Dual mechanisms of opioid-induced respiratory depression in the inspiratory rhythm-generating network
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Evaluation Summary:
Opioids are widely used as pain killers, but present the severe side-effect of respiratory depression. The study from Baertsch et al. provides a mechanistic understanding of the actions of opioids on breathing by elucidating some of the biophysical and synaptic mechanisms by which opioids depress breathing with the goal of identifying therapeutic strategies. The data suggest that opioid-induced respiratory depression (OIRD) is due to both presynaptic hyperpolarization, and reduction of synaptic efficacy. The paper is generally well written and the data presented for the most part advances understanding of the mechanisms of OIRD at the level of central respiratory neural circuits.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
The analgesic utility of opioid-based drugs is limited by the life-threatening risk of respiratory depression. Opioid-induced respiratory depression (OIRD), mediated by the μ-opioid receptor (MOR), is characterized by a pronounced decrease in the frequency and regularity of the inspiratory rhythm, which originates from the medullary preBötzinger Complex (preBötC). To unravel the cellular- and network-level consequences of MOR activation in the preBötC, MOR-expressing neurons were optogenetically identified and manipulated in transgenic mice in vitro and in vivo. Based on these results, a model of OIRD was developed in silico. We conclude that hyperpolarization of MOR - expressing preBötC neurons alone does not phenocopy OIRD. Instead, the effects of MOR activation are twofold: (1) pre-inspiratory spiking is reduced and (2) excitatory synaptic transmission is suppressed, thereby disrupting network-driven rhythmogenesis. These dual mechanisms of opioid action act synergistically to make the normally robust inspiratory rhythm-generating network particularly prone to collapse when challenged with exogenous opioids.
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Evaluation Summary:
Opioids are widely used as pain killers, but present the severe side-effect of respiratory depression. The study from Baertsch et al. provides a mechanistic understanding of the actions of opioids on breathing by elucidating some of the biophysical and synaptic mechanisms by which opioids depress breathing with the goal of identifying therapeutic strategies. The data suggest that opioid-induced respiratory depression (OIRD) is due to both presynaptic hyperpolarization, and reduction of synaptic efficacy. The paper is generally well written and the data presented for the most part advances understanding of the mechanisms of OIRD at the level of central respiratory neural circuits.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback …
Evaluation Summary:
Opioids are widely used as pain killers, but present the severe side-effect of respiratory depression. The study from Baertsch et al. provides a mechanistic understanding of the actions of opioids on breathing by elucidating some of the biophysical and synaptic mechanisms by which opioids depress breathing with the goal of identifying therapeutic strategies. The data suggest that opioid-induced respiratory depression (OIRD) is due to both presynaptic hyperpolarization, and reduction of synaptic efficacy. The paper is generally well written and the data presented for the most part advances understanding of the mechanisms of OIRD at the level of central respiratory neural circuits.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
This study identifies some of the mechanisms of action of MOR-acting opioids on the respiratory network. It determines the impacts of MOR activation of various types of neurons of the respiratory network including the preBotzinger Complex (preBotC), the site of respiratory rhythmogenesis. It shows that MOR inhibition may be due to a reduction in spiking in the pre-inspiratory phase as well as a reduction in synaptic transmission. This study uses a combination of in-vitro electrophysiology and optogenetics as well as in-vivo optogenetic experiments which are the state-of-the-art techniques in neuroscience. The experiments performed are technically challenging especially in this region of the medulla. Therefore these data are unique as they elucidate network level mechanism of opioid inhibition in relatively …
Reviewer #1 (Public Review):
This study identifies some of the mechanisms of action of MOR-acting opioids on the respiratory network. It determines the impacts of MOR activation of various types of neurons of the respiratory network including the preBotzinger Complex (preBotC), the site of respiratory rhythmogenesis. It shows that MOR inhibition may be due to a reduction in spiking in the pre-inspiratory phase as well as a reduction in synaptic transmission. This study uses a combination of in-vitro electrophysiology and optogenetics as well as in-vivo optogenetic experiments which are the state-of-the-art techniques in neuroscience. The experiments performed are technically challenging especially in this region of the medulla. Therefore these data are unique as they elucidate network level mechanism of opioid inhibition in relatively "intact" preparations. This study has limitations inherent to the animal preparations used which include in-vitro conditions, immature brain development in some conditions, and adult, anesthetized animals in others. However, these limitations are expected in these challenging animal models.
Overall, this study proposes new mechanisms of opioid inhibition that may be translated to other model systems. A better understanding of these mechanisms is critical to identify new therapies and eventually new pain killers without the respiratory side-effects of opioids.
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Reviewer #2 (Public Review):
This study leverages an emerging consensus regarding respiratory rhythmogenesis to better understand how opioids depress breathing. Since 1991, the pre-Bötzinger Complex (PBC) has been proposed as the central pattern generator for breathing, which has been extensively studied in the transverse slice, but using conventional electrophysiological techniques, definitive functional-anatomical identification of the rhythmogenic constituents of the respiratory CPG was impossible. Studies focusing on the developmental origin of these networks identified a specific subpopulation of dbx1+ neurons born between E10.5-E11.5 that were shown to generate respiratory rhythm in PBC (Bouvier et al., 2010). In earlier work by this same group, reporter genes targeting these populations, the authors of this study were able to …
Reviewer #2 (Public Review):
This study leverages an emerging consensus regarding respiratory rhythmogenesis to better understand how opioids depress breathing. Since 1991, the pre-Bötzinger Complex (PBC) has been proposed as the central pattern generator for breathing, which has been extensively studied in the transverse slice, but using conventional electrophysiological techniques, definitive functional-anatomical identification of the rhythmogenic constituents of the respiratory CPG was impossible. Studies focusing on the developmental origin of these networks identified a specific subpopulation of dbx1+ neurons born between E10.5-E11.5 that were shown to generate respiratory rhythm in PBC (Bouvier et al., 2010). In earlier work by this same group, reporter genes targeting these populations, the authors of this study were able to robustly identify the PBC both in vivo, and in their novel horizontal slice preparation (Baertsch et al., 2019), and validate the general rhythmogenic mechanism they elaborate on here. Thus, this study builds on the slow but steady progress in this field that has emerged from a collaborative, multidisciplinary approach.
Despite an emerging consensus regarding the functional anatomy of the PBC, a mechanistic account of rhythmogenesis remains elusive. Persistence of rhythmic activity following fast Cl--mediated inhibition ruled out models in which crossed inhibition generated rhythmic output (Feldman and Smith, 1989). After neurons with endogenous bursting properties were found in PBC, pacemaker-driven network models were developed (Butera et al., 1999), but these fell out of favor when it was shown that respiratory rhythm persisted following blockade of conductances essential for endogenous bursting (Del Negro et al., 2002). Currently, there is general agreement that respiratory rhythm arises out of network interactions between neurons that begin spiking hundreds of milliseconds before the inspiratory burst onset, described as the "percolation period" in this manuscript. Although recently a model in which respiratory rhythm arose out of network interaction between pre-I neurons without the requirement of pacemakers (Guerrier et al., 2015), definitive experimental evidence supporting this account has not yet emerged, and somewhat more qualitative "burstlet"-based mechanisms have been proposed. (Kam et al., 2013). Because a rigorous functional taxonomy of respiratory network constituents has not yet been completed, delineating how opioids depress breathing remains challenging.
In this study, the authors elucidate how the network respond to opioids by selective targeting of neurons directly modulated by opioids. They do this by expressing light-activated inhibitory and excitatory conductances in neurons expressing the mu-opioid receptor (MOR), via the Oprm1 gene. In addition, optogenetic manipulations were also carried out on the same mouse lines in adults, in vivo. Because light could be used to identify MOR-expressing neurons, the authors were able to robustly differentiate between direct and network-mediated effects of opioids on neuronal activity. By comparing the effect of light-induced hyperpolarization of Oprm1+ neurons with the effect of opioids, the authors also robustly establish that OIRD cannot be ascribed to hyperpolarization of MOR-expressing neurons alone, and that impaired synaptic transmission also contributes.
A less obvious and equally important finding is that the specificity and accuracy of this optogenetic approach highlights why networks that control breathing have been so difficult to study. If a specific gene can be found that enables optogenetic manipulation of a discrete, functionally homogeneous population of neurons, the reverse-engineering potential of this approach is maximized. What was found here was that among PBC neurons, taxonomized into 4 classes (pre-inspiratory, inspiratory, expiratory, tonic), roughly half of the neurons probed in each group were found to be Oprm1+. This partial overlap with all groups weakens inferences drawn from optogenetic manipulations. While incorporation of optogenetic methods offers a robust way to differentiate between direct effects of opioids on members of each group that express MORs from indirect, network-mediated changes in activity, the impact of opioids on essential rhythmogenic networks is more difficult to assess, since the methods used here cannot selectively target pre-inspiratory neurons, which are the only population proposed to have dedicated rhythmogenic function. While it is possible that other, yet-to-be-identified genes are expressed in all pre-inspiratory neurons, and only in this population, enabling a highly targeted analysis of the functional role of pre-inspiratory neurons in respiratory rhythmogenesis using an optogenetic or other gene-targeting approach, the absence of success in past efforts to selectively pick out essential respiratory rhythmogenic constituents suggests that essential constituents are themselves heterogeneous, and overlap genotypically with non-rhythmogenic neurons.
Compounding the conceptual challenges posed by the effort to delineate the impact of a drug on heterogeneous respiratory networks are the complexities of opioid pharmacodynamics (Williams et al., 2013). Within 5 minutes of initial exposure, receptor phosphorylation and arrestin binding rapidly blunt responses to opioids; on an overlapping but slower timescale, receptor endocytosis and recycling further alters the effect of opioids; all these processes vary with opioid agonist. Insofar as these factors are clinically relevant, they would suggest that OIRD is most acute in naïve subjects whose response is not blunted by desensitization or tolerance, and may contribute to the significantly lower mortality risk associated with opioid substitution programs as compared to abstinence treatment programs (Sordo et al., 2017). In this study, opioids were administered sequentially to generate dose-response curves. If it were the case that in some experiments the order in which opioid doses were administered was randomized, the impact of desensitization on OIRD could be estimated.
By including a robust, high-level low-dimensional model of respiratory rhythm generating networks to test whether their conjecture that OIRD arises out of the interaction between presynaptic hyperpolarization and synaptic depression, the authors are providing a blueprint for how empirically motivated hypotheses can be validated and tested using modeling. This multidisciplinary approach will gain in power as more detailed mechanistic accounts of respiratory rhythmogenesis continue to evolve.
Baertsch, N.A., Severs, L.J., Anderson, T.M., and Ramirez, J.M. (2019). A spatially dynamic network underlies the generation of inspiratory behaviors. Proc Natl Acad Sci U S A 116, 7493-7502.
Bouvier, J., Thoby-Brisson, M., Renier, N., Dubreuil, V., Ericson, J., Champagnat, J., Pierani, A., Chedotal, A., and Fortin, G. (2010). Hindbrain interneurons and axon guidance signaling critical for breathing. Nat Neurosci 13, 1066-1074.
Butera, R.J., Jr., Rinzel, J., and Smith, J.C. (1999). Models of respiratory rhythm generation in the pre-Botzinger complex. I. Bursting pacemaker neurons. J Neurophysiol 82, 382-397.
Del Negro, C.A., Morgado-Valle, C., and Feldman, J.L. (2002). Respiratory rhythm: an emergent network property? Neuron 34, 821-830.
Feldman, J.L., and Smith, J.C. (1989). Cellular mechanisms underlying modulation of breathing pattern in mammals. Ann N Y Acad Sci 563, 114-130.
Guerrier, C., Hayes, J.A., Fortin, G., and Holcman, D. (2015). Robust network oscillations during mammalian respiratory rhythm generation driven by synaptic dynamics. Proc Natl Acad Sci U S A 112, 9728-9733.
Kam, K., Worrell, J.W., Ventalon, C., Emiliani, V., and Feldman, J.L. (2013). Emergence of Population Bursts from Simultaneous Activation of Small Subsets of preBotzinger Complex Inspiratory Neurons. J Neurosci 33, 3332-3338.
Sordo, L., Barrio, G., Bravo, M.J., Indave, B.I., Degenhardt, L., Wiessing, L., Ferri, M., and Pastor-Barriuso, R. (2017). Mortality risk during and after opioid substitution treatment: systematic review and meta-analysis of cohort studies. BMJ 357, j1550.
Williams, J.T., Ingram, S.L., Henderson, G., Chavkin, C., von Zastrow, M., Schulz, S., Koch, T., Evans, C.J., and Christie, M.J. (2013). Regulation of mu-opioid receptors: desensitization, phosphorylation, internalization, and tolerance. Pharmacological reviews 65, 223-254.
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Reviewer #3 (Public Review):
Opioid induced respiratory depression (OIRD), characterized by a pronounced decrease in the frequency and regularity of breathing, involves suppression of neuronal activity within the brainstem inspiratory rhythm generator- the preBötzinger complex (preBötC), which contains rhythmic neurons expressing mu-opioid receptors (MOR). In this paper, Baertsch and colleagues sought to define in greater detail cellular- and network-level mechanisms of OIRD in the preBötC from in vitro and in vivo electrophysiological studies and computational modeling. The main important conclusion supported by the authors' results is that mechanisms involving hyperpolarization of preBötC excitatory neurons and depression of excitatory synaptic transmission in preBötC circuits explain perturbations of rhythmic neuron activity and the …
Reviewer #3 (Public Review):
Opioid induced respiratory depression (OIRD), characterized by a pronounced decrease in the frequency and regularity of breathing, involves suppression of neuronal activity within the brainstem inspiratory rhythm generator- the preBötzinger complex (preBötC), which contains rhythmic neurons expressing mu-opioid receptors (MOR). In this paper, Baertsch and colleagues sought to define in greater detail cellular- and network-level mechanisms of OIRD in the preBötC from in vitro and in vivo electrophysiological studies and computational modeling. The main important conclusion supported by the authors' results is that mechanisms involving hyperpolarization of preBötC excitatory neurons and depression of excitatory synaptic transmission in preBötC circuits explain perturbations of rhythmic neuron activity and the inspiratory rhythm caused by MOR activation.
Strengths of the study include: (1) the authors employ a combination of electrophysiological, optogenetic, and computational approaches to dissect mechanisms, (2) the authors employ optogenetic identification and manipulation of neurons with the MOR encoding gene Oprm1, (3) important new data on electrophysiological phenotypes of MOR-expressing neurons is presented, (4) the authors analyze effects of MOR activation on neuron spiking activity during different phases of the inspiratory rhythm, (5) the authors present novel electrophysiological evidence of MOR-induced depression of preBötC excitatory network synaptic transmission, and (6) simulations with a model of the preBötC excitatory network can reproduce experimentally observed perturbations of inspiratory rhythm when a combination of neuronal hyperpolarization and reduction in strength of excitatory synaptic transmission is implemented. Weaknesses include: (1) experimental data on neuronal hyperpolarization caused by MOR activation is required to justify some the authors conclusions, and (2) parameterization of the preBötC network model used requires further explanation.
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