Donatella Mutolo

Modulation of the cough reflex by antitussive agents within the caudal aspect of the nucleus tractus solitarii in the rabbit

We have previously shown that ionotropic glutamate receptors in the caudal portion of the nucleus tractus solitarii (NTS), especially in the commissural NTS, play a prominent role in the mediation of tracheobronchial cough and that substance P potentiates this reflex. This NTS region could be a site of action of some centrally acting antitussive agents and a component of a drug-sensitive gating mechanism of cough. To address these issues, we investigated changes in baseline respiratory activity and cough responses to tracheobronchial mechanical stimulation following microinjections (30-50 nl) of centrally acting antitussive drugs into the caudal NTS of pentobarbitone-anesthetized, spontaneously breathing rabbits. [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) and baclofen decreased baseline respiratory frequency because of increases in the inspiratory time only at the higher concentration employed (5 mM and 1 mM, respectively). DAMGO (0.5 mM) and baclofen (0.1 mM) significantly decreased cough number, peak abdominal activity, peak tracheal pressure, and increased cough-related total cycle duration. At the higher concentrations, these agents suppressed the cough reflex. The effects of these two drugs were counteracted by specific antagonists (10 mM naloxone and 25 mM CGP-35348, respectively). The neurokinin-1 (NK1) receptor antagonist CP-99,994 (10 mM) abolished cough responses, whereas the NK2 receptor antagonist MEN 10376 (5 mM) had no effect. The results indicate that the caudal NTS is a site of action of some centrally acting drugs and a likely component of a neural system involved in cough regulation. A crucial role of substance P release in the mediation of reflex cough is also suggested.

The role of excitatory amino acids and substance p in the mediation of the cough reflex within the nucleus tractus solitarii of the rabbit

We hypothesized that cough evoked by mechanical stimulation of the tracheobronchial tree in the rabbit is primarily mediated by glutamatergic neurotransmission at the level of the caudal portions of the medial subnucleus of the nucleus tractus solitarii (NTS) and the lateral commissural NTS where cough-related afferents terminate, and that this reflex is potentiated by local release of substance P. To test our hypothesis, we performed bilateral microinjections (30-50 nl) of ionotropic glutamate receptor antagonists or substance P into these locations in pentobarbitone anaesthetized, spontaneously breathing rabbits. Blockade of NMDA and non-NMDA receptors by 50mM kynurenic acid abolished the cough reflex without affecting the Breuer-Hering inflation reflex or the pulmonary chemoreflex. Blockade of non-NMDA receptors using 10mM CNQX or 5mM NBQX caused identical effects. Blockade of NMDA receptors by 10mM D-AP5 strongly reduced, but did not abolish cough responses. Microinjections of 1mM substance P increased peak and rate of rise of abdominal muscle activity as well as cough number. These results are the first to provide evidence that ionotropic glutamate receptors, especially non-NMDA receptors, located within specific regions of NTS are primarily involved in the mediation of cough evoked by mechanical stimulation of the tracheobronchial tree in the rabbit. Present findings on substance P cough-enhancing effects extend previous observations and are relevant to the tachykinin-mediated central sensitization of the cough reflex. They also may provide hints for further studies on centrally acting antitussive drugs.

Discharge patterns of Bötzinger complex neurons during cough in the cat

This study was carried out on pentobarbital sodium-anesthetized, spontaneously breathing cats to address the hypothesis that Bötzinger complex (BötC) neurons are involved in the production of the cough motor pattern induced by mechanical stimulation of the tracheobronchial tree. Phrenic nerve and abdominal muscle activities as well as intratracheal pressure were monitored; single-unit extracellular recordings from BötC neurons ( n = 87) were performed. The majority of augmenting expiratory (E-Aug) neurons encountered ( n = 47) displayed excitatory responses during the expulsive phases of coughing in parallel with the main components of the abdominal bursts and the corresponding increases in tracheal pressure. We also encountered E-Aug neurons markedly depressed up to complete inhibition during coughing ( n = 14) as well as E-Aug neurons assuming a decremental pattern without any increase or even with some reduction in their peak activity ( n = 15). During the expiratory thrusts, most decrementing expiratory neurons ( n = 7) presented excitatory responses, whereas others were depressed ( n = 3) or completely inhibited ( n = 1). The results are consistent with the view that these neurons are involved in the generation of the cough motor pattern and, in particular, that some BötC E-Aug neurons convey excitatory drive to caudal expiratory neurons and, hence, to expiratory motoneurons.

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(2)O); 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.

Opioid-induced depression in the lamprey respiratory network.

The role of opioid receptors in modulating respiratory activity was investigated in in vitro brainstem preparations of adult lampreys by bath application of agonists and antagonists. The vagal motor output was used to monitor respiratory activity. Neuronal recordings were also performed to characterize the rostrolateral trigeminal region that has been suggested to be critical for respiratory rhythmogenesis. Microinjections of the micro-opioid receptor agonist [d-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) were also made into this region and at different locations within the brainstem. Bath application of DAMGO (0.5-2 microM) caused marked decreases in respiratory frequency up to complete apnea. Bath application of the delta-opioid receptor agonist [d-Pen(2,5)]-enkephalin (DPDPE) at 10-40 microM induced less pronounced depressant respiratory effects, while no changes in respiratory activity were induced by the kappa-opioid receptor agonist trans-(1S,2S)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide (U50488) at 10-40 microM. Bath application of the opioid receptor antagonists naloxone and naltrindole did not affect baseline respiratory activity, but prevented agonist-induced effects. DAMGO microinjections (1 mM; 0.5-1 nl) at sites rostrolateral to the trigeminal motor nucleus, where respiration-related neuronal activity was recorded, abolished the respiratory rhythm. The results show that opioids may have an important role in the lamprey respiratory network and that micro-opioid receptor activation is the most effective in causing respiratory depression. They also indicate that endogenous opioids are not required for the generation of baseline respiratory activity. Apneic responses induced by DAMGO microinjections support the hypothesis that a specific opioid-sensitive region rostrolateral to the trigeminal motor nucleus, that we have termed the paratrigeminal respiratory group (pTRG), likely has a pivotal role in respiratory rhythmogenesis. Since the lamprey diverged from the main vertebrate line around 450 million years ago, our results also imply that the inhibitory role of opioids on respiration is present at an early stage of vertebrate evolution.

Respiratory responses induced by blockades of GABA and glycine receptors within the Bötzinger complex and the pre-Bötzinger complex of the rabbit

The respiratory role of GABA(A), GABA(B) and glycine receptors within the Bötzinger complex (BötC) and the pre-Bötzinger complex (preBötC) was investigated in alpha-chloralose-urethane anesthetized, vagotomized, paralysed and artificially ventilated rabbits by using bilateral microinjections (30-50 nl) of GABA and glycine receptor agonists and antagonists. GABA(A) receptor blockade by bicuculline (5mM) or gabazine (2mM) within the BötC induced strong depression of respiratory activity up to apnea. The latter was reversed by hypercapnia. Glycine receptor blockade by strychnine (5mM) within the BötC decreased the frequency and amplitude of phrenic bursts. Bicuculline microinjections into the preBötC caused decreases in respiratory frequency and the appearance of two alternating different levels of peak phrenic activity. Strychnine microinjections into the preBötC increased respiratory frequency and decreased peak phrenic amplitude. GABA(A), but not glycine receptor antagonism within the preBötC restored respiratory rhythmicity during apnea due to bicuculline or gabazine applied to the BötC. GABA(B) receptor blockade by CGP-35348 (50mM) within the BötC and the preBötC did not affect baseline respiratory activity, though microinjections of the GABA(B) receptor agonist baclofen (1mM) into the same regions altered respiratory activity. The results show that only GABA(A) and glycine receptors within the BötC and the preBötC mediate a potent control on both the intensity and frequency of inspiratory activity during eupneic breathing. This study is the first to provide evidence that these inhibitory receptors have a respiratory function within the BötC.

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.

Respiratory responses evoked by blockades of ionotropic glutamate receptors within the Bötzinger complex and the pre‐Bötzinger complex of the rabbit

The respiratory role of excitatory amino acid (EAA) receptors within the Bötzinger complex (BötC) and the pre‐Bötzinger complex (pre‐BötC) was investigated in α‐chloralose–urethane anaesthetized, vagotomized, paralysed and artificially ventilated rabbits by using bilateral microinjections (30–50 nL) of EAA receptor antagonists. Blockade of both N‐methyl‐d‐aspartic acid (NMDA) and non‐NMDA receptors by 50 mm kynurenic acid (KYN) within the BötC induced a pattern of breathing characterized by low‐amplitude, high‐frequency irregular oscillations superimposed on tonic phrenic activity and successively the disappearance of respiratory rhythmicity in the presence of intense tonic inspiratory discharges (tonic apnea). KYN microinjections into the pre‐BötC caused similar respiratory responses that, however, never led to tonic apnea. Blockade of NMDA receptors by D(–)‐2‐amino‐5‐phosphonopentanoic acid (D‐AP5; 1, 10 and 20 mm) within the BötC induced increases in respiratory frequency and decreases in peak phrenic amplitude; the highest concentrations caused tonic apnea insensitive to chemical stimuli. Blockade of non‐NMDA receptors by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 1, 10 and 20 mm) within the BötC produced only less pronounced increases in respiratory frequency. Responses to D‐AP5 in the pre‐BötC were similar, although less pronounced than those elicited in the BötC and never characterized by tonic apnea. In the same region, CNQX provoked increases in respiratory frequency similar to those elicited in the BötC, associated with slight reductions in peak phrenic activity. The results show that EAA receptors within the investigated medullary subregions mediate a potent control on both the intensity and frequency of inspiratory activity, with a major role played by NMDA receptors.

Neuronal Mechanisms of Respiratory Pattern Generation Are Evolutionary Conserved

A brainstem region, the paratrigeminal respiratory group (pTRG), has been suggested to play a crucial role in the respiratory rhythm generation in lampreys. However, a detailed characterization of the pTRG region is lacking. The present study performed on isolated brainstem preparations of adult lampreys provides a more precise localization of the pTRG region with regard to both connectivity and neurochemical markers. pTRG neurons projecting to the vagal motoneuronal pool were identified in a restricted area of the rostral rhombencephalon at the level of the isthmic Müller cell I1 close to sulcus limitans of His. Unilateral microinjections of lidocaine, muscimol, or glutamate antagonists into the pTRG inhibited completely the bilateral respiratory activity. In contrast, microinjections of glutamate agonists enhanced the respiratory activity, suggesting that this region is critical for the respiratory pattern generation. The retrogradely labeled pTRG neurons are glutamatergic and surrounded by terminals with intense substance P immunoreactivity. Cholinergic neurons were seen close to, and intermingled with, pTRG neurons. In addition, α-bungarotoxin binding sites (indicating nicotinic receptors) were found throughout the pTRG area and particularly on the soma of these neurons. During apnea, induced by blockade of ionotropic glutamate receptors within the same region, microinjections of 1 μm substance P or 1 mm nicotine into the pTRG restored rhythmic respiratory activity. The results emphasize the close similarities between the pTRG and the mammalian pre-Bötzinger complex as a crucial site for respiratory rhythmogenesis. We conclude that some basic features of the excitatory neurons proposed to generate respiratory rhythms are conserved throughout evolution.

Area postrema glutamate receptors mediate respiratory and gastric responses in the rabbit.

THE role of NMDA and non-NMDA receptors of the area postrema (AP) in the control of respiration and gastric motility was investigated in anaesthetized rabbits using microinjections (10–20 nl) of specific agonists or antagonists. NMDA (20 mM) or AMPA (10 mM) caused excitatory effects on respiration and gastric relaxation. Selective blockade of NMDA or non-NMDA receptors, respectively with D (–)-2-amino-5-phosphonopentanoic acid (D-AP5; 10 mM) and 6-nitro-7-sulphamoyl-benzo(f)quinoxaline-2,3-dione (NBQX; 5 mM), decreased respiratory frequency and increased gastric tone. Both these effects were more marked following non-NMDA receptor blockade and were prevented by vagotomy. These findings show that NMDA and non-NMDA receptors are present on AP neurones and have a role in the tonic control of respiration and gastric motility.