Opioid suppression of an excitatory pontomedullary respiratory circuit by convergent mechanisms

  1. Jordan T Bateman
  2. Erica S Levitt

  • Curated by eLife

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    Opioid-induced respiratory depression is one of the side effects of opioid drugs. Although opioid overdose deaths are highly prevalent, our knowledge of the neural circuits underlying respiratory depression in the brainstem is far from complete. The present study used a variety of sophisticated experimental techniques to convincingly reveal the identity of brainstem components that are part of the neural circuits involved in the mediation of opioid respiratory effects, together with defining potential synaptic underlying mechanisms. They focused on two regions of the brainstem, namely the Kolliker-Fuse and the preBötzinger Complex, and proposed a combination of three complementary processes at pre- and post-synaptic sites in both KF and preBötC regions to explain respiratory depression linked to opioid exposure. This study provides very important findings on the circuitry involved in opioid-induced respiratory depression, and the present results are of broad interest to the respiratory control research community, as well as medically relevant.

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Abstract

Opioids depress breathing by inhibition of interconnected respiratory nuclei in the pons and medulla. Mu opioid receptor (MOR) agonists directly hyperpolarize a population of neurons in the dorsolateral pons, particularly the Kölliker-Fuse (KF) nucleus, that are key mediators of opioid-induced respiratory depression. However, the projection target and synaptic connections of MOR-expressing KF neurons are unknown. Here, we used retrograde labeling and brain slice electrophysiology to determine that MOR-expressing KF neurons project to respiratory nuclei in the ventrolateral medulla, including the preBötzinger complex (preBötC) and rostral ventral respiratory group (rVRG). These medullary-projecting, MOR-expressing dorsolateral pontine neurons express FoxP2 and are distinct from calcitonin gene-related peptide-expressing lateral parabrachial neurons. Furthermore, dorsolateral pontine neurons release glutamate onto excitatory preBötC and rVRG neurons via monosynaptic projections, which is inhibited by presynaptic opioid receptors. Surprisingly, the majority of excitatory preBötC and rVRG neurons receiving MOR-sensitive glutamatergic synaptic input from the dorsolateral pons are themselves hyperpolarized by opioids, suggesting a selective opioid-sensitive circuit from the KF to the ventrolateral medulla. Opioids inhibit this excitatory pontomedullary respiratory circuit by three distinct mechanisms—somatodendritic MORs on dorsolateral pontine and ventrolateral medullary neurons and presynaptic MORs on dorsolateral pontine neuron terminals in the ventrolateral medulla—all of which could contribute to opioid-induced respiratory depression.

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  1. Version published to 10.7554/elife.81119 on eLife
  2. eLife

    Author Response

    Reviewer #2 (Public Review):

    This study identifies the neural circuits inhibited by activation of opioid receptors using complex experimental approaches such as electrophysiology, pharmacology, and optogenetics and combined them with retrograde and anterograde tracings. The authors characterize two key regions of the brainstem, the preBötzinger Complex, and the Kolliker-Fuse, and how these neuronal populations interact. Understanding the interactions of these circuits substantially increases our understanding of the neural circuits sensitive to opioid drugs which are critical to understand how opioids act on breathing and potentially design new therapies.

    Major strengths.

    This study maps the excitatory projections from the Kolliker-Fuse to the preBötzinger Complex and rostral ventral respiratory group and shows that these projections are inhibited by opioid drugs. These Kolliker-Fuse neurons express FoxP2, but not the calcitonin gene-related peptide, which distinguishes them from parabrachial neurons. In addition, the preBötzinger Complex is also hyperpolarized by opioid drugs. The experiments performed by the authors are challenging, complex, and the most appropriate types of approaches to understanding pre- and post-synaptic mechanisms, which cannot be studied in vivo. These experiments also used complex tracing methods using adenoassociated virus and cre-lox recombinase approaches.

    Limitations.

    (1) The roles of the mechanisms identified in this study have not been established in models recording opioid-induced respiratory depression or respiratory activity. This study does not record, modulate, or assess respiratory activity in-vitro or in-vivo, without or with opioid drugs such as fentanyl or morphine.

    (2) Experiments are performed in-vitro which do not mimic the effects of opioids observed in-vivo or in freely-moving animals. However, identification of pre- and post- synaptic mechanisms, as well as projections, cannot be performed in-vivo, so the authors use the right approaches for their experiments.

    We agree with both of these points. We hope this study lays the groundwork for future studies assessing the impact of these projections on respiratory activity in vitro and in vivo.

    (3) The type of neurons projecting from KP to preBötzinger Complex or ventral respiratory group have not been identified. Although some of these cells are glutamatergic, optogenetic experiments could have been performed in other cre-expressing cell populations, such as neurokinin-1 receptors.

    There are indeed many different cell populations that could be interrogated. In addition to the optogenetic identification of glutamatergic projections, we identified immunohistochemically that at least some opioid receptor-expressing, medullary-projecting KF neurons express FoxP2, and not CGRP. Further dissection of other cell populations, such as Lmx1b and Phox2b, are excellent future directions.

    Reviewer #3 (Public Review):

    This manuscript reveals opioid suppression of breathing could occur via multiple mechanisms and at multiple sites in the pontomedullary respiratory network. The authors show that opioids inhibit an excitatory pontomedullary respiratory circuit via three mechanisms: 1) postsynaptic MOR-mediated hyperpolarization of KF neurons that project to the ventrolateral medulla, 2) presynaptic MOR mediated inhibition of glutamate release from dorsolateral pontine terminals onto excitatory preBötC and rVRG neurons, and 3) postsynaptic MOR-mediated hyperpolarization of the preBötC and rVRG neurons that receive pontine glutamatergic input.

    This manuscript describes in detail a useful method for dissecting the relationship between the dorsolateral pons and the rostral medulla, which will be useful for various researchers. It's also great to see how many different methods have been applied to improve the accuracy of the results.

    1. Relationship between the dorsolateral pons and rostral ventrolateral medulla.

    The method of this paper is a good paper to show a very precise relationship between the presence of opioid receptors and the dorsolateral pons and rostral ventrolateral medulla, and for opioid receptors, based on the expression of Oprm1, the use of genetically modified mice with anterograde or retrograde viruses with additional fluorescent colors showed both anterograde and retrograde projections, revealing a relationship between the dorsolateral pons and rostral ventrolateral medulla.

    For example, to visualize dorsal pontine neurons expressing Oprm1, Oprm1Cre/Cre mice were crossed with Ai9tdTomato Cre reporter mice to generate Ai9tdT/+ oprm1Cre/+ mice (Oprm1Cre/tdT mice) expressing tdTomato on neurons that also express MOR at any point during development, and the retrograde virus encoding Cre-dependent expression of GFP (retrograde AAV-hSIN-DIO-eGFP was injected into the respiratory center of Oprm1Cre/+ mice and into the ventral respiratory neuron group, showing that KF neurons expressing Oprm1 project to the respiration-related nucleus of the ventrolateral medulla.

    However, although the authors have also corrected it, the virus may spread to other places as well as where they thought it would be injected, and it is important to note that it is injected accordingly to mark the injection site with an anterograde virus encoding a different fluorescent color mCherry, and the extent of the injection is quantified, which is excellent as a control experiment.

    In addition, the respiratory center seems to be related not only to preBötC but also to pFRG recently, so if the relation with it is described, it is important from the viewpoint of the effect on the respiratory center and the effect on the rhythm.

    Our injections centered in preBotC, rVRG or BötC did not spread extensively to slices containing 7N/pFRG (Figure 2C and Figure 2-supplement 1D, Bregma -6.0 to -6.4, shaded region labeled 7N).

  3. eLife

    eLife assessment

    Opioid-induced respiratory depression is one of the side effects of opioid drugs. Although opioid overdose deaths are highly prevalent, our knowledge of the neural circuits underlying respiratory depression in the brainstem is far from complete. The present study used a variety of sophisticated experimental techniques to convincingly reveal the identity of brainstem components that are part of the neural circuits involved in the mediation of opioid respiratory effects, together with defining potential synaptic underlying mechanisms. They focused on two regions of the brainstem, namely the Kolliker-Fuse and the preBötzinger Complex, and proposed a combination of three complementary processes at pre- and post-synaptic sites in both KF and preBötC regions to explain respiratory depression linked to opioid exposure. This study provides very important findings on the circuitry involved in opioid-induced respiratory depression, and the present results are of broad interest to the respiratory control research community, as well as medically relevant.

  4. eLife

    Reviewer #1 (Public Review):

    The present study investigates the anatomical connectivity between Mu opioid receptor (MOR) expressing neurons of the pontine respiratory group with down-stream targets of the respiratory network in the medulla oblongata. The study employs a variety of viral tracing approaches, optogenetic stimulation of pre-synapses of descending pontine projection neurons, and patch clamp electrophysiology. Overall the study is well conducted and the authors show that MOR expressing excitatory glutamatergic pontine neurons project to the medullary respiratory rhythm generator and adjacent ventral respiratory group. The study implies that opioids act on MOR-located somata and dendrites of the pontine and medullary respiratory groups. Importantly MOR are expressed on the pre-synapses of the descending pontine projections neurons. The authors, therefore, propose that opioids mediate respiratory depression via distinct pre- and post- synaptic mechanisms across inter-connected ponto-medullary respiratory neurons. The study advances our knowledge of network mechanisms that mediate opioid respiratory depression and may provide interesting frameworks for the development of therapies to counteract or prevent opioid respiratory depression. The study is of broad interest to the respiratory control research community, as well as medically relevant.

  5. eLife

    Reviewer #2 (Public Review):

    This study identifies the neural circuits inhibited by activation of opioid receptors using complex experimental approaches such as electrophysiology, pharmacology, and optogenetics and combined them with retrograde and anterograde tracings. The authors characterize two key regions of the brainstem, the preBötzinger Complex, and the Kolliker-Fuse, and how these neuronal populations interact. Understanding the interactions of these circuits substantially increases our understanding of the neural circuits sensitive to opioid drugs which are critical to understand how opioids act on breathing and potentially design new therapies.

    Major strengths.
    This study maps the excitatory projections from the Kolliker-Fuse to the preBötzinger Complex and rostral ventral respiratory group and shows that these projections are inhibited by opioid drugs. These Kolliker-Fuse neurons express FoxP2, but not the calcitonin gene-related peptide, which distinguishes them from parabrachial neurons. In addition, the preBötzinger Complex is also hyperpolarized by opioid drugs. The experiments performed by the authors are challenging, complex, and the most appropriate types of approaches to understanding pre- and post-synaptic mechanisms, which cannot be studied in vivo. These experiments also used complex tracing methods using adenoassociated virus and cre-lox recombinase approaches.

    Limitations.
    (1) The roles of the mechanisms identified in this study have not been established in models recording opioid-induced respiratory depression or respiratory activity. This study does not record, modulate, or assess respiratory activity in-vitro or in-vivo, without or with opioid drugs such as fentanyl or morphine.
    (2) Experiments are performed in-vitro which do not mimic the effects of opioids observed in-vivo or in freely-moving animals. However, identification of pre- and post- synaptic mechanisms, as well as projections, cannot be performed in-vivo, so the authors use the right approaches for their experiments.
    (3) The type of neurons projecting from KP to preBötzinger Complex or ventral respiratory group have not been identified. Although some of these cells are glutamatergic, optogenetic experiments could have been performed in other cre-expressing cell populations, such as neurokinin-1 receptors.

    This study provides new insights into the types of circuits inhibited by opioid drugs, and the site of actions of inhibition, such as pre- or post-synaptic, and proposes how inhibition by opioids acts at multiple sites in the brainstem through various mechanisms.

    Although many studies have recently explored the types of neurons and sites in the brain sensitive to opioids, the present study is the first to provide a clear picture of the neuronal mechanism underlying inhibition by opioids. Importantly, it provides a link between two sites known to inhibit breathing when inhibited by opioids. The results provided here combined with a complex methodology support the various conclusions reached by the authors.

  6. eLife

    Reviewer #3 (Public Review):

    This manuscript reveals opioid suppression of breathing could occur via multiple mechanisms and at multiple sites in the pontomedullary respiratory network. The authors show that opioids inhibit an excitatory pontomedullary respiratory circuit via three mechanisms: 1) postsynaptic MOR-mediated hyperpolarization of KF neurons that project to the ventrolateral medulla, 2) presynaptic MOR mediated inhibition of glutamate release from dorsolateral pontine terminals onto excitatory preBötC and rVRG neurons, and 3) postsynaptic MOR-mediated hyperpolarization of the preBötC and rVRG neurons that receive pontine glutamatergic input.

    This manuscript describes in detail a useful method for dissecting the relationship between the dorsolateral pons and the rostral medulla, which will be useful for various researchers. It's also great to see how many different methods have been applied to improve the accuracy of the results.

    1. Relationship between the dorsolateral pons and rostral ventrolateral medulla.

    The method of this paper is a good paper to show a very precise relationship between the presence of opioid receptors and the dorsolateral pons and rostral ventrolateral medulla, and for opioid receptors, based on the expression of Oprm1, the use of genetically modified mice with anterograde or retrograde viruses with additional fluorescent colors showed both anterograde and retrograde projections, revealing a relationship between the dorsolateral pons and rostral ventrolateral medulla.

    For example, to visualize dorsal pontine neurons expressing Oprm1, Oprm1Cre/Cre mice were crossed with Ai9tdTomato Cre reporter mice to generate Ai9tdT/+ oprm1Cre/+ mice (Oprm1Cre/tdT mice) expressing tdTomato on neurons that also express MOR at any point during development, and the retrograde virus encoding Cre-dependent expression of GFP (retrograde AAV-hSIN-DIO-eGFP was injected into the respiratory center of Oprm1Cre/+ mice and into the ventral respiratory neuron group, showing that KF neurons expressing Oprm1 project to the respiration-related nucleus of the ventrolateral medulla.

    However, although the authors have also corrected it, the virus may spread to other places as well as where they thought it would be injected, and it is important to note that it is injected accordingly to mark the injection site with an anterograde virus encoding a different fluorescent color mCherry, and the extent of the injection is quantified, which is excellent as a control experiment.

    In addition, the respiratory center seems to be related not only to preBötC but also to pFRG recently, so if the relation with it is described, it is important from the viewpoint of the effect on the respiratory center and the effect on the rhythm.

    2. Electrophysiological approaches and useful methods for target neurons

    Oprm1Cre/+ mice), the authors found abundant Oprm1 + projections in the preBötC region of the medulla oblongata (respiratory center) and sought to determine whether presynaptic opioid receptors inhibit glutamate release from KF terminals to excitatory preBötC and rVRG neurons, since KF neurons in the dorsolateral pons projecting to the ventrolateral medulla oblongata had been shown to be glutamatergic and to have opioid receptors. The authors injected a channelrhodopsin-2-encoding virus (AAV2-hSin-hChR2 (H134R) -EYFP-WPRE-PA) into the dorsolateral pontine KF of vglu2Cre / tdT mice and performed whole-cell voltage-clamp recordings from td tomato-expressing, excitatory vglu2-expressing preBötC and rVRG neurons, contained in acute brain slices. Moreover, both opioid-sensitive and opioid-insensitive KF neurons that project to preBötC and rVRG were visible and recorded using FluoSpheres which are much more visible in acute brain sections than retrograde tracers of viruses.

    1. Optogenetic stimulation of the KF terminus was blocked by the AMPA-type glutamate receptor antagonist DNQX. In excitatory pre-BötC and rVRG neurons, the terminals from the dorsal pontine KF were activated by optogenetic stimulation, and the KF synapses to the medullary respiratory neurons were found to be monosynaptic because oEPSCs(optical stimulated EPSCs) were removed by TTX but were subsequently restored by the application of K-channel blocker 4AP. Thus, KF neurons have been shown to send monosynaptic glutamatergic projections to excitatory ventrolateral medullary neurons using terminal optogenetic stimulation and receptor and channel inhibitors.

    2. To determine whether opioids inhibit glutamate release from KF terminals to medullary respiratory neurons, we recorded a pair of oEPSCs (50 ms stimulus interval) from excitatory preBötC and rVRG neurons and applied an endogenous opioid agonist, [Met5] enkephalin (ME), to the perfusion solution. ME is preBötC and rVRG neurons, indicating inhibition of glutamate release by presynaptic MOR PPR. Thus, presynaptic opioid receptors have been shown electrophysiologically to inhibit glutamate release from KF terminals to excitatory pre-BötC and rVRG neurons.

    3. Whether excitatory pre-BötC or rVRG neurons themselves receiving opioid-sensitive glutamatergic synaptic inputs from KF are hyperpolarized by opioids can be determined by monitoring their retention currents.

    4. Since FluoSpheres are much more visible in acute brain sections than retrograde tracers of viruses and do not spread to injection sites, they chose to record from retrogradely labeled KF neurons with FluoSpheres injected into preBötC or rVRG in wild-type mice, allowing us to label KF neurons regardless of Oprm1 expression status and determine the projection patterns of both Oprm1 + and Oprm1- neurons. Whole-cell voltage-clamp recordings from fluorescent KF neurons contained in acute brain slices show that the presence of ME-mediated outward current can identify KF neurons that express functional MORs and are opioid-sensitive compared to neurons that lack ME-mediated outward current (insensitive). This suggests that both opioid-sensitive and opioid-insensitive KF neurons project to preBötC and rVRG.

    Although much has been written about the relationship between KF neurons and medulla oblongata neurons and their being glutaminergic neurons, detailed descriptions of the recorded neuronal firing patterns are lacking. You should describe what firing pattern the recorded neurons had. If we don't do that, we won't be able to tell whether it's a respiratory neuron or another tonic firing neuron, so I don't think we can discuss whether it's involved in the respiratory rhythm.

    3. Compare the distribution of neurons

    To examine the distribution of Oprm1 + and Oprm1- dorsolateral pontine neurons projecting to the ventrolateral medulla, we injected retrograde AAV-hSin-DIO-eGFP and retrograde AAV-hSin-mCherry into preBötC and rVRG of Oprm1Cre/+ mice and found a neuronal distribution in which Oprm1-expressing projection neurons expressed GFP and mCherry, but not Oprm1-expressing projection neurons expressed only mCherry.

    In addition, rostral glutamatergic KF neurons express FoxP2, while MOR-expressing glutamatergic neurons in the lateral parabrachial region that project to the forebrain express the CGRP-encoding gene, Calca. In view of this, the authors performed immunohistochemistry for FoxP2 and CGRP on Oprm1 + KF neurons projecting to the ventrolateral medulla, and Oprm1 + medulla oblongata projecting KF neurons expressed FoxP2 but not CGRP. The expression of CGRP was not observed in rostral KF and medullary projection Oprm1 + neurons and neurites but was strong in lateral parabrachial neurons and their axonal fiber projections. Can you describe the relationship between CGRP and FoxP2 and recorded neurons?

  7. Version published to 10.1101/2022.06.24.497461v1 on bioRxiv