing a role in the generation/modulation of AE.

Suppression of the cough reflex byα2-adrenergic receptor agonists in the rabbit

The α2-adrenergic receptor agonist clonidine has been shown to inhibit citric acid-induced cough responses in guinea pigs when administered by aerosol, but not orally. In contrast, oral or inhaled clonidine had no effect on capsaicin-induced cough and reflex bronchoconstriction in humans. In addition, intravenous administration of clonidine has been shown to depress fentanyl-induced cough in humans. We investigated the effects of the α2-adrenergic receptor agonists, clonidine and tizanidine, on cough responses induced by mechanical and chemical (citric acid) stimulation of the tracheobronchial tree. Drugs were microinjected (30–50 nL) into the caudal nucleus tractus solitarii (cNTS) and the caudal ventral respiratory group (cVRG) as well as administered intravenously in pentobarbital sodium-anesthetized, spontaneously breathing rabbits. Bilateral microinjections of clonidine into the cNTS or the cVRG reduced cough responses at 0.5 mmol/L and abolished the cough reflex at 5 mmol/L. Bilateral microinjections of 0.5 mmol/L tizanidine into the cNTS completely suppressed cough responses, whereas bilateral microinjections of 5 mmol/L into the cVRG only caused mild reductions in them. Depressant effects on the cough reflex of clonidine and tizanidine were completely reverted by microinjections of 10 mmol/L yohimbine. Intravenous administration of clonidine (80–120 μg/kg) or tizanidine (150–300 μg/kg) strongly reduced or completely suppressed cough responses. These effects were reverted by intravenous administration of yohimbine (300 μg/kg). The results demonstrate that activation of α2-adrenergic receptors in the rabbit exerts potent inhibitory effects on the central mechanism generating the cough motor pattern with a clear action at the level of the cNTS and the cVRG.

Tracheal occlusion conditioning causes stress, anxiety and neural state changes in conscious rats

Evidence from human and animal studies indicates that mechanical loads to breathing are stressful stimuli and evoke compensatory behaviours. Conditioning of stressful stimuli is known to cause changes in basal stress levels and behaviour. Individuals with respiratory obstructive diseases repeatedly experience bouts of airway obstruction, which may act as a form of conditioning, and often have affective disorders, such as anxiety and depression. It is unknown whether the development of affective disorders in these individuals results from the unexpected recurring respiratory perturbations. To investigate this possibility, we developed a model to elicit tracheal occlusion (TO) in conscious rats and exposed them to 10 days of TO conditioning. We hypothesized that healthy, conscious animals exposed to TO conditioning would develop stress and anxiety and would have modulated neural activity in respiratory, stress, discriminative and affective neural regions. Following TO conditioning, rats had increased basal corticosterone levels, greater adrenal weights and elevated anxiety levels compared with animals not receiving TO. Significant increases in cytochrome oxidase staining were found in brainstem respiratory nuclei, periaqueductal grey, dorsal raphe, thalamus and insular cortex. These results suggest that healthy animals develop stress and anxiety responses to respiratory load conditioning via inescapable tracheal occlusions, which may be mediated through state changes in specific brain nuclei.

Modulation of the cough reflex by GABAA receptors in the caudal ventral respiratory group of the rabbit

We have previously shown that the caudal ventral respiratory group (cVRG) is a possible site of action of some antitussive drugs and plays a crucial role in determining both the expiratory and inspiratory components of the cough motor pattern. In addition, it has been reported that medullary expiratory neurons of the cVRG are subject to potent GABAergic gain modulation. This study was devoted to investigate the role of cVRG GABAA receptors in the control of baseline respiratory activity and cough responses to mechanical and chemical (citric acid) stimulation of the tracheobronchial tree. To this purpose, bilateral microinjections (30–50 nl) of bicuculline or muscimol were performed into the cVRG of pentobarbital sodium-anesthetized, spontaneously breathing rabbits. Bicuculline (1 mM) increased peak abdominal activity and respiratory frequency due to decreases in TE. Cough responses were potentiated mainly owing to increases in the cough number. The recovery was observed within ~2 h. On the contrary, muscimol (0.3 mM) abolished abdominal activity and decreased respiratory frequency due to increases in TE. In addition, cough responses were progressively reduced and completely suppressed within ~20 min. Partial recovery of cough responses was achieved after ~3 h or within ~5 min following bicuculline microinjections at the same locations. The sneeze reflex induced by mechanical stimulation of the nasal mucosa persisted following bicuculline and muscimol microinjections. However, the number and intensity of expiratory thrusts were enhanced by bicuculline and suppressed by muscimol. The results provide evidence that a potent GABAA-mediated inhibitory modulation is exerted at the level of the cVRG not only on respiratory activity, but also on cough and sneeze reflex responses.

Respiration-related control of abdominal motoneurons

The abdominal muscles form part of the expiratory pump in cooperation with the other expiratory muscles, primarily the internal intercostal and triangularis sterni muscles. The discharge of abdominal muscles is divided into four main patterns: augmenting, plateau, spindle and decrementing. The patterns tend to be species-specific and dependent on the state of the central nervous system. Recent studies suggest that the abdominal muscles are more active than classically thought, even under resting conditions. Expiratory bulbospinal neurons (EBSN) in the caudal ventral respiratory group are the final output pathway to abdominal motoneurons in the spinal cord. Electrophysiological and anatomical studies indicated the excitatory monosynaptic inputs from EBSN to the abdominal motoneurons, although inputs from the propriospinal neurons seemed to be necessary to produce useful motor outputs. Respiration-related sensory modulation of expiratory neurons by vagal afferents that monitor the rate of change of lung volume and the end-expiratory lung volume (EELV) play a crucial role in modulating the drive to the abdominal musculature. Studies using in vitro and in situ preparations of neonatal and juvenile rats show bi-phasic abdominal activity, characterized by bursting at the end of expiration, a silent period during the inspiratory period, and another burst that occurs abruptly after inspiratory termination. Since the abdominal muscles rarely show these post-inspiratory bursts in the adult rat, the organization of the expiratory output pathway must undergo significant development alterations.

Depression of cough reflex by microinjections of antitussive agents into caudal ventral respiratory group of the rabbit

We have previously shown that the caudal nucleus tractus solitarii is a site of action of some antitussive drugs and that the caudal ventral respiratory group (cVRG) region has a crucial role in determining both the expiratory and inspiratory components of the cough motor pattern. These findings led us to suggest that the cVRG region, and possibly other neural substrates involved in cough regulation, may be sites of action of antitussive drugs. To address this issue, we investigated changes in baseline respiratory activity and cough responses to tracheobronchial mechanical stimulation following microinjections (30-50 nl) of some antitussive drugs into the cVRG of pentobarbital-anesthetized, spontaneously breathing rabbits. [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) and baclofen at the lower concentrations (0.5 mM and 0.1 mM, respectively) decreased cough number, peak abdominal activity, and peak tracheal pressure and increased cough-related total cycle duration (Tt). At the higher concentrations (5 mM and 1 mM, respectively), both drugs abolished the cough reflex. DAMGO and baclofen also affected baseline respiratory activity. Both drugs reduced peak abdominal activity, while only DAMGO increased Tt, owing to increases in expiratory time. The neurokinin-1 (NK(1)) receptor antagonist CP-99,994 (10 mM) decreased cough number, peak abdominal activity, and peak tracheal pressure, without affecting baseline respiration. The NK(2) receptor antagonist MEN 10376 (5 mM) had no effect. The results indicate that the cVRG is a site of action of some antitussive agents and support the hypothesis that several neural substrates involved in cough regulation may share this characteristic.

Role of excitatory amino acids in the mediation of tracheobronchial cough induced by citric acid inhalation in the rabbit

We investigated the role of ionotropic glutamate receptors located within the caudal portions of the nucleus tractus solitarii (cNTS) and the caudal ventral respiratory group (cVRG) in the mediation of coughing evoked by citric acid inhalation in spontaneously breathing rabbits under pentobarbitone anaesthesia. Bilateral microinjections (30–50 nl) of 10 mM CNQX and 10 mM D-AP5 were performed to block non-NMDA and NMDA receptors, respectively. An attempt was also made to investigate the effects of ionotropic glutamate receptor blockade within the cVRG on sneezing induced by mechanical stimulation of the nasal mucosa. Blockade of non-NMDA receptors within the cNTS abolished coughing and associated tachypneic responses, while blockade of NMDA receptors only reduced cough responses. Blockade of non-NMDA receptors within the cVRG always abolished spontaneous rhythmic abdominal activity as well as coughing and associated tachypneic responses; blockade of NMDA receptors only reduced spontaneous rhythmic abdominal activity and coughing. As to sneezing, blockade of non-NMDA receptors within the cVRG suppressed the expiratory thrusts without affecting the inspiratory preparatory bursts, while blockade of NMDA receptors only strongly attenuated the expiratory thrusts. This study is the first to provide evidence that ionotropic glutamate receptors, and especially non-NMDA receptors, are involved in the mediation of coughing induced by citric acid inhalation and to suggest that citric acid-activated cough-related afferents terminate within the cNTS. Present data also corroborate the notion that the cVRG is involved in the generation of the whole cough motor pattern, but seems to represent merely an expiratory output system for sneezing.

Opioid Receptors on Bulbospinal Respiratory Neurons Are Not Activated During Neuronal Depression by Clinically Relevant Opioid Concentrations

Opioids depress the activity of brain stem respiratory-related neurons, but it is not resolved whether the mechanism at clinical concentrations consists of direct neuronal effects or network effects. We performed extracellular recordings of discharge activity of single respiratory neurons in the caudal ventral respiratory group of decerebrate dogs, which were premotor neurons with a likelihood of 90%. We used multibarrel glass microelectrodes, which allowed concomitant highly localized picoejection of opioid receptor agonists or antagonists onto the neuron. Picoejection of the mu receptor agonist [d-Ala(2), N-Me-phe(4), gly-ol(5)]-enkephalin (DAMGO, 1 mM) decreased the peak discharge frequency (mean +/- SD) of expiratory neurons to 68 +/- 22% (n = 12), the delta(1) agonist d-Pen(2,5)-enkephalin (DPDPE, 1 mM) to 95 +/- 11% (n = 15), and delta(2) receptor agonist [d-Ala(2)] deltorphin-II to 86 +/- 17% (1 mM, n = 15). The corresponding values for inspiratory neurons were: 64 +/- 12% (n = 11), 48 +/- 30% (n = 12), and 75 +/- 15% (n = 11), respectively. Naloxone fully reversed these effects. Picoejection of morphine (0.01-1 mM) depressed most neurons in a concentration dependent fashion to maximally 63% (n = 27). Picoejection of remifentanil (240-480 nM) did not cause any significant depression of inspiratory (n = 11) or expiratory neurons (n = 9). 4. Intravenous remifentanil (0.2-0.6 microg.kg(-1).min(-1)) decreased neuronal peak discharge frequency to 60 +/- 12% (inspiratory, n = 7) and 58 +/- 11% (expiratory, n = 11). However, local picoejection of naloxone did not reverse the neuronal depression. Our data suggest that mu, delta(1), and delta(2) receptors are present on canine respiratory premotor neurons. Clinical concentrations of morphine and remifentanil caused no local depression. This lack of effect and the inability of local naloxone to reverse the neuronal depression by intravenous remifentanil suggest that clinical concentrations of opioids produce their depressive effects on mechanisms upstream from respiratory bulbospinal premotor neurons.

N-methyl- d-aspartate mechanisms in depolarization of augmenting expiratory neurons during the expulsive phase of fictive cough in decerebrate cats

Cough reflex is characterized by a large expulsive phase for expelling the mucus or particles from the airway. The present study investigated the involvement of N-methyl- d-aspartate (NMDA) mechanisms in the expulsive phase of cough reflex using decerebrate and paralyzed cats. A fictive cough was induced by repetitive stimulation of the superior laryngeal nerve, which was characterized by an increased inspiratory discharge in the phrenic nerve (the stage 1 of fictive cough; SC1) and large spindle-shaped discharge in the iliohypogastric nerve (the stage 2 of fictive cough; SC2). Intravenous injection of an antagonist of NMDA receptors, dizocilpine (0.1 mg/kg), increased the threshold intensity of stimulation for inducing a fictive cough. The SC2 iliohypogastric response was more vulnerable to dizocilpine than the SC1 phrenic response. Membrane potential of augmenting expiratory (aug-E) neurons was recorded from the caudal ventral respiratory group. Aug-E neurons showed a large depolarization with a high frequency discharge during the SC2 in major cases ( n = 35) and hyperpolarization in minor cases ( n = 6). Dizocilpine inhibited the occurrence of these SC2 responses of aug-E neurons without any effect on the basal respiratory fluctuations of membrane potential. This drug had no significant effect on waves of excitatory and inhibitory postsynaptic potentials evoked in aug-E neurons by single pulse stimulation of the SLN. The present results demonstrated that NMDA mechanisms contribute preferentially to the expulsive phase response in aug-E neurons during fictive cough reflex.

Ionotropic glutamate receptors mediate excitatory drive to caudal medullary expiratory neurons in the rabbit

Most of the neurons of the caudal ventral respiratory group (cVRG) are bulbospinal expiratory neurons that receive their main excitatory drive from more rostral, but not yet defined regions. This study was devoted to investigate the functional role of ionotropic excitatory amino acid (EAA) receptors in the excitatory drive transmission to cVRG expiratory neurons during eupnoeic breathing and some respiratory reflexes including cough induced by mechanical stimulation of the tracheobronchial tree. The experiments were performed on spontaneously breathing rabbits under pentobarbitone anesthesia making use of microinjections (30–50 nl) of EAA receptor antagonists into the cVRG. Phrenic nerve and abdominal muscle activities were recorded. Bilateral microinjections of 50 mM kynurenic acid (KYN), a broad-spectrum EAA antagonist, and 10 mM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA antagonist, or 5 mM 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione (NBQX), a more specific non-NMDA antagonist, completely suppressed spontaneous rhythmic abdominal activity and reflex expiratory responses either to tracheal occlusion at end-inspiration (Breuer–Hering inflation reflex) or to expiratory threshold loading (5 cm H 2O); they also suppressed both the inspiratory and expiratory components of the cough reflex. Spontaneous rhythmic abdominal activity and the reflex respiratory responses were strongly reduced, but not completely abolished by microinjections of 10 mM d(−)-2-amino-5-phosphonopentanoic acid (D-AP5), an NMDA antagonist. The results provide evidence that the excitatory drive to cVRG bulbospinal expiratory neurons during eupnoeic breathing and the investigated respiratory reflexes is mediated by EAA receptors. They also support the view that neurons located in the cVRG are not merely elements of the expiratory output system.