Peak Phrenic Amplitude Research Articles

Differential Respiratory Effects of µ‐opioid Receptor Activation within the preBötzinger Complex and Neighbouring Respiration‐related Regions in the Rabbit

It is well known that opioids induce respiratory depression by activating µ-opioid receptors (MORs). Although MORs are expressed throughout the entire brainstem respiratory network, the effects of their activation within the different respiration-related regions are still unclear. We investigated the contribution of the preBötzinger Complex (preBötC) and the neighbouring Bötzinger Complex (BötC) and inspiratory portion of the ventral respiratory group (iVRG) to the MOR-induced respiratory depression in anesthetized, vagotomized, paralyzed and artificially ventilated adult rabbits making use of microinjections (30-50 nl) of the MOR agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO). Dose-dependent effects were observed. DAMGO microinjected at 0.1 mM into the preBötC and the BötC caused progressive reductions in peak phrenic amplitude associated with tonic phrenic activity and patterns of breathing displaying irregularities that were more marked in the preBötC (ataxic breathing). Increases or no changes in respiratory frequency accompanied these responses in the preBötC and the BötC, respectively. Tonic apneic effects developed at 0.5 mM. DAMGO microinjected into the iVRG provoked decreases in the amplitude and frequency of phrenic bursts at 0.1 mM and apnea at 0.5 mM. DAMGO-induced increases in the variability of peak phrenic amplitude and time components of the breathing pattern were more pronounced after microinjections into the preBötC. Local application of 5 mM naloxone reversed the apneic effects in all instances. The results provide further insides into the role of MORS in the genesis of respiratory depression induced by systemically administered opioids. They suggest that various areas of the respiratory network differentially contribute to the opioid-induced respiratory disorders and support the view that the preBötC may play a prominent role.

Activation of μ-opioid receptors differentially affects the preBötzinger Complex and neighbouring regions of the respiratory network in the adult rabbit

The role of the different components of the respiratory network in the mediation of opioid-induced respiratory depression is still unclear. We investigated the contribution of the preBötzinger Complex (preBötC) and the neighbouring Bötzinger Complex (BötC) and inspiratory portion of the ventral respiratory group (iVRG) in anesthetized, vagotomized, paralyzed and artificially ventilated adult rabbits making use of bilateral microinjections (30−50 nl) of the μ-opioid receptor agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO). Dose-dependent effects were observed. In the preBötC and the BötC 0.1 mM DAMGO microinjections caused mainly reductions in peak phrenic amplitude associated with tonic phrenic activity and irregular (ataxic) patterns of breathing that were more pronounced in the preBötC. Apneic effects developed at 0.5 mM. In the iVRG DAMGO provoked decreases in amplitude and frequency of phrenic bursts at 0.1 mM and apnea at 0.5 mM. Local 5 mM naloxone reversed the apneic effects. The results imply that different components of the respiratory network may contribute to opioid-induced respiratory disorders.

The contribution of endogenous glutamatergic input in the ventral respiratory column to respiratory rhythm

Glutamate is the predominant excitatory neurotransmitter in the ventral respiratory column; however, the contribution of glutamatergic excitation in the individual subregions to respiratory rhythm generation has not been fully delineated. In an adult, in vivo, decerebrate rabbit model during conditions of mild hyperoxic hypercapnia we blocked glutamatergic excitation using the receptor antagonists 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX) and d(–)-2-amino-5-phosphonopentanoic acid (AP5). Disfacilitation of the preBötzinger Complex caused a decrease in inspiratory and expiratory duration as well as peak phrenic amplitude and ultimately apnea. Disfacilitation of the Bötzinger Complex caused a decrease in inspiratory and expiratory duration; subsequent disfacilitation of the preBötzinger Complex resulted in complete loss of the respiratory pattern but maintained tonic inspiratory activity. We conclude that glutamatergic drive to the preBötzinger Complex is essential for respiratory rhythm generation. Glutamatergic drive to the Bötzinger Complex significantly affects inspiratory and expiratory phase duration. Bötzinger Complex neurons are responsible for maintaining the silent expiratory phase of the phrenic neurogram.

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 α-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 (5 mM) or gabazine (2 mM) within the BötC induced strong depression of respiratory activity up to apnea. The latter was reversed by hypercapnia. Glycine receptor blockade by strychnine (5 mM) 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 (50 mM) within the BötC and the preBötC did not affect baseline respiratory activity, though microinjections of the GABA B receptor agonist baclofen (1 mM) 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.

GABA, not glycine, mediates inhibition of latent respiratory motor pathways after spinal cord injury

Previous work has shown that latent respiratory motor pathways known as crossed phrenic pathways are inhibited via a spinal inhibitory process; however, the underlying mechanisms remain unknown. The present study investigated whether spinal GABA-A and/or glycine receptors are involved in the inhibition of the crossed phrenic pathways after a C2 spinal cord hemisection injury. Under ketamine/xylazine anesthesia, adult, female, Sprague–Dawley rats were hemisected at the C2 spinal cord level. Following 1 week post injury, rats were anesthetized with urethane, vagotomized, paralyzed and ventilated. GABA-A receptor (bicuculline and Gabazine) and glycine receptor (strychnine) antagonists were applied directly to the cervical spinal cord (C3–C7), while bilateral phrenic nerve motor output was recorded. GABA-A receptor antagonists significantly increased peak phrenic amplitude bilaterally and induced crossed phrenic activity in spinal-injured rats. Muscimol, a specific GABA-A receptor agonist, blocked these effects. Glycine receptor antagonists applied directly to the spinal cord had no significant effect on phrenic motor output. These results indicate that phrenic motor neurons are inhibited via a GABA-A mediated receptor mechanism located within the spinal cord to inhibit the expression of crossed phrenic pathways.

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 alpha-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.

Ascorbyl palmitate augments hypoxic respiratory response in the cat.

The redox signaling is germane for the hypoxia-sensing mechanisms at the carotid body. This raises the strong possibility that agents possess reducing and antioxidant attributes, such as ascorbate, could influence the hypoxic respiratory response. However, water solubility of ascorbate makes its effectiveness at membrane-associated target sites dubious. In this study, we sought to determine the effect of ascorbyl-6-palmitate (AP), a lipid-soluble derivative of ascorbate which penetrates biomembranes, on hypoxic respiration in the anesthetized, paralyzed and ventilated cat. AP was given by gavage: 600 mg/kg daily for 6 days before the beginning of the acute experiment. Respiration was then assessed from the phrenic electroneurogram, from which peak phrenic amplitude, a surrogate of tidal component, respiratory frequency, and their product, the minute phrenic output, were quantified. The response to normocapnic hypoxia, 7% O(2) in N(2), in the AP-treated cats was compared with that in controls. We found that AP augmented hypoxic respiration, delayed the appearance of hypoxic depression and decreased it, although the stimulatory/depressant character was preserved. The results suggest that the ascorbate moiety of AP interacts with the hypoxia-sensing mechanisms. Ascorbate may affect hypoxic respiration at multiple stages of chemotransduction pathways, which are subject to continuing uncertainties. The study highlights the augmentative effect of AP, a redox modulator, on hypoxic respiration, which may have a therapeutic potential.

Ascorbyl Palmitate Augments Hypoxic Respiratory Response in the Cat

The redox signaling is germane for the hypoxia-sensing mechanisms at the carotid body. This raises the strong possibility that agents possess reducing and antioxidant attributes, such as ascorbate, could influence the hypoxic respiratory response. However, water solubility of ascorbate makes its effectiveness at membrane-associated target sites dubious. In this study, we sought to determine the effect of ascorbyl-6-palmitate (AP), a lipid-soluble derivative of ascorbate which penetrates biomembranes, on hypoxic respiration in the anesthetized, paralyzed and ventilated cat. AP was given by gavage: 600 mg/kg daily for 6 days before the beginning of the acute experiment. Respiration was then assessed from the phrenic electroneurogram, from which peak phrenic amplitude, a surrogate of tidal component, respiratory frequency, and their product, the minute phrenic output, were quantified. The response to normocapnic hypoxia, 7% O₂ in N₂, in the AP-treated cats was compared with that in controls. We found that AP augmented hypoxic respiration, delayed the appearance of hypoxic depression and decreased it, although the stimulatory/depressant character was preserved. The results suggest that the ascorbate moiety of AP interacts with the hypoxia-sensing mechanisms. Ascorbate may affect hypoxic respiration at multiple stages of chemotransduction pathways, which are subject to continuing uncertainties. The study highlights the augmentative effect of AP, a redox modulator, on hypoxic respiration, which may have a therapeutic potential.

Respiratory changes induced by kainic acid lesions in rostral ventral respiratory group of rabbits.

The role played by the Bötzinger complex (BötC), the pre-Bötzinger complex (pre-BötC), and the more rostral extent of the inspiratory portion of the ventral respiratory group (iVRG) in the genesis of the eupneic pattern of breathing was investigated in anesthetized, vagotomized, paralyzed, and artificially ventilated rabbits by means of kainic acid (KA, 4.7 mM) microinjections (20-30 nl). Unilateral KA microinjections into all of the investigated VRG subregions caused increases in respiratory frequency associated with moderate decreases in peak phrenic amplitude in the BötC and pre-BötC regions. Bilateral KA microinjections into either the BötC or pre-BötC transiently eliminated respiratory rhythmicity and caused the appearance of tonic phrenic activity ("tonic apnea"), whereas injections into the rostral iVRG completely suppressed inspiratory activity. Rhythmic activity resumed as low-amplitude, high-frequency oscillations and displayed a progressive, although incomplete, recovery. Combined bilateral KA microinjections (BötC and pre-BötC) caused persistent (>3 h) tonic apnea. Results show that all of the investigated VRG subregions exert a potent control on both the intensity and frequency of inspiratory activity, thus suggesting that these areas play a major role in the genesis of the eupneic pattern of breathing.

Respiratory responses to ionotropic glutamate receptor antagonists in the ventral respiratory group of the rabbit.

Selective excitatory amino acid receptor antagonists acting on either N-methyl-D-aspartic acid (NMDA) or non-NMDA receptors were microinjected (30-50 nl) bilaterally into different subregions of the ventral respiratory group (VRG) of alpha-chloralose-urethane anaesthetized, vagotomized, paralysed and artificially ventilated rabbits. Blockade of NMDA receptors by D(-)-2-amino-5-phosphonopentanoic acid (D-AP5; 1 or 10 mM) within the inspiratory portion of the VRG (iVRG) dose-dependently decreased the peak amplitude and rate of rise of phrenic nerve activity, without significant changes in respiratory timing. Decreases in respiratory frequency and peak phrenic amplitude up to apnoea were evoked by 20 mM D-AP5; phrenic nerve activity was restored transiently by hypoxic or hypercapnic stimulation during D-AP5-induced apnoea. Microinjections of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1, 10 or 20 mM) into the iVRG provoked less intense depressant respiratory effects. No significant respiratory responses were evoked by microinjections of these antagonists into more caudal VRG subregions. The results suggest that ionotropic glutamate receptors within the iVRG are involved mainly in the control of the intensity of inspiratory activity, with a major role played by NMDA receptors. Glutamate receptor antagonism in the iVRG does not seem to impair the basic mechanisms underlying respiratory rhythm generation.

Involvement of the motor trigeminal nucleus in respiratory phase-switching in the cat.

We investigated the hypothesis that the motor trigeminal nucleus, consisting of expiratory motoneurons, might be influential in termination of inspiration. We addressed the issue by comparing the effects on neural respiration of a reversible, unilateral, pharmacologic blockade of the motor trigeminal nucleus (5M), the medial parabrachial nucleus (PB), and of other nearby structures that are neutral for respiration in anesthetized, vagotomized, paralyzed, and ventilated cats. The blockade was achieved by microinjections of 2% xylocaine, laced with Pontamine Sky Blue to identify sites of injections, from the tip of a penetrating microelectrode. Integrated phrenic neurograms were recorded to quantify the time of neural inspiration (TI), expiration (TE), and the peak phrenic amplitude. We found that blockade of the 5M caused a pattern of apneustic respiration, consisting of a selective prolongation of inspiratory phases that were interrupted by short expiratory pauses. In contrast, blockade of the PB resulted in a prolongation of both TI and TE, which corresponds to a mere slowing of respiration. The results confirmed important functions of the rostral pons in ventilatory control but pointed to the 5M rather than PB as a structure underlying the inspiratory off-switch. We conclude that the motor trigeminal nucleus may have a part in the pontine pneumotaxic mechanism.

Modulation of respiratory responses to carotid sinus nerve stimulation by brain hypoxia.

This study examines the effect of progressive isocapnic CO hypoxemia on respiratory afterdischarge and the phrenic neurogram response to supramaximal carotid sinus nerve (CSN) stimulation. Twelve anesthetized, vagotomized, peripherally chemodenervated, ventilated cats with blood pressure controlled were studied. During isocapnic hypoxemia, the amplitude of the phrenic neurogram was progressively depressed. In contrast, the increase in peak phrenic amplitude produced by CSN stimulation was unchanged, suggesting that the central respiratory response to CSN stimulation is unaffected by progressive hypoxemia. The time constant of respiratory afterdischarge (tau) was calculated from best-fit plots of phrenic amplitude vs. time after cessation of CSN stimulation. Under control conditions the value of tau was 57.7 +/- 3 (SE) s (n = 12). During progressive isocapnic hypoxemia, tau decreased as a linear function of arterial O2 content (CaO2) such that a 40% reduction of CaO2 resulted in a 48% reduction in tau. This reduction of respiratory afterdischarge may contribute to the genesis of periodic breathing during hypoxia.

GABA antagonism reverses hypoxic respiratory depression in the cat

We assessed the role of gamma-aminobutyric acid (GABA) as a potential causative agent of hypoxic respiratory depression by monitoring the response of the phrenic neurogram to systemic infusion of the GABA antagonist bicuculline (0.01 mg.kg-1.min-1) under control conditions and during isocapnic brain hypoxia produced by CO inhalation in separate groups of anesthetized, glomectomized, vagotomized, paralyzed, and ventilated cats with blood pressure held constant. The maximum effect of bicuculline in subseizure doses in control cats was to increase minute phrenic activity to 151 +/- 14% of preinfusion values. Infusion was continued until seizure activity was seen in the electroencephalogram. A 53% decrease of arterial O2 content resulted in a marked reduction of both peak phrenic amplitude and phrenic firing frequency to 16 and 64% of control values, respectively. Infusion of bicuculline while the level of hypoxia was maintained constant restored both peak phrenic amplitude and phrenic firing frequency to prehypoxic levels. The maximum effect of bicuculline was to increase minute phrenic activity to 123 +/- 13% of the prehypoxic value. These results suggest that although GABA has only a modest role in determining the output of the control phrenic neurogram, a significant portion of the phrenic depression that occurs during hypoxia can be attributed to inhibition of respiratory neurons by GABA.

CO2 sensitivity of cat phrenic neurogram during hypoxic respiratory depression

The CO2 response of the phrenic neurogram before and during CO-induced isocapnic brain hypoxia was studied in peripherally chemodenervated, vagotomized, paralyzed, ventilated cats with blood pressure held constant. During inhalation of 0.5% CO in 40% O2, arterial O2 content (CaO2) was reduced to 40% and minute phrenic activity to 38.4 +/- 9.4% (SE; n = 9) of prehypoxic levels, primarily due to depression of peak phrenic amplitude (PP). CO2 response, defined as the slope of the plot of PP vs. end-tidal PCO2 during CO2 rebreathing, was unaffected by phrenic depression even to the point of total suppression of phrenic activity in two cats. The effect of the tissue metabolic acidosis associated with hypoxia on phrenic CO2 sensitivity was assessed in a separate group of cats by blocking lactate formation during hypoxia with dichloroacetate (DCA). Preventing lactic acidosis during hypoxia did not affect the CO2 response of the phrenic activity during hypoxia. We conclude that 1) hypoxic depression does not limit the ability of central respiratory neurons to respond to CO2, and 2) the failure of DCA to affect the CO2 response of the phrenic neurogram suggests that brain intracellular lactic acidosis does not modify the phrenic response to hypercapnia.

Respiratory reflexes evoked by tracheal distension

In 13 dogs that were anesthetized and ventilated through an endotracheal tube, segments of either the intrathoracic (ITT) or extrathoracic trachea (ETT) were distended by over-inflating the sealing cuff within the first half of an inspiration and maintaining this overinflation for two breathing cycles. ITT distension caused a significant prolongation of the expiratory duration (TE) of the first challenged breath in 12 of the 13 dogs. ETT distension prolonged the TE in only 5 of the 13 dogs. An inhibition of inspiration resulting in shortened inspiratory time and reduced peak phrenic amplitude (PPA) was measured in three experiments. In six other experiments, there was only a transient decrease in phrenic discharge, which immediately resumed. In the remaining four experiments, the inspiratory burst was unaffected. After vagotomy, ITT distension shortened TE in 8 of the 13 dogs. ETT distension was ineffective in 10 out of 11 experiments; in the remaining experiment TE was significantly lengthened. No significant effects were elicited on TI and PPA by either ITT or ETT distension. These results could be explained by the known behavior of tracheal receptors and could indicate the presence of extravagal influences.