Background

Recent studies showed partial reversal of opioid-induced respiratory depression in the pre-Bötzinger complex and the parabrachial nucleus/Kölliker–Fuse complex. The hypothesis for this study was that opioid antagonism in the parabrachial nucleus/Kölliker–Fuse complex plus pre-Bötzinger complex completely reverses respiratory depression from clinically relevant opioid concentrations.

Methods

Experiments were performed in 48 adult, artificially ventilated, decerebrate rabbits. The authors decreased baseline respiratory rate ~50% with intravenous, “analgesic” remifentanil infusion or produced apnea with remifentanil boluses and investigated the reversal with naloxone microinjections (1 mM, 700 nl) into the Kölliker–Fuse nucleus, parabrachial nucleus, and pre-Bötzinger complex. In another group of animals, naloxone was injected only into the pre-Bötzinger complex to determine whether prior parabrachial nucleus/Kölliker–Fuse complex injection impacted the naloxone effect. Last, the µ-opioid receptor agonist [d-Ala,2N-MePhe,4Gly-ol]-enkephalin (100 μM, 700 nl) was injected into the parabrachial nucleus/Kölliker–Fuse complex. The data are presented as medians (25 to 75%).

Results

Remifentanil infusion reduced the respiratory rate from 36 (31 to 40) to 16 (15 to 21) breaths/min. Naloxone microinjections into the bilateral Kölliker–Fuse nucleus, parabrachial nucleus, and pre-Bötzinger complex increased the rate to 17 (16 to 22, n = 19, P = 0.005), 23 (19 to 29, n = 19, P < 0.001), and 25 (22 to 28) breaths/min (n = 11, P < 0.001), respectively. Naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex prevented apnea in 12 of 17 animals, increasing the respiratory rate to 10 (0 to 12) breaths/min (P < 0.001); subsequent pre-Bötzinger complex injection prevented apnea in all animals (13 [10 to 19] breaths/min, n = 12, P = 0.002). Naloxone injection into the pre-Bötzinger complex alone increased the respiratory rate to 21 (15 to 26) breaths/min during analgesic concentrations (n = 10, P = 0.008) but not during apnea (0 [0 to 0] breaths/min, n = 9, P = 0.500). [d-Ala,2N-MePhe,4Gly-ol]-enkephalin injection into the parabrachial nucleus/Kölliker–Fuse complex decreased respiratory rate to 3 (2 to 6) breaths/min.

Conclusions

Opioid reversal in the parabrachial nucleus/Kölliker–Fuse complex plus pre-Bötzinger complex only partially reversed respiratory depression from analgesic and even less from “apneic” opioid doses. The lack of recovery pointed to opioid-induced depression of respiratory drive that determines the activity of these areas.

Editor’s Perspective
What We Already Know about This Topic
  • Opioid-sensitive inputs to respiratory rate and rhythm originate in the pre-Bötzinger complex, the parabrachial nucleus, and the Kölliker–Fuse nucleus

What This Article Tells Us That Is New
  • The hypothesis that opioid-induced respiratory depression is due to combined depression of parabrachial nucleus/Kölliker–Fuse complex activity and pre-Bötzinger complex activity was tested in a decerebrate, hyperoxic, and moderately hypercapnic rabbit preparation at steady-state intravenous remifentanil infusions that depressed the respiratory rate by 50% and after a remifentanil bolus that produced apnea

  • Sequential naloxone microinjection into the bilateral Kölliker–Fuse nucleus, parabrachial nucleus, and pre-Bötzinger complex did not completely reverse respiratory depression produced by the steady-state remifentanil concentrations and reversed respiratory depression from apneic remifentanil doses even less effectively

  • This suggests that opioids depress respiratory drive to the para brachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex and that depression of drive reduced the activity of these areas, especially at high opioid concentrations

Recent studies of opioid-induced respiratory depression have expanded the focus from the respiratory rhythm generator in the pre-Bötzinger complex to areas that provide inputs to those pre-Bötzinger complex neurons that mediate inspiratory on- and off-switch and thus determine inspiratory and expiratory phase duration.1  We have previously shown in acute in vivo rabbit studies that naloxone injection into the parabrachial nucleus partially reversed respiratory rate depression from “analgesic” remifentanil concentrations,2  whereas injections into the pre-Bötzinger complex did not.3  These results were supported by similar studies in dogs.4,5  Our last study supported the idea of an additional opioid-sensitive source of inputs to the respiratory rhythm generator outside of the parabrachial nucleus and pre-Bötzinger complex.2  Since then, the importance of the Kölliker–Fuse nucleus for the control of respiratory phase duration,6–8  as well as for opioid-induced respiratory depression,9–11  has been highlighted by multiple investigators. In particular, a novel mouse model showed that respiratory depression including from high morphine doses was substantially attenuated when µ-opioid receptors were knocked out selectively in Kölliker–Fuse nucleus neurons.9–11 

We recently showed in our in vivo rabbit model that glutamatergic disfacilitation of the parabrachial nucleus and Kölliker–Fuse nucleus was necessary to achieve maximal respiratory rate depression.8  We thus hypothesized that opioid-induced respiratory depression was due to combined depression of parabrachial nucleus and Kölliker–Fuse nucleus activity. After initial experiments showed that the opioid effect could not be fully reversed in these areas, we extended our hypothesis to include the pre-Bötzinger complex. In contrast to our previous studies that used nonvagotomized animals,2,3  the current experiments were performed in vagotomized animals to prevent the possibility that small changes in respiratory parameters were confounded by ventilator-induced respiratory rate entrainment.

We used local microinjections of the opioid antagonist naloxone into the bilateral Kölliker–Fuse nucleus, parabrachial nucleus, and pre-Bötzinger complex to antagonize the respiratory depression from intravenous remifentanil. To determine whether the magnitude of opioid reversal depended on the opioid dose,11  we used both analgesic concentrations (i.e., steady-state remifentanil infusions that depressed respiratory rate by ~50%),2,3,11–14  as well as “apneic” concentrations (i.e., a remifentanil bolus that just resulted in apnea under control conditions).10  To clarify whether the observed pre-Bötzinger complex effect depended on prior opioid reversal in the parabrachial nucleus/Kölliker–Fuse complex, we added experiments where we injected naloxone solely into the pre-Bötzinger complex. Once we had determined that naloxone reversal of the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex prevented apnea from the apneic remifentanil bolus, we further investigated whether naloxone injection also prevented apnea from very high remifentanil concentrations (i.e., up to 10 times the threshold apneic bolus).

Last, to gauge the degree to which parabrachial nucleus/Kölliker–Fuse complex neurons could be depressed by µ-opioid receptor agonists, we injected the µ-opioid receptor agonist [D-Ala,2N-MePhe,4Gly-ol]-enkephalin (DAMGO) at high, supraclinical concentrations into the bilateral parabrachial nucleus/Kölliker–Fuse complex.

Surgical Preparation

The research was approved by the Subcommittee on Animal Studies of the Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin, and the Institutional Animal Care and Use Committee, Medical College of Wisconsin, Milwaukee, Wisconsin, in accordance with provisions of the Animal Welfare Act, the Public Health Service Guide for the Care and Use of Laboratory Animals, and Veterans Affairs policy. Experiments were carried out on adult (3 to 4 kg), pathogen-free, New Zealand White rabbits of either sex. Anesthesia was induced with 5 vol% sevoflurane via facemask and ventilated via tracheotomy with an anesthesia machine (Ohmeda CD, GE, Datex Ohmeda, USA). Anesthesia was maintained with 1.5 to 3% isoflurane. Inspiratory oxygen fraction, expiratory carbon dioxide concentration and expiratory isoflurane concentration were continuously displayed with an infrared analyzer (POET II, Criticare Systems, USA). Skin was infiltrated with lidocaine 1% before each skin incision. Femoral arterial and venous lines were used for blood pressure monitoring, infusion of solutions, and bolus drug administration, respectively. Care was taken to increase anesthetic depth for any signs of “light anesthesia” (e.g., an increase in blood pressure or lacrimation). Lactated Ringer’s solution with 3 μg/ml epinephrine was continuously infused at 1 ml/h. At this rate, the infusion did not result in appreciable changes in heart rate and blood pressure from baseline. Infusion rate was increased as needed to counteract or prevent hypotension in response to drug injections and/or from blood loss but maintained as constant as possible during the recording phase. The animal was maintained at 37.0 ± 0.5°C with a warming blanket. The animal was placed in a stereotaxic frame (David Kopf Instruments, USA), and blunt precollicular decerebration with complete removal of the forebrain was performed through a parietal craniotomy. After decerebration, isoflurane was either discontinued or continued at subanesthetic levels (0.3 to 0.4 vol%) for blood pressure control. This sedative concentration equals ~0.2 minimum alveolar concentration,15  which is associated with a decrease of 10% or less in respiratory rate and peak phrenic activity.16,17  Volatile anesthetics add to but do not amplify the remifentanil effect16  (i.e., slight variation in isoflurane concentration between experiments would affect the baseline respiratory rate and peak phrenic activity, but not the dose dependency of the remifentanil effect). Isoflurane concentration was not changed during the experimental protocol. Decerebration eliminates the need for further general anesthesia, but it may reduce forebrain and midbrain inputs to the respiratory center and thus cause a minor increase in apneic threshold.18,19  The brainstem was exposed via occipital craniotomy and partial removal of the cerebellum. The animals were paralyzed with rocuronium (15 mg/kg subcutaneous bolus), followed by pancuronium 2 mg/h infusion to avoid motion artifacts during neural/neuronal recording. Bilateral vagotomy was performed to achieve peripheral deafferentation to avoid interference of the mechanical ventilation with the underlying central respiratory rhythm and respiratory neuronal activity. Respiratory rates after vagotomy were comparable to our previous studies in nonvagotomized animals.2,3  The phrenic nerve and in some experiments the vagus nerve were recorded with fine bipolar electrodes through a posterior neck incision. The complete surgical preparation required 6 to 7 h.

Throughout the experiment, the animals were ventilated with a hyperoxic gas mixture (Fio2 0.6) to achieve functional denervation of the peripheral chemoreceptors and thus rule out that the observed drug effects were due to effects on the carotid bodies. Mild hypercapnia (expiratory carbon dioxide, 45 to 55 mmHg) was used to emulate the hypercarbia encountered clinically during opioid use in patients. Mild hypercapnia may also have compensated for the loss of respiratory drive with decerebration because respiratory rates were similar to rabbit preparations using normocapnia in anesthetized, nondecerebrate preparations.12,20  Blood pressure was maintained stable throughout the experiment by adjusting the intravenous infusion rate. At the end of the experiment, the animals were euthanized with intravenous potassium chloride.

Neuronal Recording, Microinjection Procedures, and Measured Variables

All neuronal recording and microinjection techniques have been well established by our research group and have been previously described in detail.21,22  In short, extracellular neuronal recordings were obtained using multibarrel micropipettes (20- to 40-μm tip diameter) consisting of three drug barrels and a recording barrel containing a 7-μm-thick carbon filament. The barrels were filled with the glutamatergic agonist α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 50 μM, 70 nl/injection) and the opioid receptor antagonist naloxone (1 mM, 700 nl/injection), which were dissolved in artificial cerebrospinal fluid. The microinjected volume was determined via height changes in the meniscus in the respective pipette barrel with a 100× monocular microscope and calibrated reticule (resolution ~3.5 nl). Respiratory neuronal discharge was recorded extracellularly from neuronal aggregates and individual neurons and classified by the temporal relationships relative to the phrenic neurogram. The neuronal and neural activity and pressure microejection marker signals were recorded using a digital acquisition system. These variables were also continuously displayed and recorded along with the phrenic neurogram, vagal neurogram, discharge rate meter, respiratory rate, arterial blood pressure, and airway carbon dioxide concentration on a computerized chart recorder (Powerlab/16SP; ADInstruments, Australia). Before and after drug injection, steady-state conditions were obtained for respiratory parameters. Postexperiment LabChart data were exported to SigmaPlot 11 (Systat Software, USA) for data reduction, data plotting, and statistical analysis. Between 10 and 50 consecutive respiratory cycles were averaged over 1 to 2 min with the number of cycles dependent on the respiratory rate. Using the phrenic neurogram, we determined the respiratory rate, inspiratory and expiratory duration, and peak phrenic activity. Using the vagal neurogram, we determined peak vagus activity. In rabbits, inspiratory phase timing of the vagal neurogram closely matches the phrenic neurogram without the postinspiratory activity typically observed in rats10  but with minor activity during midexpiration. Peak vagus activity was calculated as the amplitude between minimal vagus nerve activity before the start of inspiration and peak vagus nerve activity during inspiratory phase. Because changes in peak phrenic activity closely reflect changes in respiratory tidal volume but the absolute value does not correspond with the absolute tidal volume,23  peak phrenic activity and peak vagus activity were normalized to the respective control values for all calculations.

Identification of the Parabrachial Nucleus, Kölliker–Fuse Nucleus, and Pre-Bötzinger Complex

We previously characterized the locations of the Kölliker–Fuse nucleus, parabrachial nucleus,8  and pre-Bötzinger complex22  in our model through stereotaxic coordinates, neuronal recordings, and typical respiratory rate response to AMPA injection. For protocols investigating only the parabrachial nucleus and Kölliker–Fuse nucleus, we inserted the micropipette in a grid-wise fashion starting at the caudal end of the inferior collicle at 1.5 mm lateral from midline and moved lateral and caudal with 0.5-mm steps (0.47 mm rostro-caudal, corrected for the 20° angle of the stereotaxic frame). In areas where neuronal activity was encountered, we microinjected AMPA (50 µM, 70 nl) starting at the ventral limit of the tonic neuronal activity and then in 1-mm steps more dorsally. The area of maximal AMPA-induced tachypnea was labeled as “parabrachial nucleus,” and the area of maximal bradypnea was labeled as “Kölliker–Fuse nucleus” (fig. 1). The parabrachial nucleus was located 1.0 ± 0.9 mm caudal from the inferior collicle, 2.6 ± 0.7 mm lateral to midline, and 7.7 ± 1.9 mm ventral to the dorsal surface, and the Kölliker–Fuse nucleus was located 1.1 ± 0.2 mm caudal, 0.7 ± 0.3 mm lateral, and 1.8 ± 0.7 mm ventral to the parabrachial nucleus (n = 12). Because the parabrachial nucleus to Kölliker–Fuse nucleus distance was consistent and matched our previous study,8  in later experiments we functionally identified the parabrachial nucleus and used the location 1 mm caudal, 0.5 mm lateral and, 2 mm ventral to the parabrachial nucleus for Kölliker–Fuse nucleus injections.

Fig. 1.

Brainstem locations of naloxone microinjection. (A) Phrenic neurogram tracings illustrate the functional identification of the parabrachial nucleus, Kölliker–Fuse nucleus, and pre-Bötzinger complex through typical responses to injection of the glutamate receptor agonist α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; vertical arrows). (B) Dorsal view of the brainstem. Superimposed are the approximate distribution areas of the naloxone injections into the parabrachial nucleus (blue), Kölliker–Fuse nucleus (green), and pre-Bötzinger complex (red). We estimate an effective spherical diffusion radius for our injection volume of 1 to 1.2 mm.22  There is little overlap between the parabrachial nucleus and Kölliker–Fuse nucleus injections in the brainstem, because the Kölliker–Fuse nucleus is located 2 mm ventral to the parabrachial nucleus. (C) Stereotaxic coordinates of the naloxone injection sites for parabrachial nucleus injections (blue squares) and Kölliker–Fuse nucleus injections (green squares), projected over coronal slices of the rostral pons. Coordinates for the bilateral injections were averaged for each animal. For clarity, injection sites less than 1.5 mm caudal to the inferior collicle are summarized in the slice labeled “-1.0mm”; coordinates between 1.5 and 2.5 mm caudal to the inferior collicle are summarized in the slice labeled “-2.0mm”; and injection sites 2.5 mm or more caudal to the inferior collicle are summarized in the slice labeled “-3.0mm.” To account for residual cerebellar tissue covering the dorsal brainstem in our preparation, we subtracted 5 mm from the measured stereotaxic depth coordinate in all animals (i.e., the depicted depth of injection is an approximation). (D) Stereotaxic coordinates of the naloxone injections into the pre-Bötzinger complex after injection into the parabrachial nucleus and Kölliker–Fuse nucleus (yellow squares, cohort A) or solely into the pre-Bötzinger complex (red squares, cohort B), projected over coronal slices of the caudal medulla oblongata. Coordinates for the bilateral injections were averaged for each animal. Injection sites less than 1.5 mm rostral to obex are summarized in the slice labeled “+1.0mm.” Injection sites 1.5 mm or more rostral to obex are summarized in the slice labeled “+2.0mm.” Please see “Identification of the Parabrachial Nucleus, Kölliker–Fuse Nucleus, and Pre-Bötzinger Complex” for the average stereotaxic coordinates. The outlines of the maps are redrawn from histologic sections obtained for our previous studies in adult rabbits.8,22  The atlas of Meessen and Olszewski46  was used for comparison. Please note the different scale for (D).

Fig. 1.

Brainstem locations of naloxone microinjection. (A) Phrenic neurogram tracings illustrate the functional identification of the parabrachial nucleus, Kölliker–Fuse nucleus, and pre-Bötzinger complex through typical responses to injection of the glutamate receptor agonist α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; vertical arrows). (B) Dorsal view of the brainstem. Superimposed are the approximate distribution areas of the naloxone injections into the parabrachial nucleus (blue), Kölliker–Fuse nucleus (green), and pre-Bötzinger complex (red). We estimate an effective spherical diffusion radius for our injection volume of 1 to 1.2 mm.22  There is little overlap between the parabrachial nucleus and Kölliker–Fuse nucleus injections in the brainstem, because the Kölliker–Fuse nucleus is located 2 mm ventral to the parabrachial nucleus. (C) Stereotaxic coordinates of the naloxone injection sites for parabrachial nucleus injections (blue squares) and Kölliker–Fuse nucleus injections (green squares), projected over coronal slices of the rostral pons. Coordinates for the bilateral injections were averaged for each animal. For clarity, injection sites less than 1.5 mm caudal to the inferior collicle are summarized in the slice labeled “-1.0mm”; coordinates between 1.5 and 2.5 mm caudal to the inferior collicle are summarized in the slice labeled “-2.0mm”; and injection sites 2.5 mm or more caudal to the inferior collicle are summarized in the slice labeled “-3.0mm.” To account for residual cerebellar tissue covering the dorsal brainstem in our preparation, we subtracted 5 mm from the measured stereotaxic depth coordinate in all animals (i.e., the depicted depth of injection is an approximation). (D) Stereotaxic coordinates of the naloxone injections into the pre-Bötzinger complex after injection into the parabrachial nucleus and Kölliker–Fuse nucleus (yellow squares, cohort A) or solely into the pre-Bötzinger complex (red squares, cohort B), projected over coronal slices of the caudal medulla oblongata. Coordinates for the bilateral injections were averaged for each animal. Injection sites less than 1.5 mm rostral to obex are summarized in the slice labeled “+1.0mm.” Injection sites 1.5 mm or more rostral to obex are summarized in the slice labeled “+2.0mm.” Please see “Identification of the Parabrachial Nucleus, Kölliker–Fuse Nucleus, and Pre-Bötzinger Complex” for the average stereotaxic coordinates. The outlines of the maps are redrawn from histologic sections obtained for our previous studies in adult rabbits.8,22  The atlas of Meessen and Olszewski46  was used for comparison. Please note the different scale for (D).

Close modal

For protocols including the pre-Bötzinger complex, we identified the pre-Bötzinger complex as the area with inspiratory and expiratory neuronal activity where AMPA injection caused maximal tachypnea (fig. 1). The pre-Bötzinger complex was located on average 2.1 ± 0.7 mm rostral to obex, 2.7 ± 0.4 mm lateral from midline and 5.0 ± 0.5 mm ventral to the dorsal surface (n = 32). The parabrachial nucleus was located 9.6 ± 0.7 mm rostral to the pre-Bötzinger complex at the same distance lateral from midline and, dependent on the thickness of the residual cerebellar peduncle, 3.2 ± 1.2 mm ventral to the pre-Bötzinger complex (n = 23). Complete, bilateral functional identification of all areas including the time required for respiratory rate to return to baseline after each AMPA injection required 4 to 5h.

Opioid Effect Sites at Analgesic IV Remifentanil Concentrations

Experimenters were not blinded to the experimental conditions, and we did not perform formal randomization to experimental protocols. Because the effect of naloxone microinjection persists more than 2 h, only one complete protocol was performed per animal.

To determine how much opioid effects in the Kölliker–Fuse nucleus and parabrachial nucleus contributed to systemic opioid-induced respiratory depression at analgesic opioid doses, we infused remifentanil intravenously at 0.15 ± 0.08 µg · kg–1 · min–1 until the respiratory rate was depressed by approximately 50% (fig. 2). In rabbits, a ~50% respiratory rate depression was associated with loss of response to ear pinch and pedal withdrawal reflex.14,24  Its fast onset and short half-life that is independent of the duration of infusion12,25  make remifentanil the ideal drug to investigate opioid effects at steady-state concentrations. After reaching steady-state effect for 10 to 15 min, naloxone (1 mM, 700 nl) was microinjected bilaterally into the Kölliker–Fuse nucleus, and 5 min were allowed to obtain maximal effect. Subsequently, naloxone was injected into the bilateral parabrachial nucleus. After interim analysis revealed that naloxone injections into the Kölliker–Fuse nucleus and parabrachial nucleus did not lead to complete reversal of respiratory rate depression, for all subsequent protocols, we additionally injected naloxone into the bilateral pre-Bötzinger complex. Volume and timing for Kölliker–Fuse nucleus, parabrachial nucleus, and pre-Bötzinger complex microinjections had been established in previous studies.8,22  Animals in which naloxone was injected into the Kölliker–Fuse nucleus, parabrachial nucleus, and pre-Bötzinger complex are included in cohort A.

Fig. 2.

Injection sequence for intravenous remifentanil and local naloxone microinjections. The initial intravenous remifentanil bolus (red arrow) was chosen to cause apnea for more than 30 s. Once the respiratory activity returned, the remifentanil infusion was started (red line). In many animals, one or more adjustments of the infusion rate were necessary to achieve the targeted respiratory rate depression of 50%. Steady-state respiratory rate depression was confirmed for 15 min before the start of the brainstem injections. The remifentanil dose rate was continued unchanged throughout the entire injection sequence. For cohort A, naloxone (blue arrows) was microinjected into the bilateral Kölliker–Fuse nucleus and parabrachial nucleus in 19 animals and subsequently into the pre-Bötzinger complex in 12 of these animals. Apneic IV remifentanil boluses (red arrows) were given after naloxone injections into the pons and after injection into the pre-Bötzinger complex. After each IV remifentanil bolus, we awaited recovery of the respiratory rate to the prebolus level before subsequent injections. In six animals, we also tested a very high IV remifentanil dose. For cohort B, naloxone was microinjected only into the bilateral pre-Bötzinger complex. Because the subsequent apneic IV remifentanil bolus continued to cause apnea in the majority of animals, no very high IV remifentanil bolus was given in this cohort.

Fig. 2.

Injection sequence for intravenous remifentanil and local naloxone microinjections. The initial intravenous remifentanil bolus (red arrow) was chosen to cause apnea for more than 30 s. Once the respiratory activity returned, the remifentanil infusion was started (red line). In many animals, one or more adjustments of the infusion rate were necessary to achieve the targeted respiratory rate depression of 50%. Steady-state respiratory rate depression was confirmed for 15 min before the start of the brainstem injections. The remifentanil dose rate was continued unchanged throughout the entire injection sequence. For cohort A, naloxone (blue arrows) was microinjected into the bilateral Kölliker–Fuse nucleus and parabrachial nucleus in 19 animals and subsequently into the pre-Bötzinger complex in 12 of these animals. Apneic IV remifentanil boluses (red arrows) were given after naloxone injections into the pons and after injection into the pre-Bötzinger complex. After each IV remifentanil bolus, we awaited recovery of the respiratory rate to the prebolus level before subsequent injections. In six animals, we also tested a very high IV remifentanil dose. For cohort B, naloxone was microinjected only into the bilateral pre-Bötzinger complex. Because the subsequent apneic IV remifentanil bolus continued to cause apnea in the majority of animals, no very high IV remifentanil bolus was given in this cohort.

Close modal

To determine whether the effect of pre-Bötzinger complex injections depended on prior naloxone reversal in the parabrachial nucleus and Kölliker–Fuse nucleus, in a separate set of animals (cohort B), we infused remifentanil until steady-state and injected naloxone solely into the pre-Bötzinger complex. At the end of the experiments, naloxone (15 to 40 µg/kg) was injected intravenously to document that the respiratory rate returned to preremifentanil control. We have previously described that artificial cerebrospinal fluid and naloxone injections into the parabrachial nucleus or pre-Bötzinger complex did not have any independent effects and did not repeat those control injections in the current study.2,3,26 

Opioid Effect Sites at Apneic IV Remifentanil Concentrations

To determine whether the contributions of the Kölliker–Fuse nucleus, parabrachial nucleus, and pre-Bötzinger complex to opioid-induced respiratory depression depended on the opioid concentration, we injected a remifentanil bolus that was sufficient to cause apnea for more than 30 s (fig. 2). Because the exact dose to achieve apnea varied between animals, we chose 10 µg (~3 µg/kg) as a standard dose. However, boluses were repeated with larger doses when the initial bolus did not result in apnea. Because repeat doses required return of the respiratory rate to baseline, this added 30 to 60 min to the experiment. In cohort A, the apneic bolus was repeated after naloxone injection into the parabrachial nucleus/Kölliker–Fuse nucleus and again after naloxone injection into the pre-Bötzinger complex. In cohort B, the apneic bolus was repeated after naloxone injection solely into the pre-Bötzinger complex.

To compare the effect sites of analgesic and apneic remifentanil concentrations in the same animals, the entire experimental sequence consisted of the apneic intravenous remifentanil bolus, after which we started the remifentanil infusion and waited until respiratory rate reached steady-state depression (~50% control) for 10 to 15 min. If respiratory rate was substantially higher or lower than 50%, we adjusted the remifentanil infusion rate and waited until a new steady-state was obtained for more than 10 min (i.e., for an additional 20 to 30 min). Once a satisfactory infusion rate was established, the rate was not changed throughout the entire naloxone injection protocol. In cohort A, at steady-state respiratory depression, we performed bilateral naloxone microinjections into the Kölliker–Fuse nucleus and, after a 5-min wait, into the parabrachial nucleus. When respiratory parameters had reached steady state after the parabrachial nucleus injection (5 to 10 min), we repeated the apneic bolus. The concurrent remifentanil infusion meant that the plasma concentrations after the repeat apneic bolus were somewhat higher than after the initial bolus; however, the time requirement to discontinue the remifentanil infusion, inject and recover from the apneic bolus, restart the infusion, and achieve the same steady-state conditions as before would have been prohibitive. To obtain paired data for analgesic and apneic concentrations, we chose to continue the remifentanil infusion. After the apneic bolus we waited for respiratory parameters to recover to pre-bolus values and performed bilateral naloxone microinjections into the pre-Bötzinger complex. After the effects of the naloxone injection had reached steady state (5 min), we repeated the apneic remifentanil bolus. After recovery of the respiratory rate to the prebolus rate, we injected naloxone intravenously to document that the control respiratory rate had not changed. Only then was the remifentanil infusion discontinued.

In cohort B, the initial remifentanil bolus and infusion rate were determined in the same fashion, but naloxone was injected only into the pre-Bötzinger complex. The entire experiment including surgical preparation, functional identification of the injection sites, and the remifentanil/naloxone protocol required 16 to 18 h. Depending on whether the experiments required functional identification of the parabrachial nucleus and Kölliker–Fuse nucleus in addition to the pre-Bötzinger complex, the first remifentanil injection occurred between 6 and 8 pm, and the experiments often lasted past midnight. Hemodynamics, end-tidal carbon dioxide, and nerve recordings were remarkably stable throughout the entire experiment.

Effects of Very High Remifentanil Concentrations on Areas outside the Parabrachial Nucleus/Kölliker–Fuse complex and Pre-Bötzinger Complex

Initial experiments showed that naloxone injection into the parabrachial nucleus/Kölliker–Fuse nucleus and pre-Bötzinger complex reliably prevented apnea from the apneic remifentanil bolus (10 µg). We sought to determine whether naloxone reversal of these areas would be able to prevent apnea including from very high remifentanil doses or whether very high remifentanil doses would also depress other respiratory-related areas (e.g., respiratory drive to the parabrachial nucleus/Kölliker–Fuse nucleus and pre-Bötzinger complex or respiratory motor output). In a subgroup of cohort A, we completed the naloxone injection sequence into the Kölliker–Fuse nucleus, parabrachial nucleus, and pre-Bötzinger complex to test the reversal of analgesic and apneic remifentanil concentrations as described under “Opioid Effect Sites at Analgesic IV Remifentanil Concentrations” and “Opioid Effect Sites at Apneic IV Remifentanil Concentrations.” After the final apneic remifentanil bolus, we allowed the respiratory rate to recover to prebolus values with the remifentanil infusion running. We then injected 10- to 50-µg bolus doses of remifentanil intravenously in short sequence until we observed apnea in the phrenic neurogram or to a maximal dose of 100 µg of remifentanil. For this analysis, we also determined peak vagus activity.

Effects of Supraclinical Opioid Concentrations, Compared to Glutamate Receptor Blockade in the Parabrachial Nucleus and Kölliker–Fuse Nucleus

We investigated whether maximal opioid receptor activation in the parabrachial nucleus and Kölliker–Fuse nucleus resulted in similar effects as disfacilitating neuronal activity with glutamate antagonist injection.8  In a separate set of animals, we injected the µ-opioid receptor agonist DAMGO at supraclinical concentrations (100 µM, 700 nl) bilaterally into the functionally identified parabrachial nucleus and Kölliker–Fuse nucleus. We statistically compared the opioid effects with results from our previous study in which parabrachial nucleus and Kölliker–Fuse nucleus activity was eliminated using microinjections of the non–N-methyl-d-aspartate (NMDA) receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX; 1 mM, 700 nl/injection) and NMDA receptor antagonist d(–)-2-amino-5-phosphonopentanoic acid (AP5; 5 mM, 700 nl/injection).8 

Initial comparison of the two data sets showed a difference in baseline respiratory rate. Because both DAMGO and AP5/NBQX injections reduced the respiratory rate to the single digits, a difference in the change in respiratory parameters between groups could have been due to the difference in baseline rate. In response to peer review, we matched the baseline respiratory rates in both groups. We performed two additional experiments using DAMGO injections and excluded one animal with a respiratory rate of less than 20 breaths/min, and we excluded animals with respiratory rates of more than 37 breaths/min from the original AP5/NBQX data set.6–8,27  Of note, the selection of data subsets may have biased the results toward the properties of these animals. In our adult outbred rabbit model, we observe a natural variation in baseline respiratory rate between ~20 and 40 breaths/min that remains remarkably constant over the course of many hours. Individual baseline rate does not clearly correlate with sex, age, or weight and varies even between animals of the same litter when experiments are performed in the same week. However, we cannot rule out that despite our efforts at consistency in surgical preparation and experimental conditions, respiratory control and rate were influenced by unrecognized factors that may have changed since our previous study.6–8,27  It is also possible that pontine function is different in animals with very high or low baseline respiratory rates and that the interpretation of our data is limited to animals with baseline respiratory rates between 20 and 37 breaths/min.

Control Studies: Effects of Naloxone or Artificial Cerebrospinal Fluid Injections into the Parabrachial Nucleus, Kölliker–Fuse Nucleus, and Pre-Bötzinger Complex

To ensure that the effect of local naloxone microinjections we observed during remifentanil infusion represented a reversal of the extrinsic opioid effect rather than antagonism of endogenous opioid receptor activation or γ-aminobutyric acid type A (GABAA) receptor antagonism,28  we injected naloxone (1 mM, 700 nl) sequentially into the bilateral Kölliker–Fuse nucleus, parabrachial nucleus, and pre-Bötzinger complex. We also injected artificial cerebrospinal fluid (700 nl), which was used as solvent for all injected drugs, into the bilateral Kölliker–Fuse nucleus, parabrachial nucleus, and pre-Bötzinger complex to rule out an independent effect of the solvent.

Statistical Analysis

Statistical analysis was performed using SigmaPlot 11 (Systat Software, USA). We did not perform a formal power analysis, and no adjustments were made for interim analyses. Comparable studies have used 4 to 9 rats,10,29  4 to 11 mice,11,13  8 to 16 rabbits,2,3  and 10 to 21 dogs4,5  per protocol.

Because the experiments were technically very difficult and labor-intensive, we included data from a few animals where a single data point (injection of the apneic remifentanil dose into either the parabrachial nucleus/Kölliker–Fuse complex or pre-Bötzinger complex) was missing. The total number of animals is indicated for each comparison. To eliminate the problem of “missing values,” we calculated the difference (Δ) for each variable between naloxone injection into the Kölliker–Fuse nucleus and IV remifentanil, between naloxone injection into the parabrachial nucleus and Kölliker–Fuse nucleus, between naloxone injection into the pre-Bötzinger complex and parabrachial nucleus, and between naloxone injection into the pre-Bötzinger complex and IV remifentanil. Testing revealed that not all Δ values were normally distributed (Shapiro–Wilk test). For paired data, we uniformly used the Wilcoxon Signed Rank test to test each Δ against no change (null hypothesis). Within the same protocol, we used the Mann–Whitney U test to compare the effects of naloxone injection into the pre-Bötzinger complex between animals in which naloxone had been previously injected into the parabrachial nucleus/Kölliker–Fuse complex and those in which naloxone was solely injected into the pre-Bötzinger complex (unpaired data). Hypothesis testing was two-tailed. The critical value for significant differences was adjusted according to the number of comparisons for each protocol according to Bonferroni (i.e., P < 0.01 for analgesic remifentanil concentrations [five comparisons] and P < 0.0125 for apneic remifentanil concentrations [four comparisons]). Results for the different remifentanil concentrations were analyzed separately without additional correction for multiple comparisons. Inputs to inspiratory on- and off-switch were calculated from the values for inspiratory and expiratory duration as described in the appendix. For “Effects of Very High Remifentanil Concentrations on Areas outside the Parabrachial Nucleus/Kölliker–Fuse Complex and Pre-Bötzinger Complex,” the values for inspiratory duration and peak phrenic activity were log transformed, and the adjusted correlation coefficients (R2, squares of Pearson’s correlation) were compared using bootstrap analysis (R 3.5.0, R Foundation for Statistical Computing, Austria). In accordance with the exploratory nature of the study, for additional comparisons (e.g., between different remifentanil concentrations, between respiratory parameters, or between peak phrenic and peak vagus activity), we used Cohen’s d. This is defined as the difference between two means (µ1 – µ2), divided by the pooled SD (s): d = (µ1 – µ2)/s. The pooled SD is weighted for the number of samples (n) in each group: s = (s12 *(n1 – 1) + s22 *(n2 – 1))/(n1+n2 – 2))1/2. A Cohen’s d of 0.5 or more is considered a “medium” difference, 0.8 or more is considered a “large” difference, and 2 or more is considered a “huge” difference.30  In addition, the groups were compared using the Mann–Whitney U test without corrections for multiple comparisons. The parameters are presented as medians (25 to 75% range).

Primary outcomes were the changes in respiratory parameters with local microinjection of naloxone at one remifentanil concentrations or microinjection of DAMGO, respectively. Secondary outcomes were comparisons between respiratory parameters (e.g., inspiratory and expiratory duration) at the same remifentanil concentration, between the effects on different areas (i.e., parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex) at the same remifentanil concentration, between different opioid concentrations, and between phrenic and vagal nerve activity.

In total, 48 animals were used in our studies. Three animals died during the surgical preparation, and four animals developed significant respiratory slowing during AMPA mapping and were removed from further study. No animal died during the remifentanil/naloxone injection sequence.

Opioid Effect Sites at Analgesic IV Remifentanil Concentrations

To determine the effect of analgesic remifentanil concentrations on the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex, we microinjected naloxone into the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex (cohort A) or the pre-Bötzinger complex alone (cohort B) during remifentanil infusion, dosed to depress the baseline respiratory rate by 50%. Cohort A consisted of eight female (3.5 ± 0.9 kg) and 11 male (2.8 ± 0.4 kg) animals, and cohort B consisted of four female (3.7 ± 0.6 kg) and six male (3.3 ± 0.4 kg) animals. In cohort A, the continuous remifentanil infusion depressed the respiratory rate from 36 to 16 breaths/min (fig. 3B, left). Sequential, bilateral microinjections of naloxone into the bilateral Kölliker–Fuse nucleus, parabrachial nucleus, and pre-Bötzinger complex (n = 12) increased the respiratory rate to 17 (P = 0.005), 23 (P <0.001), and 25 breaths/min (P <0.001), respectively. For the 25 to 75% range, please see table 1. In Cohort B, remifentanil infusion decreased the respiratory rate from 33.5 to 17 breaths/min (fig. 3B, right). Naloxone microinjection solely into the pre-Bötzinger complex increased the respiratory rate to 20.5 breaths/min (P = 0.005). The effect of naloxone injection into the pre-Bötzinger complex was similar with or without prior naloxone injection into the parabrachial nucleus/Kölliker–Fuse nucleus (P = 0.242; fig. 3B, blue bracket).

Table 1.

Summary Data for Analgesic and Apneic Remifentanil Concentrations in Cohorts A and B

Summary Data for Analgesic and Apneic Remifentanil Concentrations in Cohorts A and B
Summary Data for Analgesic and Apneic Remifentanil Concentrations in Cohorts A and B
Fig. 3.

Analgesic remifentanil (REMI) concentrations. Bilateral naloxone (NAL) injections into the Kölliker–Fuse (KF) nucleus, the parabrachial nucleus (PBN), and the pre-Bötzinger complex (preBotC) significantly reversed the respiratory rate depression from intravenous remifentanil. The analgesic remifentanil dose rate was chosen to achieve ~50% respiratory rate depression. (A) Phrenic neurogram tracings during control conditions and sequential drug injections in an individual rabbit. (B–E) Pooled data for changes in respiratory rate and other respiratory parameters. (F, G) Values for inputs to inspiratory on- and off-switch were derived from the values for inspiratory and expiratory duration and are presented as percentages of apneic threshold with the apneic threshold equal to 100% (appendix). The data for cohort A are presented on the left side of each panel. Sequential naloxone injections into the Kölliker–Fuse nucleus and the parabrachial nucleus were performed in 19 animals. In 12 of these animals, naloxone was subsequently injected into the pre-Bötzinger complex. The data for cohort B are presented on the right side of each panel (shaded). In a separate group of 10 animals, naloxone was injected only into the pre-Bötzinger complex. Black brackets indicate that the difference (Δ) between values from two sequential injections was tested against no change (Wilcoxon signed rank test). Blue brackets indicate comparison of the Δ values from pre-Bötzinger complex injection with and without prior naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex (Mann–Whitney rank sum test). The levels of significance below the critical P = 0.01 are highlighted in red. The phrenic neurogram traces and pooled data are color-coded for the same condition to facilitate reader orientation.

Fig. 3.

Analgesic remifentanil (REMI) concentrations. Bilateral naloxone (NAL) injections into the Kölliker–Fuse (KF) nucleus, the parabrachial nucleus (PBN), and the pre-Bötzinger complex (preBotC) significantly reversed the respiratory rate depression from intravenous remifentanil. The analgesic remifentanil dose rate was chosen to achieve ~50% respiratory rate depression. (A) Phrenic neurogram tracings during control conditions and sequential drug injections in an individual rabbit. (B–E) Pooled data for changes in respiratory rate and other respiratory parameters. (F, G) Values for inputs to inspiratory on- and off-switch were derived from the values for inspiratory and expiratory duration and are presented as percentages of apneic threshold with the apneic threshold equal to 100% (appendix). The data for cohort A are presented on the left side of each panel. Sequential naloxone injections into the Kölliker–Fuse nucleus and the parabrachial nucleus were performed in 19 animals. In 12 of these animals, naloxone was subsequently injected into the pre-Bötzinger complex. The data for cohort B are presented on the right side of each panel (shaded). In a separate group of 10 animals, naloxone was injected only into the pre-Bötzinger complex. Black brackets indicate that the difference (Δ) between values from two sequential injections was tested against no change (Wilcoxon signed rank test). Blue brackets indicate comparison of the Δ values from pre-Bötzinger complex injection with and without prior naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex (Mann–Whitney rank sum test). The levels of significance below the critical P = 0.01 are highlighted in red. The phrenic neurogram traces and pooled data are color-coded for the same condition to facilitate reader orientation.

Close modal

In cohort A, remifentanil infusion decreased peak phrenic activity from 100 to 84% of control (fig. 3C, left). Naloxone injections into the Kölliker–Fuse nucleus, parabrachial nucleus, and pre-Bötzinger complex increased peak phrenic activity to 87 (P = 0.022), 88 (P <0.001), and 91% (P = 0.898), respectively. In Cohort B, remifentanil infusion decreased peak phrenic activity from 100 to 89%, and naloxone injection into the pre-Bötzinger complex did not reverse the depression (96%, P = 0.039; fig. 3C, right). The effect of naloxone injection into the pre-Bötzinger complex was similar with or without prior naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex (P = 0.193; fig. 3C, blue bracket).

In cohort A, remifentanil infusion increased inspiratory duration from 0.78 to 1.5 s (fig. 3D, left). Naloxone injections into the Kölliker–Fuse nucleus, parabrachial nucleus, and pre-Bötzinger complex decreased inspiratory duration to 1.43 (P = 0.012), 1.2 (P <0.001), and 1.16 s (P = 0.007), respectively. In cohort B, remifentanil increased inspiratory duration from 0.84 to 1.32 s, which naloxone injection into the pre-Bötzinger complex did not change (1.35 s, P = 0.375; fig. 3D, right). The effect of naloxone injection into the pre-Bötzinger complex was similar with and without prior naloxone injection into the parabrachial nucleus/Kölliker–Fuse nucleus (P = 0.149; fig. 3D, blue bracket).

In cohort A, remifentanil infusion increased expiratory duration from 0.84 to 2.22 s (fig. 3E, left). Naloxone injections into the Kölliker–Fuse nucleus, parabrachial nucleus, and pre-Bötzinger complex decreased expiratory duration to 2.01 (P = 0.008), 1.50 (P <0.001), and 1.28 s (P <0.001), respectively. In cohort B, remifentanil infusion increased expiratory duration from 1.08 to 2.26 s, which was decreased by naloxone injection into the pre-Bötzinger complex to 1.68 s (P = 0.002; fig. 3E, right). The decrease in expiratory duration with naloxone injection into the pre-Bötzinger complex alone was greater than the decrease after prior naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex (P < 0.001; fig. 3E, blue bracket).

In cohort A, remifentanil infusion decreased inputs to inspiratory off-switch from 185 to 129% of apneic threshold (fig. 3F, left). Naloxone injections into the Kölliker–Fuse nucleus, parabrachial nucleus, and pre-Bötzinger complex increased these inputs to 131% (P = 0.002), 143% (P < 0.001), and 146% (P = 0.024), respectively. In cohort B, remifentanil decreased inputs to inspiratory off-switch from 161 to 136%, which naloxone injection into the pre-Bötzinger complex did not change (134%, P = 0.064; fig. 3F, right). The effect of naloxone injection into the pre-Bötzinger complex was similar with and without prior naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex (P = 0.460; fig. 3F, blue bracket).

In cohort A, remifentanil decreased inspiratory on-switch from 175 to 112% of apneic threshold (fig. 3G, left). Naloxone injections into the Kölliker–Fuse nucleus, parabrachial nucleus, and pre-Bötzinger complex increased these inputs to 116 (P = 0.007), 129 (P <0.001), and 138% of apneic threshold (P <0.001), respectively. In cohort B, inputs to inspiratory on-switch decreased with remifentanil infusion from 152 to 112% of apneic threshold and increased with naloxone injection into the pre-Bötzinger complex to 122% (P = 0.002; fig. 3G, right). The effect of naloxone injection into the pre-Bötzinger complex was similar with and without prior naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex (P = 0.699; fig. 3G, blue bracket), suggesting that the observed difference in expiratory duration may have been due to the slower respiratory rate and longer expiratory duration before naloxone injection (see the appendix).

Additional analysis showed that naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex (fig. 3B, left) increased the respiratory rate more than injection into the pre-Bötzinger complex (data pooled for cohorts A+B; fig. 3B, left and right; Cohen’s d = 0.8; P = 0.033). Naloxone microinjection into the parabrachial nucleus/Kölliker–Fuse nucleus decreased expiratory duration (fig. 3E, left) more than inspiratory duration (fig. 3D, left, Cohen’s d = 0.9, P = 0.008), as did the subsequent injection into the pre-Bötzinger complex (fig. 3, D and E, left; Cohen’s d = 1.6; P = 0.004). Naloxone injection into the pre-Bötzinger complex alone decreased expiratory duration (fig. 3E, right) more than inspiratory duration (fig. 3D, right; Cohen’s d = 1.6; P < 0.001).

Opioid Effect Sites at Apneic IV Remifentanil Concentrations

To determine the effect of apneic remifentanil concentrations on the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex, we administered an intravenous remifentanil bolus that caused apnea under control conditions before and after microinjection of naloxone into the parabrachial nucleus/Kölliker–Fuse complex and after additional naloxone injection into the pre-Bötzinger complex (cohort A, 17 animals), or after naloxone injection into the pre-Bötzinger complex alone (cohort B, nine animals). In cohort A, in 12 of 17 animals, naloxone microinjection into the parabrachial nucleus/Kölliker–Fuse nucleus prevented apnea from the intravenous remifentanil bolus that had caused apnea under control conditions. Respiratory rate increased from 0 to 10 breaths/min (P < 0.001; fig. 4B, left). For the 25 to 75% range, please see table 1. After additional naloxone microinjection into the pre-Bötzinger complex (n = 12), the repeat remifentanil bolus did not cause apnea in any animal, and the respiratory rate was increased to 13 breaths/min (P = 0.002). In cohort B, naloxone microinjection into the pre-Bötzinger complex alone prevented apnea from the IV remifentanil bolus only in 2 of 9 animals (fig. 4B, right), and the median respiratory rate was not increased (0 breaths/min, P = 0.500). Compared to the effect of naloxone microinjection into the pre-Bötzinger complex alone, pre-Bötzinger complex injection after naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex caused a larger increase in the respiratory rate (P = 0.006; fig. 4B, blue bracket).

Fig. 4.

Bilateral naloxone (NAL) injections into the Kölliker–Fuse nucleus (KF), parabrachial nucleus (PBN), and pre-Bötzinger complex (preBotC) prevented respiratory rate depression and apnea from an intravenous remifentanil (REMI) bolus (~10 μg). (A) Phrenic neurogram tracings from the same rabbit shown in figure 3 show that the same remifentanil bolus (red arrow) that elicited apnea during control conditions (left) only moderately depressed respiratory rhythm after naloxone injection into the Kölliker–Fuse nucleus and parabrachial nucleus (middle) and even less after subsequent naloxone injection into the pre-Bötzinger complex (right). (B–E) Pooled data for changes in respiratory rate and other respiratory parameters. Please note the different time scale for inspiratory (D) and expiratory (E) duration. (F, G) Values for inputs to inspiratory on- and off-switch were derived from the values for inspiratory and expiratory duration and are presented as percentages of apneic threshold with an apneic threshold of 100% (appendix). The data for cohort A are presented on the left side of each panel. The data were available for apneic bolus after naloxone injection into the Kölliker–Fuse nucleus and parabrachial nucleus in 17 animals and for 12 animals after additional injection into the pre-Bötzinger complex. The data for cohort B are presented on the right side of each panel (shaded). In a separate group of nine animals, naloxone was injected only into the pre-Bötzinger complex. Black brackets indicate that the difference (Δ) between values from two sequential injections was tested against no change (Wilcoxon signed rank test). Blue brackets indicate comparison of the Δ values from pre-Bötzinger complex injection with and without prior naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex (Mann–Whitney rank sum test). Levels of significance below the critical P = 0.0125 are highlighted in red.

Fig. 4.

Bilateral naloxone (NAL) injections into the Kölliker–Fuse nucleus (KF), parabrachial nucleus (PBN), and pre-Bötzinger complex (preBotC) prevented respiratory rate depression and apnea from an intravenous remifentanil (REMI) bolus (~10 μg). (A) Phrenic neurogram tracings from the same rabbit shown in figure 3 show that the same remifentanil bolus (red arrow) that elicited apnea during control conditions (left) only moderately depressed respiratory rhythm after naloxone injection into the Kölliker–Fuse nucleus and parabrachial nucleus (middle) and even less after subsequent naloxone injection into the pre-Bötzinger complex (right). (B–E) Pooled data for changes in respiratory rate and other respiratory parameters. Please note the different time scale for inspiratory (D) and expiratory (E) duration. (F, G) Values for inputs to inspiratory on- and off-switch were derived from the values for inspiratory and expiratory duration and are presented as percentages of apneic threshold with an apneic threshold of 100% (appendix). The data for cohort A are presented on the left side of each panel. The data were available for apneic bolus after naloxone injection into the Kölliker–Fuse nucleus and parabrachial nucleus in 17 animals and for 12 animals after additional injection into the pre-Bötzinger complex. The data for cohort B are presented on the right side of each panel (shaded). In a separate group of nine animals, naloxone was injected only into the pre-Bötzinger complex. Black brackets indicate that the difference (Δ) between values from two sequential injections was tested against no change (Wilcoxon signed rank test). Blue brackets indicate comparison of the Δ values from pre-Bötzinger complex injection with and without prior naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex (Mann–Whitney rank sum test). Levels of significance below the critical P = 0.0125 are highlighted in red.

Close modal

Because inspiratory duration, expiratory duration, and peak phrenic activity cannot be measured during apnea, we extrapolated these variables from the first breath after apnea (see fig. 4A, control). If no apnea was observed, we averaged parameters for approximately six to eight breaths at the lowest respiratory rate after the bolus (fig. 4A, NAL KF+PBN and NAL KF+PBN+preBötC). In cohort A, the apneic remifentanil bolus reduced peak phrenic activity from 100 to 47% (fig. 4C, left). Naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex (56%, P = 0.030) and the pre-Bötzinger complex (63%, P = 0.064) did not significantly increase peak phrenic activity. In cohort B, the remifentanil bolus depressed peak phrenic activity from 100 to 61% (fig. 4C, right), and naloxone injection into the pre-Bötzinger complex did not change peak phrenic activity (69%, P = 0.250). The naloxone effect was similar with and without prior naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex (P = 0.596; fig. 4C, blue bracket).

The apneic remifentanil bolus increased inspiratory duration from 0.78 to 2.96 s (fig. 4D, left). Naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex reduced the increase to 1.87 s (P = 0.009), whereas naloxone injection into the pre-Bötzinger complex caused no further change (1.92 s, P = 0.375). In cohort B, the apneic remifentanil bolus increased inspiratory duration from 0.84 to 2.20 s (fig. 4D, right). Naloxone injection into the pre-Bötzinger complex further increased inspiratory duration to 4.65 s (P = 0.008). The naloxone effect on inspiratory duration was not significantly different with prior parabrachial nucleus/Kölliker–Fuse complex reversal (P = 0.016; fig. 4D, blue bracket).

In cohort A, the apneic remifentanil bolus increased expiratory duration from 0.85 to 17.48 s (fig. 4E, left). Naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex reduced the increase to 3.73 s (P = 0.007), and injection into the pre-Bötzinger complex further reduced the increase to 2.73 s (P = 0.002). In cohort B, the apneic remifentanil bolus increased expiratory duration from 1.14 to 21.2 s (fig. 4E, right), and naloxone injection into the pre-Bötzinger complex did not change it (20.58 s, P = 0.426). The naloxone effect on expiratory duration was not different after prior parabrachial nucleus/Kölliker–Fuse complex reversal (P = 0.079; fig. 4E, blue bracket).

In cohort A, the apneic remifentanil bolus decreased inspiratory off-switch from 185 to 105% of apneic threshold (fig. 4F, left). Naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex (118%, P = 0.268) and pre-Bötzinger complex (117%, P = 0.625) did not significantly change these inputs. In cohort B, remifentanil decreased inputs to inspiratory off-switch from 176 to 112%, and naloxone injection into the pre-Bötzinger complex further decreased inputs to inspiratory off-switch to 101% (P = 0.039; fig. 4F, right). The naloxone effect on inputs to inspiratory off-switch was not significantly different with prior parabrachial nucleus/Kölliker–Fuse complex reversal (P = 0.066; fig. 4F, blue bracket).

In cohort A, the apneic remifentanil bolus decreased inputs to inspiratory on-switch from 175 to 100% of apneic threshold (fig. 4G, left). Naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex increased inputs to 102% (P = 0.020), and subsequent injection into the pre-Bötzinger complex increased inputs to 107% (P = 0.002). In cohort B, inputs to inspiratory on-switch decreased with the remifentanil bolus from 147 to 100% of apneic threshold (fig. 4G, right) and did not change with naloxone injection (100%, P = 0.82). Inputs to inspiratory on-switch increased more after prior parabrachial nucleus/Kölliker–Fuse complex reversal (P = 0.002; fig. 4G, blue bracket).

Additional analysis showed that naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex decreased expiratory duration (fig. 4E, left) more than inspiratory duration (fig. 4D, left; Cohen’s d = 0.9, P = 0.057), as did the subsequent injection into the pre-Bötzinger complex (fig. 4, D and E, left; Cohen’s d = 1.6; P < 0.001). Naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex increased inputs to inspiratory on-switch more at analgesic (fig. 3G, left) than apneic remifentanil concentrations (fig. 4G, left; Cohen’s d = 1.2; P <0.001), as did the subsequent injection into the pre-Bötzinger complex (figs. 3 and 4G, left; Cohen’s d = 2.4; P < 0.001). Inputs to inspiratory off-switch also increased more with naloxone injection into the pre-Bötzinger complex at analgesic than at apneic concentrations (fig. 3 and 4F, left; Cohen’s d = 1.3; P = 0.003). After naloxone injection solely into the pre-Bötzinger complex, there was a substantial difference between the lack of effect on expiratory duration (fig. 4E, right) and the increase in inspiratory duration (fig. 4D, right; Cohen’s d = 1.1; P = 0.093) and also between the corresponding decrease in inputs to inspiratory off-switch (fig. 4F, right) and the lack of effect on inspiratory on-switch (fig. 4G, right; Cohen’s d = 1.0; P = 0.005). The percentage of control inputs to inspiratory on- and off-switch above the apneic threshold that could be recovered with naloxone injections into the respective brainstem areas at analgesic and apneic concentrations are presented in table 2.

Table 2.

Percentage of Inputs to Respiratory Phase Duration Restored with Naloxone Injections into the Parabrachial Nucleus/Kölliker–Fuse Complex and the Pre-Bötzinger Complex at Analgesic and Apneic Remifentanil Concentrations

Percentage of Inputs to Respiratory Phase Duration Restored with Naloxone Injections into the Parabrachial Nucleus/Kölliker–Fuse Complex and the Pre-Bötzinger Complex at Analgesic and Apneic Remifentanil Concentrations
Percentage of Inputs to Respiratory Phase Duration Restored with Naloxone Injections into the Parabrachial Nucleus/Kölliker–Fuse Complex and the Pre-Bötzinger Complex at Analgesic and Apneic Remifentanil Concentrations

The Quotient of Peak Phrenic Activity/Inspiratory Duration and Respiratory Drive

To determine whether the quotient of peak phrenic activity and inspiratory duration (PPA/TI) is an adequate surrogate for “respiratory drive,” we analyzed how intravenous remifentanil bolus injections affected peak phrenic activity and inspiratory duration. As illustrated for a single animal in fig. 5 (A through C), inspiratory duration and peak phrenic activity changed concomitantly after an apneic remifentanil bolus; however, the remifentanil-induced increase in inspiratory duration was substantially reduced with naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex and the pre-Bötzinger complex, whereas the maximal depression of peak phrenic activity did not change much. Correlation analysis using data from all animals, log-transformed to allow linear regression analysis and analyzed separately for opioid dose and naloxone injection (fig. 5, D through F), showed that the predictive properties of ln(inspiratory duration) for ln(peak phrenic activity/inspiratory duration) were consistently higher than ln(peak phrenic activity) for all but one data set. We conclude that systemic opioids affect excitatory inputs to phase switching mechanisms differently from peak phrenic activity. Consequently, the quotient of peak phrenic activity and inspiratory duration is not a reliable reflection of drive to all components of the respiratory rhythm and pattern generator. In the following analysis and discussion, we will use the term respiratory drive more broadly as inputs to the mechanisms of phase switch and motor activity and not limited to the concept of drive as the quotient of peak phrenic activity and inspiratory duration.

Fig. 5.

Opioids depress inspiratory phase timing differently from peak phrenic activity. (A–C) Data from the rabbit shown in figs. 3A and 4A. (A) The increase in inspiratory duration from the apneic remifentanil bolus (red arrow), plotted for each breath versus time, was smaller after naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex (PBN/KF) (blue; lowest respiratory rate, 12 breaths/min) and pre-Bötzinger complex (preBotC; red; lowest respiratory rate, 19 breaths/min). (B) The decrease in peak phrenic activity, normalized to control, was attenuated less. Please note that inspiratory duration was increased, and peak phrenic activity was decreased before remifentanil injection after naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex and the pre-Bötzinger complex because of the continuous remifentanil infusion (see “Opioid Effect Sites at Apneic IV Remifentanil Concentrations”). (C) Naloxone reversal greatly decreased the prolongation of inspiratory duration from the apneic remifentanil bolus from 4 to ~1.5 s, whereas peak phrenic activity was always depressed 30 to 40%. (D–F) Pooled data from all remifentanil protocols (n = 171) illustrate the correlation between inspiratory duration, peak phrenic activity, and respiratory drive, here defined as the quotient of peak phrenic activity and inspiratory duration. The predictive properties of ln(inspiratory duration) for ln(peak phrenic activity/inspiratory duration) were higher than ln(peak phrenic activity) in all but one data set. (F) Correlation coefficients (R2, squares of Pearson’s correlation) for each data set, and bootstrap analysis for adjusted correlation coefficients.

Fig. 5.

Opioids depress inspiratory phase timing differently from peak phrenic activity. (A–C) Data from the rabbit shown in figs. 3A and 4A. (A) The increase in inspiratory duration from the apneic remifentanil bolus (red arrow), plotted for each breath versus time, was smaller after naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex (PBN/KF) (blue; lowest respiratory rate, 12 breaths/min) and pre-Bötzinger complex (preBotC; red; lowest respiratory rate, 19 breaths/min). (B) The decrease in peak phrenic activity, normalized to control, was attenuated less. Please note that inspiratory duration was increased, and peak phrenic activity was decreased before remifentanil injection after naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex and the pre-Bötzinger complex because of the continuous remifentanil infusion (see “Opioid Effect Sites at Apneic IV Remifentanil Concentrations”). (C) Naloxone reversal greatly decreased the prolongation of inspiratory duration from the apneic remifentanil bolus from 4 to ~1.5 s, whereas peak phrenic activity was always depressed 30 to 40%. (D–F) Pooled data from all remifentanil protocols (n = 171) illustrate the correlation between inspiratory duration, peak phrenic activity, and respiratory drive, here defined as the quotient of peak phrenic activity and inspiratory duration. The predictive properties of ln(inspiratory duration) for ln(peak phrenic activity/inspiratory duration) were higher than ln(peak phrenic activity) in all but one data set. (F) Correlation coefficients (R2, squares of Pearson’s correlation) for each data set, and bootstrap analysis for adjusted correlation coefficients.

Close modal

Effects of Very High Remifentanil Concentrations on Areas outside the Parabrachial Nucleus/Kölliker–Fuse Complex and Pre-Bötzinger Complex

To determine whether very high remifentanil concentrations affect respiratory rate and tidal volume outside the respiratory rhythm generator, we injected up to 100 µg of remifentanil IV after naloxone reversal of the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex. In six animals, the very high remifentanil bolus substantially decreased respiratory rate from 23.5 (20.5 to 28) breaths/min to 11 (7.2 to 12.5) breaths/min (P = 0.031). Peak phrenic activity was depressed to 5 (0 to 38)% of the prebolus amplitude (P = 0.031). In 3 of 6 animals, peak phrenic activity was completely abolished by the remifentanil bolus (phrenic apnea; fig. 6A). The very high remifentanil bolus depressed peak vagal activity to 50 (41 to 55)% of prebolus amplitude (P = 0.031), but rhythmic vagal respiratory activity continued during phrenic apnea (fig. 6B). In comparison, the initial apneic remifentanil bolus before naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex (see 2.5.) generated apnea in the phrenic and vagal neurogram (fig. 6C). At baseline, vagal inspiratory activity closely matched phrenic activity, which confirms that the central respiratory pattern generator controls the phase timing of respiratory pump muscles as well as airway motor activity.31  The concomitant phrenic and vagal apnea observed after the initial apneic remifentanil bolus indicated that remifentanil completely depressed the respiratory rhythm generator (i.e., parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex function). The observation that after naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex, the very high remifentanil bolus still completely depressed phrenic activity (fig. 6A) but that the respiratory rhythm continued as reflected in the continued phasic vagal activity (fig. 6B) suggests (1) that naloxone successfully prevented complete depression of the respiratory rhythm generator and (2) that the very high remifentanil concentration directly depressed inspiratory premotor and/or phrenic motoneurons. Vagal premotor and motoneurons appeared to be less sensitive to direct opioid depression. Before naloxone microinjections into the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex, the initial apneic remifentanil bolus depressed peak phrenic activity more than peak vagus activity (both calculated from the first breath after apnea, relative to the respective peak activity before the bolus; Cohen’s d = 2.1; P = 0.015; fig. 6C). This difference was also observed at the maximal remifentanil effect after the very high remifentanil bolus (Cohen’s d = 1.1; P = 0.065; fig. 6D).

Fig. 6.

Effect of very high remifentanil concentrations on areas outside the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex. (A) Phrenic neurogram tracings from an individual rabbit show that high bolus doses of intravenous remifentanil (total of 100 μg, red arrows) after naloxone microinjection into the bilateral parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex decreased peak phrenic activity to 0. (B) However, during phrenic apnea, the respiratory rhythm continued in the vagus neurogram. The continued rhythm confirmed that opioid antagonism in the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex successfully prevented inhibition of the respiratory rhythm generator even by very high remifentanil concentrations. The depression of peak phrenic activity (A) was likely due to direct inhibition of inspiratory premotor and/or motoneurons. In all animals, respiratory rate decreased by 15 (14 to 16) breaths/min (n = 6). The slowing of the respiratory rate after the initial remifentanil bolus suggests that respiratory drive to the respiratory rhythm generator was decreased by very high remifentanil concentrations. (C, D) We performed additional analysis to determine whether opioids depressed peak phrenic activity more than peak vagus activity. Peak phrenic and peak vagus activity was calculated relative to peak activity before the intravenous remifentanil bolus and pooled for six animals (means ± SD). (C) Peak phrenic and peak vagus activity for each breath after recovery from apnea from the 10-μg remifentanil bolus before naloxone injection into the brainstem showed that phrenic activity was more depressed by remifentanil. (D) Peak phrenic and peak vagus activity for each breath starting at maximal depression from the 100-μg remifentanil bolus after naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex again showed that phrenic activity was more depressed. Complete loss of phrenic motor output (apnea) was observed in 3 of 6 animals. The statistical difference between pooled peak phrenic and peak vagus activity for the first breath after apnea at maximal depression: #Cohen’s d = 2.1, P = 0.015; *Cohen’s d = 1.1, P = 0.065. Mann–Whitney U test.

Fig. 6.

Effect of very high remifentanil concentrations on areas outside the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex. (A) Phrenic neurogram tracings from an individual rabbit show that high bolus doses of intravenous remifentanil (total of 100 μg, red arrows) after naloxone microinjection into the bilateral parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex decreased peak phrenic activity to 0. (B) However, during phrenic apnea, the respiratory rhythm continued in the vagus neurogram. The continued rhythm confirmed that opioid antagonism in the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex successfully prevented inhibition of the respiratory rhythm generator even by very high remifentanil concentrations. The depression of peak phrenic activity (A) was likely due to direct inhibition of inspiratory premotor and/or motoneurons. In all animals, respiratory rate decreased by 15 (14 to 16) breaths/min (n = 6). The slowing of the respiratory rate after the initial remifentanil bolus suggests that respiratory drive to the respiratory rhythm generator was decreased by very high remifentanil concentrations. (C, D) We performed additional analysis to determine whether opioids depressed peak phrenic activity more than peak vagus activity. Peak phrenic and peak vagus activity was calculated relative to peak activity before the intravenous remifentanil bolus and pooled for six animals (means ± SD). (C) Peak phrenic and peak vagus activity for each breath after recovery from apnea from the 10-μg remifentanil bolus before naloxone injection into the brainstem showed that phrenic activity was more depressed by remifentanil. (D) Peak phrenic and peak vagus activity for each breath starting at maximal depression from the 100-μg remifentanil bolus after naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex again showed that phrenic activity was more depressed. Complete loss of phrenic motor output (apnea) was observed in 3 of 6 animals. The statistical difference between pooled peak phrenic and peak vagus activity for the first breath after apnea at maximal depression: #Cohen’s d = 2.1, P = 0.015; *Cohen’s d = 1.1, P = 0.065. Mann–Whitney U test.

Close modal

We also observed that although naloxone reversal in the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex prevented cessation of the respiratory rhythm, the very high remifentanil bolus still caused significant depression of the respiratory rate. Assuming that the naloxone injections were sufficient to locally antagonize all opioid effects, this suggests that remifentanil depressed the activity of the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex by decreasing the respiratory drive to these areas.

Effects of Supraclinical Opioid Concentrations, Compared to Glutamate Receptor Blockade in the Parabrachial Nucleus and Kölliker–Fuse Nucleus

To determine the effect of maximal opioid receptor activation, in six animals, the µ-opioid agonist DAMGO was injected into the bilateral parabrachial nucleus and Kölliker–Fuse nucleus (fig. 7). These data were compared to glutamate antagonist injections in a subset of 13 animals from our previous study,8  which were selected to match the average respiratory rate at control. The levels of significance for comparisons between the effects of DAMGO and glutamate antagonists in the two studies are indicated by blue brackets in fig. 7. DAMGO injection depressed respiratory rate from 29 (28 to 31) to 6 (4 to 7) breaths/min (P = 0.031; fig. 7B, left). This was similar to the effect of the glutamate receptor antagonists NBQX and AP5, which depressed the respiratory rate from 29 (26 to 33) to 2 (1 to 2) breaths/min (P = 0.160; fig. 7B), blue bracket). DAMGO injection did not depress peak phrenic activity (100% to 96 [81 to 105]%, P = 0.688; fig. 7C, left), which was similar to NBQX and AP5 (100% to 97 [84 to 99]%; P = 0.511; fig. 7C, blue bracket). DAMGO increased inspiratory duration from 0.98 (0.90 to 1.04) to 5.03 (4.45 to 6.66) s (P = 0.031; fig. 7D, left), which was less than the increase from the glutamate antagonists (0.81 [0.77 to 0.90) to 17.5 [11.4 to 22.6] s; P = 0.020; fig. 7D, blue bracket). DAMGO increased expiratory duration from 1.32 (1.08 to 1.64) to 5.41 (4.53 to 9.83) s (P = 0.031; fig. 7E, left), which was less than the increase from the glutamate antagonists (1.16 [0.98 to 1.59] to 22.1 [17.1 to 29.3] s; P = 0.007; fig. 7E, blue bracket). Similar to inspiratory duration, the inputs to inspiratory off-switch were decreased by DAMGO from 161 (155 to 169) to 101 (100 to 101)% of apneic threshold (P = 0.031; fig. 7F, left), which was less than the decrease from the glutamate antagonists (180 [170 to 188] to 100 [100]% of apneic threshold; P = 0.010; fig. 7F, blue bracket). DAMGO decreased inputs to inspiratory on-switch from 151 (130 to 154) to 101 (100 to 101)% of apneic threshold (P = 0.031; fig. 7G, left), which was similar to the decrease from glutamate antagonists (146 [126 to 162] to 100 [100 to 100]%; P = 0.965; fig. 7G, blue bracket). The discrepancy between the significant difference in effect on expiratory duration and no difference in effect on inputs to inspiratory on-switch may have been because with very long respiratory phases, large differences in phase duration can be caused by only very small differences in inputs to phase duration (appendix).

Fig. 7.

Microinjection of supraclinical concentrations of the μ-opioid agonist [d-Ala,2N-MePhe,4Gly-ol]-enkephalin (DAMGO; 100 μM, 700 nl) into the bilateral parabrachial nucleus/Kölliker–Fuse complex severely depressed the respiratory rate. We compared the effect size with data from 13 animals from our previous study using microinjections of the non–N-methyl-d-aspartate receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX; 1 mM, 700 nl) and N-methyl-d-aspartate receptor antagonist d(–)-2-amino-5-phosphonopentanoic acid (APS; 5 mM, 700 nl).9  Animals were selected to match the control respiratory rate. (A) Phrenic neurogram tracings from one individual animal. (B–E) Pooled data for changes in respiratory rate and other respiratory parameters. Respiratory rate depression was similar between [d-Ala,2N-MePhe,4Gly-ol]-enkephalin (n = 6) and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione/2-amino-5-phosphonopentanoic acid injection (n = 13), as was the effect on peak phrenic activity. (F, G) Values for inputs to inspiratory on- and off-switch were derived from the values for inspiratory and expiratory duration and are presented as percentages of apneic threshold with an apneic threshold of 100% (appendix). Black brackets indicate that the difference (Δ) between [d-Ala,2N-MePhe,4Gly-ol]-enkephalin injection and control was tested against no change (Wilcoxon signed rank test). Blue brackets indicate comparison of the Δ values from [d-Ala,2N-MePhe,4Gly-ol]-enkephalin injection versus control with 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione/2-amino-5-phosphonopentanoic acid injection versus control (Mann–Whitney rank sum test). Levels of significance below the critical P = 0.025 are highlighted in red.

Fig. 7.

Microinjection of supraclinical concentrations of the μ-opioid agonist [d-Ala,2N-MePhe,4Gly-ol]-enkephalin (DAMGO; 100 μM, 700 nl) into the bilateral parabrachial nucleus/Kölliker–Fuse complex severely depressed the respiratory rate. We compared the effect size with data from 13 animals from our previous study using microinjections of the non–N-methyl-d-aspartate receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX; 1 mM, 700 nl) and N-methyl-d-aspartate receptor antagonist d(–)-2-amino-5-phosphonopentanoic acid (APS; 5 mM, 700 nl).9  Animals were selected to match the control respiratory rate. (A) Phrenic neurogram tracings from one individual animal. (B–E) Pooled data for changes in respiratory rate and other respiratory parameters. Respiratory rate depression was similar between [d-Ala,2N-MePhe,4Gly-ol]-enkephalin (n = 6) and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione/2-amino-5-phosphonopentanoic acid injection (n = 13), as was the effect on peak phrenic activity. (F, G) Values for inputs to inspiratory on- and off-switch were derived from the values for inspiratory and expiratory duration and are presented as percentages of apneic threshold with an apneic threshold of 100% (appendix). Black brackets indicate that the difference (Δ) between [d-Ala,2N-MePhe,4Gly-ol]-enkephalin injection and control was tested against no change (Wilcoxon signed rank test). Blue brackets indicate comparison of the Δ values from [d-Ala,2N-MePhe,4Gly-ol]-enkephalin injection versus control with 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione/2-amino-5-phosphonopentanoic acid injection versus control (Mann–Whitney rank sum test). Levels of significance below the critical P = 0.025 are highlighted in red.

Close modal

The prominent respiratory rate depression by DAMGO injection into the parabrachial nucleus/Kölliker–Fuse complex that is similar to the effects of glutamate antagonists in scale and pattern (i.e., significant prolongation of inspiratory and expiratory duration) suggests that many of the parabrachial nucleus/Kölliker–Fuse complex neurons that contribute to respiratory phase timing are opioid-sensitive. As described under “Effects of Supraclinical Opioid Concentrations, Compared to Glutamate Receptor Blockade in the Parabrachial Nucleus and Kölliker–Fuse Nucleus,” this may apply only for animals with baseline respiratory rates between 20 and 37 breaths/min.

Control Studies: Effects of Naloxone or Artificial Cerebrospinal Fluid Injections; Potential Desensitization to Remifentanil

To rule out endogenous opioid receptor activation, in four animals, naloxone was microinjected into the bilateral parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex without remifentanil infusion. Naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex or pre-Bötzinger complex, respectively, did not have any effect on respiratory rate (vs. control, P > 0.999 or P > 0.999, respectively; data not shown), inspiratory duration (P = 0.625 or P = 0.625, respectively), expiratory duration (P = 0.250 and P = 0.250, respectively), or peak phrenic activity (P = 0.375 and P = 0.250, respectively). At high concentrations (more than 100 µM), naloxone can act as a GABAA receptor antagonist.28  However, we did not observe any changes in respiratory rate with any naloxone injections into the parabrachial nucleus/Kölliker–Fuse complex or pre-Bötzinger complex (i.e., respiratory nuclei, which are under GABAA-mediated control). Therefore, naloxone-mediated GABAA receptor antagonism did not appear to be a confounder in our experiments.

Injection of artificial cerebrospinal fluid, which was used as solvent, into the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex did not affect respiratory rate (parabrachial nucleus/Kölliker–Fuse complex vs. control, P = 0.500; pre-Bötzinger complex vs. control, P > 0.999; n = 3; data not shown), inspiratory duration (P = 0.250 and P = 0.750, respectively), expiratory duration (P = 0.250 and 0.500, respectively), or peak phrenic activity (P = 0.500 and P = 0.500, respectively).

Post hoc, we reviewed all of our experimental data for signs of desensitization to the remifentanil effect caused by prolonged remifentanil infusion and repeated bolus injections. We found that even after long remifentanil infusions when multiple adjustments (i.e., increases or decreases), in infusion rate were required to achieve 50% respiratory rate depression, the respiratory rate did not change during the 15 min of steady state before the first naloxone injection. After apneic bolus injections of remifentanil that followed naloxone injections into the parabrachial nucleus/Kölliker–Fuse complex or pre-Bötzinger complex, the respiratory rate reliably returned to prebolus values (difference pre- to postbolus rate 0 [–1 to 1] breaths/min, n = 26), including after the last remifentanil bolus injection in Cohort A, which was the third remifentanil bolus per animal (difference 0 [–1 to 1] breaths/min, n = 8). Other authors have shown a decrease in respiratory rate depression during continuous remifentanil infusion starting after 90 min and resulting in only ~60% of the maximal depression after 300 min in freely behaving rabbits with variable partial pressure of carbon dioxide and decreasing sedation levels.24  However, in our decerebrate rabbit model with controlled partial pressure of carbon dioxide and partial pressure of oxygen, there was no obvious attenuation of the respiratory rate depression after prolonged remifentanil infusion and repeated bolus injections. We conclude that desensitization to the respiratory depressant effects of remifentanil was not a relevant confounding factor in our experiments. The complete recovery of the respiratory rate to control values with intravenous naloxone injection at the end of the experiment confirmed that there was also no systematic decrease in the respiratory rate caused by a deterioration of the preparation.

This study explored the contributions of the parabrachial nucleus/Kölliker–Fuse complex and the pre-Bötzinger complex to opioid-induced respiratory depression in an acute, in vivo rabbit model. Remifentanil decreased the respiratory rate by depressing inspiratory on-switch through effects in the parabrachial nucleus/Kölliker–Fuse complex and the pre-Bötzinger complex. Remifentanil also depressed inspiratory off-switch through effects on the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex. However, apneic concentrations decreased inspiratory duration through an effect in the pre-Bötzinger complex. Sequential naloxone injection into the Kölliker–Fuse nucleus, parabrachial nucleus, and pre-Bötzinger complex could not completely reverse the respiratory depression from analgesic remifentanil concentrations and even less so from apneic doses (table 2). This suggests that opioids significantly depressed respiratory drive to the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex and that the depression of drive reduced the activity of these areas, especially at high opioid concentrations (fig. 8).

Fig. 8.

Schematic of opioid effects on respiratory rate and tidal volume. Tonic respiratory drive determines the activity of the parabrachial nucleus/Kölliker–Fuse complex, the pre-Bötzinger complex, and inspiratory premotor and motoneurons. Part of the drive that determines respiratory phase duration (blue) is relayed by the parabrachial nucleus/Kölliker–Fuse complex, whereas other drive projects directly to the respiratory rhythm generator in the pre-Bötzinger complex. Drive that determines the magnitude of the tidal volume (green) is partially relayed through the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex but also directly projects to respiratory premotor neurons and phrenic motoneurons. Expiratory motoneurons were not recorded in this study. Analgesic opioid doses depress mostly parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex activity (bold red frames) and thus the respiratory rate. The magnitude of the opioid effect that can be reversed in each area is presented in table 2. Higher opioid doses directly affect respiratory drive and premotor and motoneurons, resulting in an additional decrease in tidal volume.

Fig. 8.

Schematic of opioid effects on respiratory rate and tidal volume. Tonic respiratory drive determines the activity of the parabrachial nucleus/Kölliker–Fuse complex, the pre-Bötzinger complex, and inspiratory premotor and motoneurons. Part of the drive that determines respiratory phase duration (blue) is relayed by the parabrachial nucleus/Kölliker–Fuse complex, whereas other drive projects directly to the respiratory rhythm generator in the pre-Bötzinger complex. Drive that determines the magnitude of the tidal volume (green) is partially relayed through the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex but also directly projects to respiratory premotor neurons and phrenic motoneurons. Expiratory motoneurons were not recorded in this study. Analgesic opioid doses depress mostly parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex activity (bold red frames) and thus the respiratory rate. The magnitude of the opioid effect that can be reversed in each area is presented in table 2. Higher opioid doses directly affect respiratory drive and premotor and motoneurons, resulting in an additional decrease in tidal volume.

Close modal

Opioids Depress Drive to the Parabrachial Nucleus/Kölliker–Fuse Complex and pre-Bötzinger Complex

We used changes in inspiratory and expiratory duration with naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex or pre-Bötzinger complex to calculate the relative inputs to inspiratory on- and off-switch from these areas (table 2). Two observations stood out: naloxone injection into the parabrachial nucleus/Kölliker–Fuse complex or pre-Bötzinger complex restored inputs more at analgesic than apneic remifentanil concentrations (table 2), suggesting that the level of parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex activity that can be recovered with naloxone injection depends on respiratory drive to these areas and that this drive is opioid-sensitive (fig. 8).

Second, naloxone reversal of the parabrachial nucleus/Kölliker–Fuse complex did not always prevent apnea from the apneic remifentanil bolus, and reversal of the pre-Bötzinger complex rarely did. However, reversal of both areas together reliably prevented apnea, and respiratory rhythm persisted even after very high remifentanil bolus doses, albeit with a decreased rate. This suggests that the inputs from both areas to inspiratory on-switch are additive and that respiratory drive to these areas is opioid-sensitive but still sufficient to sustain respiratory rhythm even at very high remifentanil concentrations (see “Effects of Very High Remifentanil Concentrations on Areas outside the Parabrachial Nucleus/Kölliker–Fuse Complex and Pre-Bötzinger Complex”; fig. 6).

In a freely behaving mouse model, morphine dose-dependently depressed the respiratory rate. µ-Opioid receptor deletion in the Kölliker–Fuse nucleus prevented respiratory rate depression by ~20% of baseline rate at every dose, and µ-opioid receptor deletion in the pre-Bötzinger complex did not reduce respiratory depression at doses of 30 mg/kg or more (see fig. 3 in the study by Varga et al.11 ), suggesting that the increasing depression was due to a reduction in respiratory drive. Apneic doses were not tested. A similar study found no additional reduction of respiratory depression after µ-opioid receptor deletion in the pre-Bötzinger complex and parabrachial nucleus/Kölliker–Fuse complex in the same animals; however, the study was likely underpowered (see fig. 3 in the study by Bachmutsky et al.13 ). Species differences may exist between mammals; for example, in dogs naloxone injection into the parabrachial nucleus fully reversed respiratory rate depression,5  whereas no reversal was observed in the pre-Bötzinger complex.4 

Potential Sources of Opioid-sensitive Respiratory Drive

In our decerebrate, hyperoxic, and moderately hypercapnic preparation, both supratentorial “awake” drive32  and carotid body inputs were eliminated. This left the prevailing hypercapnia (tissue carbon dioxide tension) and the medullary and pontine arousal centers of the raphe as the main source of respiratory drive (for review, see the article by Palkovic et al.1 ). Chemosensitive neurons in the retrotrapezoid nucleus, considered the main source and integrator of respiratory chemodrive,33,34  were unaffected even by apneic morphine doses.35  However, in rats, injection of the opioid antagonist D-Phe-Cys-Tyr-D-Typ-Arg-Thr-Pen-Thr-NH2 (CTAP) into the caudal medullary raphe reduced respiratory rate depression by intravenous DAMGO from 30 to 15%,36  making this area a promising candidate for future research.

Opioid Effects on Postinspiratory Activity

Vagal nerve recordings in rats display prominent postinspiratory activity, which controls motor output to airway muscles.31,37  It also allows gauging the contribution of postinspiratory activity to respiratory phase timing. Volumetric mapping of the brainstem respiratory network showed highly synchronized activity during respiratory phase transitions in the areas relevant for phase timing.31  Prominent activity during inspiratory on- and off-switch was observed in the pre-Bötzinger complex, and the main postinspiratory–expiratory phase transition was in the dorsal respiratory group.31  In the in-situ rat model, vagal postinspiratory activity was completely abolished by systemic opioids, suggesting a mechanism for the observed increase in inspiratory duration.10  In rabbits, vagal activity is mostly inspiratory, and postinspiratory activity, although present in the pre-Bötzinger complex, cannot be determined from vagal recordings. We thus limit our discussion of opioid effects to inspiratory–expiratory phase timing.

Potential Neuronal Targets for Respiratory Opioid Effects

Opioids prolong expiratory duration, and apnea always results from a failure of inspiratory on-switch.1 In vitro, µ-opioid receptor agonists depressed more than 60% of Kölliker–Fuse complex neurons.9,11  In our preparation, DAMGO injection into the parabrachial nucleus/Kölliker–Fuse complex resulted in severe depression of inspiratory on-switch (fig. 7), suggesting that opioids depressed neurons that promote inspiratory on-switch in the pre-Bötzinger complex.7  DAMGO also directly inhibited Dbx+ pre-Bötzinger complex neurons in medullary slices38  (i.e., pre-inspiratory and inspiratory neurons whose stimulation generates inspiratory bursts in vivo).39  Both mechanisms, depression of pontine inputs to the pre-Bötzinger complex and direct inhibition of pre-Bötzinger complex neurons, result in prolonged expiratory duration.

DAMGO injection into the parabrachial nucleus/Kölliker–Fuse complex increased inspiratory duration (fig. 7D), and naloxone in the parabrachial nucleus/Kölliker–Fuse complex reversed the increase in inspiratory duration from systemic remifentanil, suggesting that remifentanil depressed inputs to pre-Bötzinger complex neurons that promote inspiratory off-switch7,8  (e.g., SST+ postinspiratory neurons).39  Naloxone injection into the pre-Bötzinger complex decreased inspiratory duration (fig. 3), as did µ-opioid receptor deletion in the pre-Bötzinger complex in mice,11  pointing to additional, direct inhibition of postinspiratory neurons.

In contrast, at apneic remifentanil concentrations, naloxone injection solely into the pre-Bötzinger complex increased inspiratory duration (fig. 4). Opioids directly depressed inspiratory pre-Bötzinger complex neurons in vitro,29  and DAMGO injection into the pre-Bötzinger complex shortened inspiratory duration in rabbits in vivo.26,40  We hypothesize that the moderate increase in inspiratory duration at apneic remifentanil doses (fig. 4) was the net effect of depressed pontine inputs to inspiratory off-switch and direct inhibition of pre-Bötzinger complex inspiratory neurons. All neuron types have been described in the pre-Bötzinger complex in multiple species in vivo,7,39,41–43  but opioid effects on individual neuron types have yet to be systematically investigated.

Remifentanil Effects on Phrenic Nerve Motor Output

We found that depression of peak phrenic activity did not closely correlate with changes in respiratory phase timing (fig. 5). After opioid reversal in the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex, very high remifentanil doses depressed phrenic motor output completely, while rhythmic respiratory activity continued (fig. 6). Single neuron recordings in vivo showed no direct depression of respiratory premotor neurons at analgesic systemic opioid concentrations26,44  but neurons were directly depressed at near-apneic fentanyl doses.44  Direct depression of spinal motoneurons was observed in vitro at “clinical” DAMGO concentrations (100 nM).45  Direct depression of motor output from very high opioid doses may reduce the effectiveness of drugs designed specifically to stimulate respiratory rhythm.

Methodologic Considerations

Naloxone Injections

Modeling suggests that the spread of our injection volume (700 nl) resulted in an effective naloxone concentration of 50 µM (5% of 1 mM barrel concentration) within a spherical radius of 1 and 1.2 mm at 5 min after injection.22  We believe that this concentration was sufficient to fully antagonize the remifentanil effect in the entire parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex area at analgesic (~50 nM25 ) and apneic plasma concentrations. We have discussed elsewhere that the neuronal population of the parabrachial nucleus/Kölliker–Fuse complex that contributes to phase timing may be located between our injection sites, i.e., in the medial parabrachial nucleus,8,46  which matches other studies.13  Projections from this area to the pre-Bötzinger complex were shown functionally and histologically.7,47,48  Pilot data showed an unchanged naloxone effect 2 h after microinjection, suggesting that sequential injections completely blocked the remifentanil effect in all areas. The lack of change in respiratory rate starting 3 to 5 min after naloxone injection indicated that the area of effective naloxone concentration did not increase after that point. In a subset of nine animals in the current study, naloxone injection 0.5 mm caudal to the pre-Bötzinger complex caused minor increases in respiratory rate (1 [0 to 3] breaths/min), whereas more caudal injections did not, suggesting that our pre-Bötzinger complex injections did not reach the rostral ventral respiratory group.

Opioid Dosing

We chose a 50% depression of respiratory rate as surrogate for an analgesic remifentanil dose because veterinary14  and respiratory studies13,24  showed a ~50% rate depression at opioid doses that suppressed pain responses in spontaneously breathing animals. These doses are likely higher than analgesic doses in humans where analgesics can be dosed to “acceptable” pain levels, whereas animal studies generally measure complete lack of movement to pain stimulus. However, a 40 to 60% depression of minute ventilation was observed in human volunteers after 0.15 mg/kg49  or 0.2 mg/kg50  morphine (i.e., a dose sufficient to provide effective analgesia for patients after major surgery).

Conclusions

Opioid reversal in the parabrachial nucleus/Kölliker–Fuse complex and pre-Bötzinger complex does not completely reverse opioid-induced respiratory depression, suggesting that depression of respiratory drive limits the activity that can be recovered in these areas. This mechanism must be taken into account during the development of drugs designed to stimulate the respiratory rhythm generator.

Acknowledgments

The authors thank Andrew Williams, Medical College of Wisconsin, Milwaukee, Wisconsin, for his outstanding support with the experimental setup.

Research Support

Supported by National Institutes of Health (Bethesda, Maryland) grant No. R01-GM112960 (to Dr. Stucke). The statistical analysis was aided by the CTSI Biostatistics Program with funding from the National Center for Advancing Translational Sciences, National Institutes of Health grant No. UL1TR001436. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Competing Interests

The authors declare no competing interests.

The determinants of inspiratory and expiratory phase duration can be understood using a “leaky integrator” model.1,51,52  In short, populations of phase switching neurons located in the pre-Bötzinger complex promote the switch from expiration to inspiration (preinspiratory neurons)53,54  and, to a degree, the switch from inspiration to expiration (postinspiratory neurons).39  The sum of inputs to these neuron populations determines the time to phase switch threshold and thus the duration of the preceding phase. Using the actual values for inspiratory duration the sum of inputs (σ) to inspiratory off-switch and for expiratory duration, the sum of inputs to inspiratory on-switch can be calculated from LI(σ) = σ *(1 – e–t/τ), where LI(σ) is the leaky integrator function, σ is the sum of inputs to LI(σ), e = 2.71828 is the base of the natural logarithm, and τ is the time constant of LI(σ). Phase switching occurs when leaky integrator output LI(σ) = threshold, where the threshold is set at 1.0 input units or as a percent = 100. The time constant of the leaky integrator determines the rate of rise of the sum of inputs (σ) toward the threshold for phase off-switch. Conversely, the inputs can be calculated from σ= 1/(1 – e–T/τ), where T is the time of threshold crossing.

Fig. A1.

Actual values for inspiratory and expiratory duration measured at (A) analgesic and (B) apneic remifentanil concentrations before and after local naloxone injections for the data presented in fig. 3A (analgesic) and fig. 4A (apneic) and their corresponding input values; naloxone injection into the parabrachial nucleus/Kölliker–Fuse decreased expiratory duration after apneic remifentanil bolus from 10.3 to 3 s, which reflected an increase in inputs to expiratory duration from 100.01 to 107% (a 7% increase) of the apneic threshold (purple arrow). σ is a step input pattern with onset at t = 0, and LI(σ) is the output step response of the leaky integrator. In contrast, naloxone injection into the parabrachial nucleus/Kölliker–Fuse decreased expiratory duration during analgesic remifentanil concentrations from 2.2 to 1.3 s, reflecting an increase in inputs to expiratory duration from 114 to 143% of apneic threshold (red arrow). This illustrates that at high input levels (short respiratory phase durations), the same change in the sum of inputs results in a much smaller change in phase duration than at low input levels, for which small changes in the sum of inputs can change phase duration by several seconds. Calculating the inputs to phase duration allows comparison of opioid effects, as determined by the amount of naloxone reversal, from multiple injections without bias resulting from the respiratory rate at the time of injection. NAL, naloxone; PBN/KF, parabrachial nucleus/Kölliker–Fuse complex; preBotC, pre-Bötzinger complex.

Fig. A1.

Actual values for inspiratory and expiratory duration measured at (A) analgesic and (B) apneic remifentanil concentrations before and after local naloxone injections for the data presented in fig. 3A (analgesic) and fig. 4A (apneic) and their corresponding input values; naloxone injection into the parabrachial nucleus/Kölliker–Fuse decreased expiratory duration after apneic remifentanil bolus from 10.3 to 3 s, which reflected an increase in inputs to expiratory duration from 100.01 to 107% (a 7% increase) of the apneic threshold (purple arrow). σ is a step input pattern with onset at t = 0, and LI(σ) is the output step response of the leaky integrator. In contrast, naloxone injection into the parabrachial nucleus/Kölliker–Fuse decreased expiratory duration during analgesic remifentanil concentrations from 2.2 to 1.3 s, reflecting an increase in inputs to expiratory duration from 114 to 143% of apneic threshold (red arrow). This illustrates that at high input levels (short respiratory phase durations), the same change in the sum of inputs results in a much smaller change in phase duration than at low input levels, for which small changes in the sum of inputs can change phase duration by several seconds. Calculating the inputs to phase duration allows comparison of opioid effects, as determined by the amount of naloxone reversal, from multiple injections without bias resulting from the respiratory rate at the time of injection. NAL, naloxone; PBN/KF, parabrachial nucleus/Kölliker–Fuse complex; preBotC, pre-Bötzinger complex.

Close modal

Time constants for inspiratory on-switch have been determined for rabbits (τ= 1.07 s52 ) and dogs (τ= 0.8 s51 ). D’Angelo55  described a τ of 0.21 s for inspiratory duration; however, the latter appeared very different from our present results, where a decrease in central (parabrachial nucleus/Kölliker–Fuse) input to phase duration changed inspiratory and expiratory duration similarly (fig. 2). Bradley et al.56  stated a τ of 1.0 s for inspiratory duration for cats. To simplify calculation and comparison between inspiratory and expiratory duration, we used a τ of 1.0 s to calculate inputs to inspiratory on- and off-switch.

1.
Palkovic
B
,
Marchenko
V
,
Zuperku
EJ
,
Stuth
EAE
,
Stucke
AG
:
Multi-level regulation of opioid-induced respiratory depression.
Physiology (Bethesda)
2020
;
35
:
391
404
2.
Miller
JR
,
Zuperku
EJ
,
Stuth
EAE
,
Banerjee
A
,
Hopp
FA
,
Stucke
AG
:
A subregion of the parabrachial nucleus partially mediates respiratory rate depression from intravenous remifentanil in young and adult rabbits.
Anesthesiology
2017
;
127
:
502
14
3.
Stucke
AG
,
Miller
JR
,
Prkic
I
,
Zuperku
EJ
,
Hopp
FA
,
Stuth
EA
:
Opioid-induced respiratory depression is only partially mediated by the preBötzinger complex in young and adult rabbits in vivo.
Anesthesiology
2015
;
122
:
1288
98
4.
Mustapic
S
,
Radocaj
T
,
Sanchez
A
,
Dogas
Z
,
Stucke
AG
,
Hopp
FA
,
Stuth
EA
,
Zuperku
EJ
:
Clinically relevant infusion rates of µ-opioid agonist remifentanil cause bradypnea in decerebrate dogs but not via direct effects in the pre-Bötzinger complex region.
J Neurophysiol
2010
;
103
:
409
18
5.
Prkic
I
,
Mustapic
S
,
Radocaj
T
,
Stucke
AG
,
Stuth
EA
,
Hopp
FA
,
Dean
C
,
Zuperku
EJ
:
Pontine µ-opioid receptors mediate bradypnea caused by intravenous remifentanil infusions at clinically relevant concentrations in dogs.
J Neurophysiol
2012
;
108
:
2430
41
6.
Dutschmann
M
,
Dick
TE
:
Pontine mechanisms of respiratory control.
Compr Physiol
2012
;
2
:
2443
69
7.
Zuperku
EJ
,
Stucke
AG
,
Krolikowski
JG
,
Tomlinson
J
,
Hopp
FA
,
Stuth
EA
:
Inputs to medullary respiratory neurons from a pontine subregion that controls breathing frequency.
Respir Physiol Neurobiol
2019
;
265
:
127
40
8.
Navarrete-Opazo
A
,
Cook-Snyder
D
,
Miller
J
,
Callison
J
,
McCarthy
N
,
Palkovic
B
et al
:
Endogenous glutamatergic inputs to the parabrachial nucleus/Kölliker–Fuse complex determine respiratory rate
.
Resp Physiol Neurobiol
2020
;
277
:
103401
.
9.
Levitt
ES
,
Abdala
AP
,
Paton
JF
,
Bissonnette
JM
,
Williams
JT
:
µ-Opioid receptor activation hyperpolarizes respiratory-controlling Kölliker–Fuse neurons and suppresses post-inspiratory drive.
J Physiol
2015
;
593
:
4453
69
10.
Saunders
SE
,
Levitt
ES
:
Kölliker–Fuse/Parabrachial complex µ-opioid receptors contribute to fentanyl-induced apnea and respiratory rate depression.
Respir Physiol Neurobiol
2020
;
275
:
103388
11.
Varga
AG
,
Reid
BT
,
Kieffer
BL
,
Levitt
ES
:
Differential impact of two critical respiratory centres in opioid-induced respiratory depression in awake mice.
J Physiol
2020
;
598
:
189
205
12.
Ma
D
,
Chakrabarti
MK
,
Whitwam
JG
:
Effects of propofol and remifentanil on phrenic nerve activity and nociceptive cardiovascular responses in rabbits.
Anesthesiology
1999
;
91
:
1470
80
13.
Bachmutsky
I
,
Wei
X
,
Kish
E
,
Yackle
K
:
Opioids depress breathing through two small brainstem sites.
eLife
2020
;
9
:
e52694
.
14.
Flecknell
PA
,
Mitchell
M
:
Midazolam and fentanyl-fluanisone: Assessment of anaesthetic effects in laboratory rodents and rabbits.
Lab Anim
1984
;
18
:
143
6
15.
Drummond
JC
:
MAC for halothane, enflurane, and isoflurane in the New Zealand white rabbit: And a test for the validity of MAC determinations.
Anesthesiology
1985
;
62
:
336
8
16.
Ma
D
,
Chakrabarti
MK
,
Whitwam
JG
:
The combined effects of sevoflurane and remifentanil on central respiratory activity and nociceptive cardiovascular responses in anesthetized rabbits.
Anesth Analg
1999
;
89
:
453
61
17.
Stucke
AG
,
Stuth
EA
,
Tonkovic-Capin
V
,
Tonkovic-Capin
M
,
Hopp
FA
,
Kampine
JP
,
Zuperku
EJ
:
Effects of sevoflurane on excitatory neurotransmission to medullary expiratory neurons and on phrenic nerve activity in a decerebrate dog model.
Anesthesiology
2001
;
95
:
485
91
18.
Hugelin
A
.
Forebrain and midbrain influences on respiration.
In Handbook of Physiology: The Respiratory System
, 2nd edition. Edited by
Geiger
SR
.
Bethesda, Maryland
,
American Physiological Society
,
1986
,
pp. 69
91
.
19.
Stuth
EA
,
Krolo
M
,
Stucke
AG
,
Tonkovic-Capin
M
,
Tonkovic-Capin
V
,
Hopp
FA
,
Kampine
JP
,
Zuperku
EJ
:
Effects of halothane on excitatory neurotransmission to medullary expiratory neurons in a decerebrate dog model.
Anesthesiology
2000
;
93
:
1474
81
20.
Mutolo
D
,
Bongianni
F
,
Nardone
F
,
Pantaleo
T
:
Respiratory responses evoked by blockades of ionotropic glutamate receptors within the Bötzinger complex and the pre-Bötzinger complex of the rabbit.
Eur J Neurosci
2005
;
21
:
122
34
21.
Dogas
Z
,
Krolo
M
,
Stuth
EA
,
Tonkovic-Capin
M
,
Hopp
FA
,
McCrimmon
DR
,
Zuperku
EJ
:
Differential effects of GABAA receptor antagonists in the control of respiratory neuronal discharge patterns.
J Neurophysiol
1998
;
80
:
2368
77
22.
Cook-Snyder
DR
,
Miller
JR
,
Navarrete-Opazo
AA
,
Callison
JJ
,
Peterson
RC
,
Hopp
FA
,
Stuth
EAE
,
Zuperku
EJ
,
Stucke
AG
:
The contribution of endogenous glutamatergic input in the ventral respiratory column to respiratory rhythm.
Respir Physiol Neurobiol
2019
;
260
:
37
52
23.
Eldridge
FL
:
Expiratory effects of brief carotid sinus nerve and carotid body stimulations.
Respir Physiol
1976
;
26
:
395
410
24.
Hayashida
M
,
Fukunaga
A
,
Hanaoka
K
:
Detection of acute tolerance to the analgesic and nonanalgesic effects of remifentanil infusion in a rabbit model.
Anesth Analg
2003
;
97
:
1347
52
25.
Michelsen
LG
,
Salmenperä
M
,
Hug
CC
, Jr
,
Szlam
F
,
VanderMeer
D
:
Anesthetic potency of remifentanil in dogs.
Anesthesiology
1996
;
84
:
865
72
26.
Stucke
AG
,
Zuperku
EJ
,
Sanchez
A
,
Tonkovic-Capin
M
,
Tonkovic-Capin
V
,
Mustapic
S
,
Stuth
EA
:
Opioid receptors on bulbospinal respiratory neurons are not activated during neuronal depression by clinically relevant opioid concentrations.
J Neurophysiol
2008
;
100
:
2878
88
27.
Barnett
WH
,
Jenkin
SEM
,
Milsom
WK
,
Paton
JFR
,
Abdala
AP
,
Molkov
YI
,
Zoccal
DB
:
The Kölliker–Fuse nucleus orchestrates the timing of expiratory abdominal nerve bursting.
J Neurophysiol
2018
;
119
:
401
12
28.
Gruol
DL
,
Barker
JL
,
Smith
TG
:
Naloxone antagonism of GABA-evoked membrane polarizations in cultured mouse spinal cord neurons.
Brain Res
1980
;
198
:
323
32
29.
Montandon
G
,
Qin
W
,
Liu
H
,
Ren
J
,
Greer
JJ
,
Horner
RL
:
PreBotzinger complex neurokinin-1 receptor–expressing neurons mediate opioid-induced respiratory depression.
J Neurosci
2011
;
31
:
1292
301
30.
Sawilowsky
S
:
New effect size rules of thumb
.
J Mod Appl Stat Methods
2009
;
8
:
597
99
.
31.
Dhingra
RR
,
Dick
TE
,
Furuya
WI
,
Galán
RF
,
Dutschmann
M
:
Volumetric mapping of the functional neuroanatomy of the respiratory network in the perfused brainstem preparation of rats.
J Physiol
2020
;
598
:
2061
79
32.
Montandon
G
,
Cushing
SL
,
Campbell
F
,
Propst
EJ
,
Horner
RL
,
Narang
I
:
Distinct cortical signatures associated with sedation and respiratory rate depression by morphine in a pediatric population.
Anesthesiology
2016
;
125
:
889
903
33.
Silva
JN
,
Lucena
EV
,
Silva
TM
,
Damasceno
RS
,
Takakura
AC
,
Moreira
TS
:
Inhibition of the pontine Kölliker–Fuse nucleus reduces genioglossal activity elicited by stimulation of the retrotrapezoid chemoreceptor neurons.
Neuroscience
2016
;
328
:
9
21
34.
Wu
Y
,
Proch
KL
,
Teran
FA
,
Lechtenberg
RJ
,
Kothari
H
,
Richerson
GB
:
Chemosensitivity of Phox2b-expressing retrotrapezoid neurons is mediated in part by input from 5-HT neurons.
J Physiol
2019
;
597
:
2741
66
35.
Mulkey
DK
,
Stornetta
RL
,
Weston
MC
,
Simmons
JR
,
Parker
A
,
Bayliss
DA
,
Guyenet
PG
:
Respiratory control by ventral surface chemoreceptor neurons in rats.
Nat Neurosci
2004
;
7
:
1360
9
36.
Zhang
Z
,
Xu
F
,
Zhang
C
,
Liang
X
:
Activation of opioid mu receptors in caudal medullary raphe region inhibits the ventilatory response to hypercapnia in anesthetized rats.
Anesthesiology
2007
;
107
:
288
97
37.
Dutschmann
M
,
Herbert
H
:
The Kölliker–Fuse nucleus gates the postinspiratory phase of the respiratory cycle to control inspiratory off-switch and upper airway resistance in rat.
Eur J Neurosci
2006
;
24
:
1071
84
38.
Sun
X
,
Thörn Perez
C
,
Halemani
N
,
Shao
X
,
Greenwood
M
,
Heath
S
et al
:
Opioids modulate an emergent rhythmogenic process to depress breathing.
eLife
2019
;
8
:
e50613
.
39.
Cui
Y
,
Kam
K
,
Sherman
D
,
Janczewski
WA
,
Zheng
Y
,
Feldman
JL
:
Defining preBötzinger complex rhythm- and pattern-generating neural microcircuits in vivo.
Neuron
2016
;
91
:
602
14
40.
Cinelli
E
,
Bongianni
F
,
Pantaleo
T
,
Mutolo
D
:
Activation of µ-opioid receptors differentially affects the preBötzinger complex and neighbouring regions of the respiratory network in the adult rabbit
.
Resp Physiol Neurobiol
2020
;
280
:
103482
41.
Segers
LS
,
Nuding
SC
,
Dick
TE
,
Shannon
R
,
Baekey
DM
,
Solomon
IC
,
Morris
KF
,
Lindsey
BG
:
Functional connectivity in the pontomedullary respiratory network.
J Neurophysiol
2008
;
100
:
1749
69
42.
Ezure
K
:
Synaptic connections between medullary respiratory neurons and considerations on the genesis of respiratory rhythm.
Prog Neurobiol
1990
;
35
:
429
50
43.
Krolo
M
,
Tonkovic-Capin
V
,
Stucke
AG
,
Stuth
EA
,
Hopp
FA
,
Dean
C
,
Zuperku
EJ
:
Subtype composition and responses of respiratory neurons in the pre-Botzinger region to pulmonary afferent inputs in dogs.
J Neurophysiol
2005
;
93
:
2674
87
44.
Lalley
PM
:
µ-Opioid receptor agonist effects on medullary respiratory neurons in the cat: Evidence for involvement in certain types of ventilatory disturbances.
Am J Physiol Regul Integr Comp Physiol
2003
;
285
:
R1287
304
45.
Honda
H
,
Kawasaki
Y
,
Baba
H
,
Kohno
T
:
The μ opioid receptor modulates neurotransmission in the rat spinal ventral horn.
Anesth Analg
2012
;
115
:
703
12
46.
Meessen
H
,
Olszewski
J
.
Cytoarchitectonischer Atlas des Rautenhirns des Kaninchens
.
Basel
:
Karger
,
1949
.
47.
Gang
S
,
Sato
Y
,
Kohama
I
,
Aoki
M
:
Afferent projections to the Bötzinger complex from the upper cervical cord and other respiratory related structures in the brainstem in cats: Retrograde WGA-HRP tracing.
J Auton Nerv Syst
1995
;
56
:
1
7
48.
Smith
JC
,
Morrison
DE
,
Ellenberger
HH
,
Otto
MR
,
Feldman
JL
:
Brainstem projections to the major respiratory neuron populations in cat medulla
.
J Comp Neurol
1989
;
281
:
69
96
.
49.
Olofsen
E
,
van Dorp
E
,
Teppema
L
,
Aarts
L
,
Smith
TW
,
Dahan
A
,
Sarton
E
:
Naloxone reversal of morphine- and morphine-6-glucuronide–induced respiratory depression in healthy volunteers: A mechanism-based pharmacokinetic–pharmacodynamic modeling study.
Anesthesiology
2010
;
112
:
1417
27
50.
Dahan
A
,
Romberg
R
,
Teppema
L
,
Sarton
E
,
Bijl
H
,
Olofsen
E
:
Simultaneous measurement and integrated analysis of analgesia and respiration after an intravenous morphine infusion.
Anesthesiology
2004
;
101
:
1201
9
51.
Zuperku
EJ
,
Hopp
FA
,
Kampine
JP
:
Central integration of pulmonary stretch receptor input in the control of expiration.
J Appl Physiol Respir Environ Exerc Physiol
1982
;
52
:
1296
315
52.
D’Angelo
E
:
Verification of a model for the mechanisms controlling expiratory duration in rabbits under various conditions.
Respir Physiol
1985
;
59
:
239
64
53.
Butera
RJ
, Jr
,
Rinzel
J
,
Smith
JC
:
Models of respiratory rhythm generation in the pre-Bötzinger complex: I. Bursting pacemaker neurons.
J Neurophysiol
1999
;
82
:
382
97
54.
Butera
RJ
, Jr
,
Rinzel
J
,
Smith
JC
:
Models of respiratory rhythm generation in the pre-Bötzinger complex: II. Populations of coupled pacemaker neurons.
J Neurophysiol
1999
;
82
:
398
415
55.
D’Angelo
E
:
Mechanisms controlling inspiration studied by electrical vagal stimulations in rabbits.
Respir Physiol
1979
;
38
:
185
202
56.
Bradley
GW
,
von Euler
C
,
Marttila
I
,
Roos
B
:
A model of the central and reflex inhibition of inspiration in the cat.
Biol Cybern
1975
;
19
:
105
16