Elenia Cinelli

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.

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.

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.

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 (30-50nl) of 10mM CNQX and 10mM 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.

Neurokinin receptor modulation of respiratory activity in the rabbit

The respiratory role of neurokinin (NK) receptors was investigated in α‐chloralose‐urethane‐anaesthetized, vagotomized, paralysed and artificially ventilated rabbits by using bilateral microinjections (30–50 nL) of NK receptor agonists and antagonists. Microinjections were performed in a region located just caudal to the rostral expiratory neurons. This region displayed features similar to those of the pre‐Bötzinger complex (pre‐BötC) of adult cats and rats, and proved to produce excitatory respiratory effects in response to microinjections of d,l‐homocysteic acid. We used as agonists (0.1, 0.5 and 5 mm) substance P (SP), the NK1 receptor agonists [Sar9, Met(O2)11]‐SP and GR 73632, the NK2 receptor agonist NKA, the NK3 receptor agonist senktide, and as antagonists (5 mm) the NK1 receptor antagonist CP‐99,994 and the NK2 receptor antagonist MEN 10376. SP always increased respiratory frequency, but NK1 receptor agonists did not change respiratory variables. NKA and senktide at 5 mm increased respiratory frequency. CP‐99,994 caused increases in respiratory frequency and did not antagonize the effects of SP. MEN 10376 prevented the respiratory responses induced by NKA and reduced those provoked by SP. SP or the NK1 receptor agonists (5 mm) injected (1 μL) into the IV ventricle caused marked excitatory effects on respiration. The results suggest that NK2 and NK3, but not NK1, receptors are involved in the excitatory modulation of inspiratory activity within the investigated region and are consistent with the notion that the pre‐BötC neurons are important components of the inspiratory rhythm‐generating mechanisms.

GABAergic and glycinergic inputs modulate rhythmogenic mechanisms in the lamprey respiratory network

In this study we investigated the role of GABA and glycine receptors within the respiratory central pattern generator, i.e. the paratrigeminal respiratory group (pTRG), and the vagal motoneuron region of the lamprey. Only GABA‐mediated inhibition modulates the pTRG both during apnoea induced by blockade of glutamatergic transmission and under basal conditions. Both GABA‐ and glycine‐mediated inhibition within the vagal region are involved in the regulation of respiratory frequency via ascending excitatory projections to the pTRG. Projecting neurons are retrogradely labelled from the pTRG, and intense GABA immunoreactivity is present within the pTRG and the vagal motoneuron region. Inhibitory mechanisms, which appear to be evolutionarily conserved, regulate network excitability and may provide an important contribution to rhythmic activities, such as respiration and locomotion.

Identification of a Cholinergic Modulatory and Rhythmogenic Mechanism within the Lamprey Respiratory Network

Acetylcholine (ACh) is well known to be involved in the control of breathing. However, no information is available on the role of ACh receptors (AChRs) within the lamprey respiratory network. The present study was performed on in vitro brainstem preparations of adult lampreys to investigate whether ACh affects respiratory activity possibly through an action on the paratrigeminal respiratory group (pTRG) that has been identified as an essential component of the respiratory network. Respiratory activity was monitored as vagal motor output. Bath application of 100 μm physostigmine or 1 μm nicotine increased respiratory frequency, while bath application of 100 μm d-tubocurarine or 0.25 μm α-bungarotoxin reduced respiratory frequency and increased the duration of vagal bursts. Since these effects were mimicked by microinjections of the same drugs into the pTRG, ACh proved to influence respiratory activity by acting on α7 nicotinic AChRs located within the pTRG. During apnea caused by partial blockade of ionotropic glutamate receptors at the level of the pTRG, bath application of bicuculline and strychnine restored the respiratory rhythm, although at reduced frequency. Similar results were obtained by the concurrent removal of both fast synaptic excitatory and inhibitory transmission. Blockade of pTRG α7 nicotinic AChRs suppressed this respiratory activity, thus indicating that pTRG neurons expressing these receptors contribute to respiratory rhythm generation. Together, these findings identify a novel cholinergic modulatory and possibly subsidiary rhythmogenic mechanism within the respiratory network of the adult lamprey and encourage further studies on the respiratory role of cholinergic receptors in different animal species.

Neural mechanisms underlying respiratory rhythm generation in the lamprey

The isolated brainstem of the adult lamprey spontaneously generates respiratory activity. The paratrigeminal respiratory group (pTRG), the proposed respiratory central pattern generator, has been anatomically and functionally characterized. It is sensitive to opioids, neurokinins and acetylcholine. Excitatory amino acids, but not GABA and glycine, play a crucial role in the respiratory rhythmogenesis. These results are corroborated by immunohistochemical data. While only GABA exerts an important modulatory control on the pTRG, both GABA and glycine markedly influence the respiratory frequency via neurons projecting from the vagal motoneuron region to the pTRG. Noticeably, the removal of GABAergic transmission within the pTRG causes the resumption of rhythmic activity during apnea induced by blockade of glutamatergic transmission. The same result is obtained by microinjections of substance P or nicotine into the pTRG during apnea. The results prompted us to present some considerations on the phylogenesis of respiratory pattern generation. They may also encourage comparative studies on the basic mechanisms underlying respiratory rhythmogenesis of vertebrates.

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.