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

As stated by Korpás 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.

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.

Central nervous mechanisms in the generation of the pattern of breathing.

The role of the Bötzinger complex (BötC) and the pre-Bötzinger complex (pre-BötC) in the genesis of the breathing pattern was investigated in anesthetized, vagotomized, paralysed and artificially ventilated rabbits making use of bilateral microinjections of kainic acid (KA) and excitatory amino acid (EAA) receptor antagonists. KA microinjections into either the BötC or the pre-BötC transiently eliminated respiratory rhythmicity in the presence of tonic phrenic activity (tonic apnea). Rhythmic activity resumed as low-amplitude, high-frequency irregular oscillations, superimposed on tonic inspiratory activity and displayed a progressive, although incomplete recovery. Microinjections of kynurenic acid (KYN) and D(-)-2-amino-5-phosphonopentanoic acid (D-AP5) into the BötC caused a pattern of breathing characterized by low-amplitude, high-frequency irregular oscillations and subsequently tonic apnea. Responses to KYN and D-AP5 in the pre-BötC were similar, although less pronounced than those elicited by these drugs in the BötC and never characterized by tonic apnea. Microinjections of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) into the BötC and the pre-BötC induced much less intense responses mainly consisting of increases in respiratory frequency. The results show that the investigated medullary regions play a prominent role in the genesis of the normal pattern of breathing through the endogenous activation of EAA receptors.

Dorsal root and dorsal column mediated synaptic inputs to reticulospinal neurons in lampreys: involvement of glutamatergic, glycinergic, and GABAergic transmission.

This study was aimed at characterizing the inputs from dorsal roots and dorsal columns to reticulospinal neurons within the posterior rhombencephalic reticular nucleus in the lamprey. The in vitro isolated brainstem and spinal cord preparation was used. Microstimulation of dorsal roots and columns on both sides induced, in identified reticulospinal neurons, synaptic responses which consisted of large IPSPs mixed with excitation, particularly from stimulation on the ipsilateral side. When the spinal cord was selectively exposed to kynurenic acid or to Ca2+ free Ringer's containing 2mM Mn2+, synaptic responses to stimulation of dorsal roots and columns were not modified, whereas the same responses were abolished when the brainstem was exposed selectively to kynurenic acid, thus suggesting that the responses were carried by long fibres ascending directly to the brainstem. The excitatory and inhibitory synaptic responses are relayed by interneurons located in the brainstem. The ascending excitatory inputs to inhibitory interneurons and, most likely, also to excitatory interneurons, use excitatory amino acid transmission. Inhibitory responses were abolished by adding the glycinergic antagonist strychnine (5 microM) to the physiological solution, thus suggesting that inhibitory interneurons use glycine transmission. The synaptic transmission was depressed by (-)-baclofen, a GABAB agonist, probably acting at a presynaptic site. Taken together, the present results suggest that dorsal root and dorsal column stimulations give rise to disynaptic inhibition and excitation of reticulospinal neurons mediated by excitatory and inhibitory amino acid transmission via brainstem interneurons.

Anatomical and physiological study of brainstem nuclei relaying dorsal column inputs in lampreys.

The course and sites of termination of dorsal column fibres in the lamprey brainstem are described along with their brainstem relays projecting to reticulospinal neurons. Dorsal column fibres ascend to the brainstem level where they intermingle with cells located in the alar plate close to the obex, a location that is analogous to that of the dorsal column nucleus in other vertebrates. Some dorsal column fibres continue further rostrally where they reach the octavolateralis and octavomotorii nuclei. Finally, a small contingent of fibres reach the cerebellum. Injections of cobalt-lysine into the posterior rhombencephalic reticular nucleus retrogradely label neurons within the dorsal column nucleus and within the octavolateralis and octavomotorii nuclei. Microstimulation of the dorsal column nucleus on either side elicits monosynaptic inhibitory responses in reticulospinal neurons while stimulation of octavolateralis and octavomotorius nuclei elicits excitation. By using intracellular recordings, it was shown that neurons within these alar plate nuclei receive monosynaptic inputs from the dorsal columns. It is thus proposed that disynaptic inputs from dorsal columns to reticulospinal neurons are relayed by these alar plate neurons: inhibition is relayed mainly by neurons in dorsal column nuclei and excitation by neurons in the octavolateralis and octavomotorii nuclei.

Calcium channel inhibitors suppress the morphine-withdrawal syndrome in rats.

The effects of the Ca2+-channel blockers verapamil and nimodipine, on the behavioural signs of naloxone (1 mg kg-1)-induced abstinence syndrome in morphine-dependent rats, were evaluated. The content of noradrenaline (NA) and of its metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) was measured, using high performance liquid chromatography and electrochemical detection or gas chromatography-mass spectrometry, in various brain regions of these animals. Possible interactions of nimodipine and verapamil with opioid receptors were evaluated by examining their ability to displace [3H]-naloxone binding to brain membranes. Verapamil (5, 10 and 50 mg kg-1) and nimodipine (1, 5 and 10 mg kg-1) dose-dependently reduced most of the signs of morphine abstinence. Naloxone-precipitated abstinence decreased the NA content in the cortex, hippocampus, brainstem and cerebellum. In the same brain regions the content of MHPG increased, suggesting an increased release of the amine during morphine abstinence. Nimodipine (10 mg kg-1 i.v.) did not change the content of NA or MHPG in the cortex, hippocampus and brainstem. However, nimodipine pre-treatment markedly reduced the changes in NA and MHPG content induced by the abstinence syndrome. Neither verapamil nor nimodipine displaced [3H]-naloxone from its binding sites. These results suggest that Ca2+-channel blockers suppress the behavioural and neurochemical expressions of morphine abstinence by a mechanism that differs from those of opioids or alpha 2-adrenoceptor agonists.

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-sulphamoylbenzo(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.

Influences of superior laryngeal afferent stimulation on expiratory activity in cats.

The effects of superior laryngeal nerve (SLN) stimulation on the activity of the expiratory muscles and medullary expiration-related (ER) neurons were investigated in 24 pentobarbital-anesthetized cats. In some experiments the animals were also paralyzed and artificially ventilated. Sustained tetanic stimulation of SLN consistently caused an apneic response associated with the appearance of tonic CO2-dependent activity in the expiratory muscles and in ER neurons located in the caudal ventral respiratory group (VRG) and the Bötzinger complex. Single shocks or brief tetani at the same stimulation intensities failed to evoke excitatory responses in the expiratory muscles and in the vast majority of ER neurons tested. At higher stimulation strengths, single shocks or short tetani elicited excitatory responses in the expiratory muscles (20- to 35-ms latency) and in the majority of ER neurons of the caudal VRG (7.5- to 15.5-ms latency). These responses were obtained only during the expiratory phase and proved to be CO2 independent. On the contrary, only inhibitory responses were evoked in the activity of Bötzinger complex neurons. The observed tonic expiratory activity most likely represents a disinhibition phenomenon due to the suppression of inspiratory activity; activation of expiratory muscles at higher stimulation intensities appears to be a polysynaptic reflex mediated by ER neurons of the caudal VRG but not by Bötzinger complex neurons.

Respiratory neuronal activity during apnea and poststimulatory effects of laryngeal origin in the cat.

We investigated the behavior of medullary respiratory neurons in cats under pentobarbitone anesthesia, vagotomized, paralysed, and artificially ventilated to elucidate neural mechanisms underlying apnea and poststimulatory respiratory depression induced by superior laryngeal nerve (SLN) stimulation. Inspiratory neurons were completely inhibited during SLN stimulation and poststimulatory apnea. During recovery of inspiratory activity, augmenting inspiratory neurons were depressed, decrementing inspiratory neurons were excited, and late inspiratory neurons displayed unchanged bursts closely locked to the end of the inspiratory phase. Augmenting expiratory neurons were either silenced or displayed different levels of tonic activity during SLN stimulation; some of them were clearly activated. These expiratory neurons displayed activity during poststimulatory apnea, before the onset of the first recovery phrenic burst. Postinspiratory or decrementing expiratory neurons were activated during SLN stimulation; their discharge continued with a decreasing trend during poststimulatory apnea. The results support the three-phase theory of rhythm generation and the view that SLN stimulation provokes a postinspiratory apnea that could represent the inhibitory component of respiratory reflexes of laryngeal origin, such as swallowing. In addition, because a subpopulation of augmenting expiratory neurons displays activation during SLN stimulation, the hypothesis can be advanced that not only postinspiratory, or decrementing expiratory neurons, but also augmenting expiratory neurons may be involved in the genesis of apnea and poststimulatory phenomena. Finally, the increase in the activity of decrementing inspiratory neurons after the end of SLN stimulation may contribute to the generation of poststimulatory respiratory depression by providing an inhibitory input to bulbospinal augmenting inspiratory neurons.

Respiratory responses induced by the activation of somatic nociceptive afferents in humans.

To further investigate the role of somatic nociceptive afferents in the neural control of breathing, we studied the respiratory effects of their activation by means of either electrical stimulation or ischemic pain in 14 healthy volunteers. Painful electrical cutaneous stimulation increased respiratory frequency (f), mean inspiratory flow (VT/TI), and rate of rise (XP/TI) of integrated electromyographic activity of diaphragm (IEMGdi). Painful muscular electrical stimulation caused similar but larger changes accompanied by increases in tidal volume (VT), peak XP of IEMGdi, and ventilation (VE); it also entrained respiratory rhythm. Ischemic pain, which was characterized by a progressively increasing intensity, caused augmentation in respiratory activity that displayed an increasing trend: VE, f, VT, XP, VT/TI, and XP/TI increased. In the light of available literature, it seems conceivable to suggest that respiratory responses to painful electrical stimulation are mediated through the activation of cutaneous (A delta) and muscular (group III) fine-myelinated afferents, and responses to ischemic pain are mediated by the activation of both fine myelinated (group III) and unmyelinated (group IV) muscular afferents. The input conveyed by these afferents may constitute an effective stimulus to respiration in humans.

Changes in respiratory activity induced by mastication in humans.

We studied the influence of mastication on respiratory activity in nine healthy volunteers who were requested to masticate a 5-g chewing gum bolus at a spontaneous rate (SR) for 5 min and "at the maximum possible rate" (MPR) for 1 min. Significant increases in respiratory frequency were induced by SR mastication due to a decrease in both the inspiratory and expiratory time. Tidal volume displayed slight nonsignificant decreases, but minute ventilation and mean inspiratory flow significantly increased. The duty cycle (TI/TT) did not change significantly. Total airway resistance significantly increased. Both peak and rate of rise of the integrated electromyographic activity of inspiratory muscles presented marked increases, accompanied by the appearance of a low level of tonic muscular activity. Similar but more intense effects on respiratory activity were induced by MPR mastication; in addition, a significant decrease in tidal volume and a significant increase in TI/TT were observed. Rhythmic handgrip exercise performed at metabolic rates comparable to those attained during SR or MPR mastication induced similar changes in the drive and time components of the breathing pattern, although accompanied respectively by nonsignificant or significant increases in tidal volume. Furthermore, the frequency of SR mastication significantly entrained the respiratory rhythm. The results suggest that mastication-induced hyperpnea does not merely represent a ventilatory response to exercise but also reflects complex interactions between respiratory and nonrespiratory functions of the upper airway and chest wall muscles.

Prostaglandin synthesis blockade by ketoprofen attenuates respiratory and cardiovascular responses to static handgrip.

We investigated the effects of prostaglandin synthesis blockade on the changes in breathing pattern, mean blood pressure (MBP), and heart rate (HR) elicited by 3 min of static handgrip at 30% of the maximum voluntary contraction in 12 healthy volunteers. Before each handgrip trial, subjects were treated with intravenous administration of either saline placebo (control) or 1 mg/kg of ketoprofen. Muscle tension and integrated electromyographic activity of exercising muscles remained fairly constant during each trial. In agreement with our earlier findings, during control handgrip minute ventilation progressively increased (P < 0.01) due to a rise in tidal volume and, to a lesser extent, in respiratory frequency. Mean inspiratory flow, MBP, and HR also increased (P < 0.01). End-tidal PCO2 decreased (P < 0.05) during the late phases of control handgrip bouts. Ketoprofen administration reduced serum thromboxane B2 levels (from 57.5 +/- 7.0 to 1.6 +/- 0.4 pg/ml; P < 0.01) and significantly attenuated mean increases in minute ventilation (40.25 +/- 0.60%), tidal volume (37.78 +/- 7.48%), respiratory frequency (55.94 +/- 17.92%), inspiratory flow (42.66 +/- 5.11%), MBP (22.33 +/- 6.82%), and HR (11.04 +/- 2.75%) during the 3rd min of handgrip. End-tidal PCO2 remained close to normocapnic levels. In agreement with previous animal investigations, the present results show that arachidonic acid metabolites are involved in the regulation of the cardiovascular responses to static efforts in humans, possibly through a stimulatory action on muscle receptors. Furthermore, they provide the first experimental evidence that products of the cyclooxygenase metabolic pathway play a role in the mediation of the respiratory adjustments elicited by this form of exercise.

Respiratory and cardiovascular responses to static handgrip exercise in humans.

We studied the time course of respiratory and cardiovascular responses by evaluating changes in the breathing pattern, mean blood pressure (MBP), and heart rate elicited by 3 min of static handgrip at 15, 25, and 30% of the maximum voluntary contraction (MVC) in 15 healthy volunteers. Muscle tension and integrated electromyographic activity remained fairly constant during each trial. During 15% MVC bouts, initially only mean inspiratory flow increased; then, tidal volume and minute ventilation (VI) also rose progressively. No significant changes in MBP and heart rate were observed. During 25 and 30% MVC bouts, not only did mean inspiratory flow, VT, and VI increase but MBP and heart rate increased as well. A slight and delayed rise in respiratory rate was also observed. Unlike 15 and 25% MVC handgrip, 30% MVC handgrip caused a small decrease in end-tidal PCO2. Changes in the pattern of breathing occurred more promptly than those in cardiovascular variables in the majority of subjects. Furthermore, we found a positive correlation between changes in VI and those in cardiovascular variables at the end of 25 and 30% MVC trials. This study indicates that respiratory and cardiovascular responses to static handgrip exercise are controlled independently.

Depressant effects on inspiratory and expiratory activity produced by chemical activation of Bötzinger complex neurons in the rabbit.

The respiratory role of the Bötzinger complex (Böt. c.) was investigated in alpha-chloralose-urethane or pentobarbitone anesthetized rabbits by means of microinjections of DL-homocysteic acid (DLH). The animals were either spontaneously breathing or vagotomized, paralysed and artificially ventilated. Both phrenic and abdominal activities were monitored; extracellular recordings from medullary respiration-related neurons were performed. Unilateral microinjections (5-30 nl) of DLH (160 mM) into the Böt. c., at sites where intense expiratory activity with an augmenting discharge pattern was encountered, provoked mild or moderate depressant effects on inspiratory activity characterized by decreases in frequency as well as in peak amplitude and rate of rise of phrenic nerve discharge. Stronger depressant effects up to complete apnea were consistently obtained in response to bilateral microinjections. Concomitant depressant effects on the activity of both expiratory motoneurons and expiration-related (ER) neurons of the caudal ventral respiratory group (cVRG) were observed. At variance with previous findings in the cat, the results indicate that chemical activation of Böt. c. augmenting ER neurons may exert inhibitory influences not only on inspiratory activity, but also on cVRG ER neurons and, hence, on expiratory motoneurons. The functional role of the Böt. c. in the control of respiration deserves further investigations; present findings suggest that the rabbit may profitably be used for such a purpose.

Effects of electrical and chemical stimulation of the Bötzinger complex on respiratory activity in the cat.

The effects of electrical and chemical stimulation of the expiratory neuronal population in the region of the retrofacial nucleus, the so called 'Bötzinger complex' (Böt. c.), on respiratory activity were investigated in vagotomized cats under pentobarbitone anaesthesia. Some of the experiments were performed on paralyzed or bilaterally thoracotomized, artificially ventilated animals. Sustained tetanic electrical stimulation (20 to 100-Hz, 0.5-ms current pulses at intensities of 5-60 microA) induced strong depressant effects on the inspiratory motor output which could lead to complete apnoea. The apnoeic response was accompanied by tonic activation of expiratory muscles; the appearance and the strength of tonic expiratory activity were dependent upon the frequency of stimulation. Brief tetani (40 to 100 ms trains of 0.5-ms rectangular pulses at 100-300 Hz) timed either during the inspiratory or the expiratory phase caused depression of inspiratory activity and prolongation of expiratory time, respectively. These effects increased gradually as the onset of stimulation was progressively delayed during each respiratory phase. The effects of sustained tetanic stimulation were mimicked by microinjections (25-100 nl) of 0.5 M L-glutamate or 0.16 M DL-homocysteic acid in the same region, thus indicating that they were the result of the stimulation of cell bodies and not of axons of passage. The present results support the hypothesis that Böt. c. neurons play an important role in the control of the breathing pattern by exerting inhibitory influences on inspiratory activity and, possibly, by contributing to the off-switch mechanisms. Furthermore, they suggest that these neurons are involved in the central control of expiratory activity.

Expiration-related neurons in the caudal ventral respiratory group of the cat: influences of the activation of Bötzinger complex neurons.

The functional role of Bötzinger complex (Böt. c.) projections to the expiration-related (ER) area of the caudal ventral respiratory group (cVRG) was investigated in anesthetized, vagotomized, paralyzed and artificially ventilated cats. ER neurons in both the ipsi- and the contralateral cVRG displayed excitatory responses to Böt. c. electrical microstimulation. They were also activated by microinjections of D,L-homocysteic acid into the Böt. c. region. We propose that at least part of the Böt. c. projections to the cVRG have an excitatory function.

Neuropeptide Y-immunoreactive spinal neurons make close appositions on axons of primary sensory afferents.

The dorsal cells in the lamprey are primary sensory neurons located in a parasagittal region of the spinal cord. In the same plane neuropeptide Y (NPY) immunoreactivity has been described. To investigate if the dorsal cells were in contact with NPY-immunoreactive (ir) fibers and cell bodies, intracellular injections of Lucifer yellow into identified dorsal cells were combined with NPY immunohistochemistry. NPY-ir varicosities were found to be in close apposition to both the descending and the ascending axon of dorsal cells. No NPY-ir boutons were seen in close contact with the dorsal cell bodies.

Putative GABAergic input to axons of spinal interneurons and primary sensory neurons in the lamprey spinal cord as shown by intracellular Lucifer yellow and GABA immunohistochemistry.

GABAergic phasic modulation of the membrane potential occurs in spinal interneurons during fictive locomotion in lamprey presumably indicating a presynaptic inhibition. GABA also modulates synaptic transmission from primary sensory neurons (dorsal cells) at a presynaptic site. From these findings GABA terminals would be expected to be in close contact with phasically modulated axons of spinal interneurons and/or dorsal cells and their axons. To test this supposition intracellular injections of Lucifer yellow into spinal interneurons or dorsal cells were combined with GABA immunohistochemistry. GABA-immunoreactive (ir) varicosities were found to be in close contact (less than 1 micron distance) with axons modulated during fictive locomotion as well as dorsal cell axons. Small GABA-ir bipolar neurons form processes, which are in close contact with the axons of dorsal cells.

Role of glutamate receptor subtypes in the lamprey respiratory network.

The respiratory role of glutamate receptors was investigated in the isolated lamprey brain preparation by analyzing the changes in respiratory activity induced by bath application of specific antagonists of ionotropic and metabotropic glutamate receptors. The results show that these antagonists differentially affect the pattern of breathing and provide the first evidence that both ionotropic and metabotropic glutamate receptors are involved in neurotransmission within the lamprey respiratory network.

Respiratory responses to chemical stimulation of the parabrachial nuclear complex in the rabbit.

The respiratory role of the parabrachial nuclear complex (PNC) was investigated in alpha-chloralose-urethane anesthetized, vagotomized, paralysed and artificially ventilated rabbits by means of unilateral microinjections (10-20 nl) of 20 mM dl-homocysteic acid. Chemical stimulation elicited three main types of site-specific respiratory effects: excitatory, apneustic and inhibitory responses. The results suggest that the PNC plays a complex role in the control of breathing.