Opioids such as morphine or fentanyl are powerful substances used to relieve pain in medical settings. However, taken in too high a dose they can depress breathing – in other words, they can lead to slow, shallow breaths that cannot sustain life. In the United States, where the misuse of these drugs has been soaring in the past decades, about 130 people die each day from opioid overdose. Pinpointing the exact brain areas and neurons that opioids act on to depress breathing could help to create safer painkillers that do not have this deadly effect. While previous studies have proposed several brain regions that could be involved, they have not been able to confirm these results, or determine which area plays the biggest role.

Opioids influence the brain of animals (including humans) by attaching to proteins known as opioid receptors that are present at the surface of neurons. Here, Bachmutsky et al. genetically engineered mice that lack these receptors in specific brain regions that control breathing. The animals were then exposed to opioids, and their breathing was closely monitored.

The experiments showed that two small brain areas were responsible for breathing becoming depressed under the influence of opioids. The region with the most critical impact also happens to be where the breathing rhythms originate. There, a small group of 50 to 140 neurons were used by opioids to depress breathing. Crucially, these cells were not necessary for the drugs’ ability to relieve pain.

Overall, the work by Bachmutsky et al. highlights a group of neurons whose role in creating breathing rhythms deserves further attention. It also opens the possibility that targeting these neurons would help to create safer painkillers.

Nearly 400,000 people in the United States died from a drug overdose involving a prescription or illicit opioid between 1999 and 2017 (Scholl et al., 2018). This epidemic is not unique to the United States and with the increasing distribution of highly potent synthetic opioids like fentanyl, it has become a global public health emergency (Rudd et al., 2016). Death from opioid overdose results from slow and shallow breathing, also known as opioid induced respiratory depression (OIRD, Pattinson, 2008). Like humans, breathing in mice is severely depressed by opioids and this response is eliminated when the µ-Opioid receptor (Oprm1) is globally deleted (Dahan et al., 2001). Oprm1 is broadly expressed, in both the central and peripheral nervous systems, including sites that could modulate breathing such as: the cerebral cortex, brainstem respiratory control centers, primary motor neurons, solitary nucleus, and oxygen sensing afferents (Mansour et al., 1994; Kirby and McQueen, 1986). Therefore, either one or multiple sites could be mediating the depressive effects of opioids on breathing.

Indeed, multiple brain regions have been shown to independently slow breathing after local injection of opioid agonists (Kirby and McQueen, 1986; Montandon et al., 2011; Mustapic et al., 2010; Prkic et al., 2012). Although informative, doubts remain for which of these sites are necessary and sufficient to induce OIRD from systemic opioids for three reasons. First, injection of opioid agonists or antagonists into candidate areas modulates µ-opioid receptors on the cell body (post-synaptic) as well as receptors on incoming terminals (pre-synaptic). Second, these studies necessitate anesthetized and reduced animal preparations, which alter brain activity in many of the candidate Oprm1 expressing sites. And third, there is not a standard and quantitative definition for how breathing changes in OIRD, and this makes comparing studies that use different breathing metrics measured in different experimental paradigms challenging.

To address these limitations, we conducted a detailed quantitative analysis of OIRD in awake animals and identify two key changes to the breath that drive the depressive effects of opioids. These two metrics thereafter define OIRD in our study and can serve as a rubric for others. We then locally eliminate the µ-opioid receptor in awake mice, disambiguating pre and post-synaptic effects, and use these metrics to define two key brain sites that mediate OIRD. Recently, a similar approach demonstrated some role for these sites in OIRD (Varga et al., 2020). Among these two sites in our study, we find that one is dominant and driven by just 140 critical neurons in vitro and, importantly, these neurons are not required for opioid-induced analgesia, suggesting a neutral target for developing safer opioids or rescue strategies for opioid overdose.

Up to now, OIRD has generally been described as a slowing and shallowing of breath (Pattinson, 2008). We therefore felt it was important to more precisely, quantitatively describe the changes in breathing in hopes of elucidating potential mechanisms of respiratory depression. We began by asking whether specific parameters of the breath are affected by opioids. We monitored breathing in awake, behaving mice by whole body plethysmography after intraperitoneal injection (IP) of saline for control and then 20 mg/kg morphine at least 24 hr later (Figure 1A). Compared to saline, breathing after morphine administration (in normoxia) became much slower and inspiratory airflow decreased, each by 60% (Figure 1B,C). This culminated in ~50% decrease in overall minute ventilation (MV = approximated tidal volume x respiratory rate, Figure 1C), demonstrating that 20 mg/kg morphine is, indeed, a suitable dose to model OIRD.

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Average frequency, peak flow, minute ventilation, tidal volume and pause duration for 31 animals after intraperitoneal saline or morphine in normoxia and hypercapnia.

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Breath morphology in normoxia after IP saline versus morphine cannot be directly compared since activity of the mouse is different (exploring vs. sedated, Supplementary file 1), which significantly influences the types of breaths taken. This prevented a precise characterization of breath parameters that dictate OIRD. To overcome this, we measured breathing in hypercapnic air (21% O₂, 5% CO₂) which normalizes behavior and thus breathing (Figure 1D, Supplementary file 1). As in normoxia, morphine depressed respiratory rate (by 50%, Figure 1E,F), peak inspiratory airflow (by 60%, Figure 1E,F), and minute ventilation (by 60%, Figure 1F). Hypercapnic breaths after saline exhibited two phases, inspiration and expiration, each lasting about 50 msec. (Figure 1G,H). After morphine, only the inspiratory phase (measured as inspiratory time, Ti) became substantially longer (Figure 1G,H). Additionally, hypercapnic breaths showed a new, third phase after the initial expiration (measured as expiratory time, Te, Figure 1G,H) that was characterized by prolonged little to no airflow (<0.5 mL/sec.) preceding hypercapnia induced active expiration (Pisanski and Pagliardini, 2019). We define this new phase as a pause (low airflow + active expiration, Figure 1G, Figure 1—figure supplement 1). Such pauses lasted up to several hundred milliseconds (Figure 1I), accounting for about one-third of the average breath length (Figure 1J). Thus, the 50% decrease in respiratory rate after morphine administration is primarily due to prolonging of Ti and pause phases, and the increased prevalence of time spent in pause significantly contributes to the decrease in minute ventilation.

Typically the length of inspiratory time is determined by a stretch-evoked feedback signal from the lung which terminates inspiration (West, 2005). This reflex is represented by the correlation observed between Ti and peak inspiratory airflow (Figure 1K). Breaths in morphine still maintain this correlation despite having a longer Ti and decreased inspiratory airflow (Figure 1K). As a result, morphine breaths have a similar approximated tidal volume (TV) compared to saline control (Figure 1L,M). In other words, as opioids decrease inspiratory airflow, Ti displays a compensatory increase to preserve TV (Figures 1N and Hill et al., 2018). In summary, opioids cause only two primary changes to the breath, namely, 1) decreased inspiratory airflow and 2) addition of a pause phase that delays initiation of subsequent breaths (Figure 1N). These two parameters can both be controlled by the breathing central pattern generator, the preBötzinger Complex (preBötC), in the brainstem and suggest that this may be a key locus affected during OIRD (Smith et al., 1991; Feldman et al., 2013; Cui et al., 2016).

Indeed, the preBötC has been proposed to play a key role in OIRD since localized injection of opioids results in respiratory depression and localized naloxone reverses decreased breathing after administration of systemic opioids (Montandon et al., 2011; Montandon and Horner, 2014). However, such experiments fail to distinguish between the action of opioids on presynaptic terminals (Mudge et al., 1979) of distant neurons projecting into the preBötC versus direct action on preBötC neurons themselves (Figure 2A; Montandon et al., 2011; Dobbins and Feldman, 1994; Gray et al., 1999) To overcome this, we genetically eliminated the µ-Opioid receptor (Oprm1) from preBötC cells exclusively, sparing projecting inputs, by stereotaxic injection of adeno-associated virus constitutively expressing Cre (AAV-Cre-GFP) into the preBötC of Oprm1 flox/flox (Oprm1^f/f) adult mice (Figure 2B). Injection site specificity was confirmed by the restricted expression of Cre-GFP (Figure 2—figure supplement 1), and subsequent Oprm1 deletion, was inferred. To establish a baseline, we first measured breathing after administration of saline and morphine in normoxia and hypercapnia in intact animals, as described above. At least one month after bilateral injection of virus into the preBötC, we then re-analyzed breathing (Figure 2C). With this protocol, each animal’s unique breathing and OIRD response serves as its own internal control, which is necessary due to the variability in OIRD severity between mice (Figure 1F). Deletion of Oprm1 in the preBötC did not affect breathing observed after saline injection (Figure 2D,E), suggesting that in this context, opioids do not exert an endogenous effect. In contrast, breathing was markedly less depressed by morphine administration (Figure 2D,E) compared to the intact control state: breaths were twice as fast (3 to 6 Hz, Figure 2F,H), the peak inspiratory flow was larger (Figure 2F,I), and pauses were nearly eliminated (Figure 2G,J). Notably, histological analysis confirmed that AAV-Cre-GFP expression was localized to the preBötC (Figure 2—figure supplement 1), and AAV-GFP or tdTomato injected control mice without removal of Oprm1 showed no change in OIRD compared to the pre-injected control state (Figure 2H–J), demonstrating that animals do not develop tolerance to opioids within our experimental timeline. Importantly, rescue of OIRD similarly occurred in normoxia (Figure 2—figure supplement 2) and was also specific to breathing since opioids induced analgesia in tail-flick assay after deletion of Oprm1 in the preBötC (Figure 2—figure supplement 3).

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Average frequency, peak flow and pause duration after intraperitoneal saline or morphine in hypercapnia at baseline or after Oprm1 deletion from the preBötC.

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Average frequency, peak flow and pause duration after intraperitoneal saline or morphine in hypercapnia at baseline or after sham viral injection into the preBötC.

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Although key features of OIRD (inspiratory airflow and pause) were attenuated by preBötC AAV-Cre injection, rescue was incomplete. This could be explained by incomplete Oprm1 deletion within the preBötC, or participation of another brain site in OIRD. Injection of opioids into the parabrachial (PBN)/Kolliker-Fuse (KF) nucleus can also slow breathing, making it a candidate second site (Mustapic et al., 2010; Prkic et al., 2012). In fact, the PBN/KF has been proposed to be the key site mediating OIRD (Eguchi et al., 1987; Lalley et al., 2014). We therefore took a similar approach to test the role of the PBN/KF in OIRD (Figure 3A). AAV-Cre injection into the PBN/KF (Figure 3—figure supplement 1) produced a slight increase in the morphine-evoked respiratory rate (Figure 3—figure supplement 2, Figure 3D,E) and inspiratory airflow (Figure 3—figure supplement 2, Figure 3F,G), but had a more moderate effect than injection into the preBötC.

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Average frequency, peak flow and pause duration after intraperitoneal saline or morphine in hypercapnia at baseline or after Oprm1 deletion from the preBötC or PBN/KF and then the preBötC and PBN/KF.

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To determine if the preBötC and PBN/KF can completely account for OIRD (Figure 3A), we genetically deleted Oprm1 from the preBötC and then from the PBN/KF (Cohort 1) or vice versa (Cohort 2, Figure 3B). In either cohort, double deletion breathing after morphine administration looked nearly identical to that of saline control animals (Figure 3C), with breathing rate and inspiratory airflow depressed by only ~20% compared to saline (Figure 3D–G). Moreover, changes in breathing after viral injection at the second site appeared additive (Figure 3D–G) and equivalent to individual preBötC (Figure 2) or PBN/KF (Figure 3—figure supplement 2, Figure 3D–G) effects for each cognate cohort. The double deletion OIRD rescue was similar in normoxia (Figure 3—figure supplement 3). To our surprise, rescues also occurred in animals which happened to have mostly unilateral PBN/KF AAV-cre transduction (Figure 3—figure supplement 4), therefore these animals were still included in our double deletion analysis (Figure 3E,G). Breathing in double-deleted animals was even resilient to super-saturating doses of opioid that severely slow breathing in control animals (150 mg/kg fentanyl, Figure 3H,I). Taken together, our data are consistent with a model in which both the preBötC and PBN/KF contribute to opioid respiratory depression, with the former being predominant, and together account for OIRD.

Given the relative importance of the preBötC to OIRD, we sought to identify which Oprm1 expressing cells within this region depress breathing. Single cell transcriptome profiling of the ventral lateral brainstem of P0 mice (Figure 4A) showed that Oprm1 (mRNA) is expressed almost exclusively by neurons (Figure 4—figure supplement 1) and is remarkably restricted to just 8% of presumed preBötC neurons (Figure 4B). This alone is interesting, as it suggests that modulation of only a small subset of neurons with the preBötC is enough to significantly impact its ability to generate a rhythm. We also determined that within the preBötC, Oprm1 (mRNA) was expressed by glycinergic (Slc6a5 expressing), gabaergic (Gad2/Slc32a1 expressing), and glutamatergic (Slc17a6 expressing) neural types alike (Figure 4C) and therefore Oprm1 (mRNA) expression is not exclusive to any known rhythmogenic preBötC subpopulation.

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Slices containing the preBötC autonomously generate respiratory-like rhythmic activity in vitro which is depressed in both rate and amplitude by bath administration of opioid agonists (Gray et al., 1999; Lorier et al., 2010), similar to opioid effects we observed on breathing in vivo. To determine which neural class mediates the depression of preBötC activity, we measured rhythmic bursting activity in vitro (Figure 4D) after selectively genetically deleting Oprm1 from each neural class. We achieved this deletion by crossing Oprm1^f/f mice with each of the following: Slc17a6-Cre, Slc32a1-Cre, Gad2-Cre, or Slc6a5-Cre transgenic animals. preBötC slices from control mice (Oprm1^f/f, Oprm1^f/+, or Oprm1^+/+) burst every 5–10 s and this activity was eliminated in 100% of slices by bath application of the selective µ-Opioid receptor agonist [D-Ala², NMe-Phe⁴, Gly-ol⁵]-enkephalin (DAMGO, 50 nM), and subsequently rescued by opioid antagonist naloxone (Figure 4E,F). Strikingly, the bursting rhythm of Slc17a6-Cre;Oprm1^f/f slices was not slowed by DAMGO, whereas the rhythm in Gad2-, Slc32a1-, and Slc6a5-Cre; Oprm1^f/f slices was entirely eliminated, akin to wild type controls (Figure 4E,F, Figure 4—figure supplement 2). This demonstrates that glutamatergic excitatory neurons, representing ~50% of all preBötC Oprm1-expressing neurons and therefore 4% of preBötC neurons, mediate OIRD in vitro.

Next, we dissected the glutamatergic Oprm1 preBötC neurons by two developmental transcription factors, Dbx1 or Foxp2 (Gray et al., 2010; Bouvier et al., 2010; Gray, 2013), to determine if a subset can rescue rhythm depression (Figure 4G). Triple-labeling of Dbx1-YFP, OPRM1 fused to mCherry (OPRM1-mCherry), and FOXP2 protein quantified within a single preBötC revealed three molecular subtypes of P0 Oprm1 glutamatergic neurons: 92 ± 9 Dbx1, 50 ± 2 Dbx1/FOXP2, and 20 ± 4 FOXP2 (Figure 4H,I). We selectively eliminated the µ-Opioid receptor in these two lineages (Dbx1-Cre;Oprm1^f/f or Foxp2-Cre;Oprm1^f/f) and measured preBötC slice activity at increasing concentrations of DAMGO, exceeding the dose necessary to silence the control rhythm (500 nM vs. 50 nM). Elimination of Oprm1 from both genotypes was sufficient to rescue the frequency and amplitude of preBötC bursting in DAMGO, and the Dbx1 rescue was comparable to elimination of Oprm1 from all glutamatergic neurons, while the Foxp2 rescue was substantial, but partial (~50–60%, Figure 4J). This shows that opioids silence a small cohort (~140) of glutamatergic neurons to depress preBötC activity, and that a molecularly defined subpopulation, about half, can be targeted to rescue these effects.

Here we show that two small brainstem sites are sufficient to rescue opioid induced respiratory depression in vivo. Between them, the preBötC is the critical site and we molecularly define ~140 Oprm1 glutamatergic neurons within that are responsible for this effect in vitro. Future study of these neurons will provide the first example of endogenous opioid modulation of breathing. Furthermore, characterization of these neurons and their molecular response to opioids may extend existing strategies (Manzke et al., 2003) or reveal a novel strategy for separating respiratory depression from analgesia and therefore enable the development of novel opioids or related compounds that relieve pain without risk of overdose.

Summary data generated in this study are included as a supplemental supporting file. All Matlab code and an example data are posted on Github: https://github.com/YackleLab/Opioids-depress-breathing-through-two-small-brainstem-sites (copy archived at https://github.com/elifesciences-publications/Opioids-depress-breathing-through-two-small-brainstem-sites).

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Acceptance summary:

This is a technically sophisticated study employing mouse transgenic approaches, viral vector-based neuronal targeting, in vivo behavioral analyses, single cell transcriptomics, and in vitro electrophysiological measurements to explore the mechanisms underlying opioid respiratory depression. The tour-de-force of impressive tools and the clear presentation make this paper especially appealing to a wide audience beyond those who study respiration.

The authors determine that mu-opioid receptors (Oprm1) in neurons in two circumscribed brainstem sites- the preBötzinger Complex (preBötC) and parabrachial (PBN)/Kolliker-Fuse (KF) nucleus, account for opioid-induced respiratory depression (OIRD) in awake behaving adult mice in vivo as well as in rhythmically-active preBötC circuits in slices from neonatal mice in vitro. Importantly between the preBötC and PBN/KF, the preBötC is the more critical site in vivo, and that at least in the slices in vitro, a small subpopulation (~140) Oprm1-expressing glutamatergic neurons, including Dbx1and Foxp2 expressing glutamatergic neurons, within the preBötC are responsible for the opioid suppression of rhythmic activity.

Decision letter after peer review:

Thank you for submitting your article "Opioids depress breathing through two small brainstem sites" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Jan-Marino Ramirez as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Eve Marder as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Gaspard Montandon (Reviewer #2); Jeffrey C. Smith (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

This is a technically sophisticated study employing mouse transgenic approaches, viral vector-based neuronal targeting, in vivo behavioral analyses, single cell transcriptomics, and in vitro electrophysiological measurements demonstrating that mu-opioid receptors (Oprm1) in neurons in two circumscribed brainstem sites- the preBötC and PBN/KF, account for opioid-induced respiratory depression (OIRD) in awake behaving adult mice in vivo as well as in rhythmically-active preBötC circuits in slices from neonatal mice in vitro. The experimental approaches employed are novel, providing a number of important results that more clearly define the critical brainstem sites and molecular phenotypes of neurons involved in OIRD. The authors' strategy of Cre-viral injection into the preBötC and/or PBN/KF of Oprm1 (f/f) mice to genetically delete Oprm1 together with the in vivo behavioral analyses to measure the individual and combined contribution of these two functionally defined regions to OIRD proved to be very effective. Also very effective is their use of Vglut2-Cre, Vgat-Cre, Gad2-Cre, or Glyt2-Cre transgenic mouse lines crossed with the Oprm1 (f/f) mice to selectively genetically delete Oprm1 from different neuronal classes, as well as the dissection of the glutamatergic Oprm1 preBötC neurons by two developmental transcription factors, Dbx1 or Foxp2, all combined with electrophysiological analyses of the rhythmic activity responses to opioid receptor agonists in slices in vitro. The important results are that between the preBötC and PBN/KF, the preBötC is the more critical site in vivo, and that at least in the slices in vitro, a small subpopulation (~140) Oprm1-expressing glutamatergic neurons, including Dbx1and Foxp2 expressing glutamatergic neurons, within the preBötC are responsible for the opioid suppression of rhythmic activity. The paper is well written and the figures and supplementary material very nicely illustrate the essential experimental results. However, there are several concerns that need to be addressed.

Essential revisions:

1) The authors try to introduce a "gold standard" definition for how breathing changes in OIRD. Yes, it would be nice if there were a simple gold standard. However, this is not the reality. Gold standard for OIRD should come from a consensus of studies showing similar results. The research presented here show one example of respiratory depression with only one type of opioids, not highly specific for the µ-opioid receptors, at only one dosage. Also the "gold standard" proposed here is based upon flow which cannot be reliably measured with whole-body plethysmography. So don't state that you are introducing a gold standard. OIRD is very state dependent and shows huge interindividual variability. Trying to imply that the respiratory rhythm is simply depressed is an oversimplification. Just look at the huge variability in Te (Figure 1H). Plethysmograph recordings are extremely difficult to evaluate, because animals behave, morphine will be sedating before measuring the OIRD, this will not be a static response. We don't understand the definition of inspiratory time, expiratory time, and pause. It seems in Figure 1G that flow doesn't cross the zero at the end of expiration as shown in the figure, but rather at the end of the pause. Is the "pause" only a longer expiration? How was this pause detected or defined? If those pauses are part of expiration, then expiratory time is also increase by morphine. How was the criterion for pause of 0.5mL/sec defined? Knowing that flow is highly variable from one animal to the other it is very likely an unreliable threshold.

Along the same lines: The authors respiratory assessments in freely-behaving rodents limited for a few reasons: a) using an arbitrary threshold based on flow to define the end of expiration is not reliable. Flow can change from one animal to another, so the duration of their "pause" may differ depending upon the initial baseline of the animal. How do you determine the amplitude, where do you set the threshold = look at Figure 1A, what do you consider a breath and what not?

b) Using tidal volume or flow as measurements in whole-body plethysmography in rodents is problematic. Unless the right formula is used, it is very difficult to evaluate tidal volume in rodents. In addition, mL cannot be a correct measurement in rodents without normalizing the value according to body weight. Unless all animals have the exact body weight, flow and tidal volume need to be measured as ml/10g or other units. You need to carefully consider these caveats

c) One of the difficult of measuring breathing with plethysmography is to control for arousal levels. It is well know that sedation by opioids may affect the severity of respiratory depression so arousal levels before morphine may be an issue. Please discuss this important caveat.

2) While using hypercapnia as a tool may simplify the approach, it will dramatically change the behavioral state of the animal. Under hypercapnia, arousal levels may be severely disrupted and it has been shown that arousal levels are tightly linked to respiratory depression. Hypercapnia will also affect the opiate sensitivity, and this effect will not be uniform in different areas. Carefully discuss this important caveat, and don't imply that this is an advantage, other than the fact that it regularizes the rhythm.

3) Figure 2 characterizes the necessity of PreBötC. Without quantitative immunohistochemical confirmation (Figure 2—figure supplement 1) it is indeed difficult to know whether the injections were restricted to the preBötC, same applies to the KF injections. (Figure 3—figure supplement 1).

4) Oprm1-/- animals were not compared with other mice that received injection of scrambled AAV, but were compared to themselves before surgery, microinjection, and recovery. It is possible that the inflammation induced by AAV, and the damage done by the microinjection may change the response to opioids. We recommend that this should be verified with proper controls.

5) From the transcriptome profiling and neonatal in vitro results, the authors propose that opioids silence a small subset (~140) of glutamatergic neurons to depress preBötC activity, and that a molecularly defined subpopulation (~70 neurons) can be targeted to rescue these effects. It would be important to emphasize in the Discussion that tests need to be performed in the awake behaving mice in vivo with the transgenic lines utilized for the in vitro studies to determine if so few neurons are also responsible for OIRD in the adult system in vivo.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your article "Opioids depress breathing through two small brainstem sites" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by Ronald Calabrese as the Senior Editor and Reviewing Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Gaspard Montandon (Reviewer #2); Jeffrey C Smith (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

This paper has been previously reviewed and requires some further revisions. All these revisions can be accomplished without any further experiments. Some new analysis in needed but the vast majority of the changes involve rewriting.

Essential revisions:

There are still some outstanding issues that must be addressed.

Anatomy a) Regarding PBN/KF injection sites, it is clear that these encompassed a much larger area than the targeted region, which is not surprising for targeting these small areas in the mouse brainstem, although it looks like (at least from the examples shown) that the most concentrated fluorescent protein expression may typically be in and immediately around the targeted areas. So it is clear that the authors have included the targeted areas with their stereotaxic injection approach.

b) Regarding preBötC targeting, the reconstructions shown in Figure 2—figure supplement 1, part A and the two anterior sites in part B are reasonable for demonstrating preBötC targeting in the mouse, but the labeling for the posterior site reconstructed extends too caudal as the authors have represented the anatomy, implying some receptor deletion in the rVRG. The authors thusly should modify statements in the manuscript regarding site-specificity and that injection sites were confirmed by the restricted expression of Cre-GFP (subsection “Stereotaxic injection”, and figure legends), and they need to discuss any caveats associated with this issue.

c) Given the results showing functional rescue from depressed breathing frequency and peak inspiratory flow obtained with their Oprm1f/f transgenic and Cre vector construct, their results with control vector injections, and the injection site anatomical reconstructions, it is likely that the authors have deleted some opioid receptors in the regions of interest. We ask the authors to further clarify in the manuscript if/how they have validated deletion of opioid receptors with their experimental approach and to address any associated caveats. The authors could confirm the absence of MOR in knockout animals with Immunohistochemistry.

Hypercapnia

Measuring breathing in 5% CO2 likely limits the authors' conclusions. They need to mention that these data are valid in animals that are exposed to CO2, and discuss the issues related to high CO2, low pH, hyperventilation induced by CO2, and reduced arousal, which may limit the role of the preBotC in opioid-induced respiratory depression. The authors don't mention any papers in the discussion related to hypercapnia, medullary sites sensitive to CO2, arousal states, opioid receptor knockout animals etc.

Pauses

Definition of pause versus expiration. The definition of a pause according to the authors is when volume goes below 0.5mL during the expiratory period. According to Figure 1G, the last part of the pause with morphine seems to be above this threshold. This arbitrary threshold is concerning. The threshold is likely detecting different pauses in saline versus morphine considering that volume is considerably reduced by morphine. For instance, in Figure 1G, peak flow with saline condition is -6 mL/sec versus -2ml/sec for morphine, with corresponding differences in volume. The use of an absolute value (0.5mL) as a threshold means that the detection of the "pause" will likely be different between saline and morphine conditions, so not due to different timings but different detections. It would be easier to use the simple definitions of inspiratory and expiratory times, which would be consistent with previous reports showing that expiratory duration is affected by opioids. Also, we are not sure what the physiological relevance of a pause is. Is it controlled by different brainstem circuits? The long expirations are likely due to a combination of hypercapnia and hypoxia with morphine.

Discussion

The authors do not discuss previous studies about MOR-/-, preBötC, etc. There are many studies investigating this topic that could be discussed in the Discussion. Dahan et al., 2001, Montandon et al., 2011, 2016, Varga et al., 2020, Stucke et al., Mustapic et al., Lalley et al. etc.

Essential revisions:

1) The authors try to introduce a "gold standard" definition for how breathing changes in OIRD. Yes, it would be nice if there were a simple gold standard. However, this is not the reality. Gold standard for OIRD should come from a consensus of studies showing similar results. The research presented here show one example of respiratory depression with only one type of opioids, not highly specific for the µ-opioid receptors, at only one dosage. Also the "gold standard" proposed here is based upon flow which cannot be reliably measured with whole-body plethysmography. So don't state that you are introducing a gold standard. OIRD is very state dependent and shows huge interindividual variability. Trying to imply that the respiratory rhythm is simply depressed is an oversimplification. Just look at the huge variability in Te (Figure 1H). Plethysmograph recordings are extremely difficult to evaluate, because animals behave, morphine will be sedating before measuring the OIRD, this will not be a static response.

We don't understand the definition of inspiratory time, expiratory time, and pause.

1) Inspiratory time (Ti) is defined as the period of negative airflow at the beginning of the breath, such that airflow is less than 0mL/sec. Once the 0mL/sec. threshold is crossed to positive values, inspiration is considered to be terminated.

2) Expiratory time (Te) is defined as the period of positive airflow immediately after inspiration, when the 0mL/sec. threshold is crossed until the airflow decreases below a second threshold of 0.5mL/sec. We use this second threshold to distinguish Te from the pause. We have now clarified in the manuscript that this is the initial portion of expiration (Results, second paragraph, subsection “A rubric for studying opioid and other respiratory depressants”, subsection “Plethysmography, respiratory and behavioral analysis”, first paragraph).

3) The pause is defined as the airflow after Te until the onset of the next inspiration. The specific details of the pause are discussed below.

It seems in Figure 1G that flow doesn't cross the zero at the end of expiration as shown in the figure, but rather at the end of the pause. Is the "pause" only a longer expiration?

The pause is an expiratory period that occurs after the initial large-amplitude expiratory pulse wherein the majority of tidal volume has been expelled. The pause is characterized by very low airflow and ends with a final brief expulsion of air. Since this phenomenon is being described in hypercapnia, we interpret this terminating expiratory airflow to be "active expiration" (Pisanski and Pagliardini, 2019). The low airflow pause period can vary in length from tens to hundreds of milliseconds. We have now clarified this definition in the second paragraph of the Results, subsection “A rubric for studying opioid and other respiratory depressants” and in the first paragraph of the subsection “Plethysmography, respiratory and behavioral analysis” and have added a supplementary figure (Figure 1—figure supplement 1) which shows breaths with different pause lengths aligned by the onset of the pause to better qualitatively demonstrate this phenomenon. Additionally, we have added a description in the Materials and methods and Figure 1—figure supplement 2 to clarify how the airflow threshold was chosen to define pause onset.

How was this pause detected or defined?

The pause is an apneic period of low airflow which we believe represents a failure or delay to initiate a subsequent breath. This period is qualitatively defined as the point during expiration when expiratory airflow crosses below 0.5mL/sec. All airflow after this point, until the next inspiration begins (continuous airflow below 0mL/sec.) is defined as the pause. In control hypercapnic breathing fewer than 2% of breaths have a pause longer than 5msec. by this definition (Figure 1—figure supplement 2). After morphine, ~40% of breaths have a pause between 5 and several hundred milliseconds (Figure 1—figure supplement 2). This definition is now clarified in the first paragraph of the subsection “Plethysmography, respiratory and behavioral analysis”and we have added a supplementary figure (Figure 1—figure supplement 2).

If those pauses are part of expiration, then expiratory time is also increase by morphine.

It is certainly true that the overall time between two inspirations does increases. However, the initial portion of expiration (before our pause) does not change in length. This is the data displayed in Figure 1H. We have now clarified our language to include the pause as part of the overall expiratory period in the second paragraph of the Results, subsection “A rubric for studying opioid and other respiratory depressants” and in the first paragraph of the subsection “Plethysmography, respiratory and behavioral analysis”.

How was the criterion for pause of 0.5mL/sec defined?

Please see discussion above and Figure 1—figure supplement 2.

Knowing that flow is highly variable from one animal to the other it is very likely an unreliable threshold.

Since the pause analysis was conducted on data from adult animals in hypercapnia, airflow recordings are very reproducible between animals and not "highly variable". For example, for 29 animals recorded, the mean+/-SD for average peak inspiratory airflow after saline injection was 7.2+/-0.57mL/sec. and after morphine injection was 3.60+/-0.62mL/sec. Furthermore, in Figure 1—figure supplement 2, where the pause threshold is defined, the data in the scatter plot was generated from 100 breaths from 15 different animals and all data points are intermixed and not clustering as individual animals. Therefore, our pause threshold is chosen to be a reliable metric between animals.

Along the same lines: The authors respiratory assessments in freely-behaving rodents limited for a few reasons: a) Using an arbitrary threshold based on flow to define the end of expiration is not reliable. Flow can change from one animal to another, so the duration of their "pause" may differ depending upon the initial baseline of the animal. How do you determine the amplitude, where do you set the threshold = look at Figure 1A, what do you consider a breath and what not?

Please see explanations above and Figure 1—figure supplement 2. Briefly, data and pause threshold between animals is highly reproducible and data from 29 animals was used to define the pause threshold.

b) Using tidal volume or flow as measurements in whole-body plethysmography in rodents is problematic. Unless the right formula is used, it is very difficult to evaluate tidal volume in rodents.

We appreciate the criticism that whole-body plethysmography inaccurately measures tidal volume and that a more correct estimate necessitates the incorporation of chamber humidity and temperature via the Drorbaugh and Fenn formula. We have now indicated that the displayed tidal volumes are approximates in the first and third paragraphs of the Results, the first paragraph of the subsection “Plethysmography, respiratory and behavioral analysis”, and in the legend of Figure 1C.

In addition, mL cannot be a correct measurement in rodents without normalizing the value according to body weight. Unless all animals have the exact body weight, flow and tidal volume need to be measured as ml/10g or other units. You need to carefully consider these caveats.

It is certainly important to normalize the airflow and tidal volume to body weight when animals of very different sizes are being compared. For example, this correction is particularly important when comparing developing rodents where lung size strongly correlates with size. However, we feel that this correction is not appropriate in our manuscript for four reasons:

1) When different animals are being compared, the respiratory parameters after morphine injection are normalized to those after saline injection recordings. These two recordings are performed within several days of each other (i.e. without significant weight change). All statistical tests are performed on normalized data.

2) As mentioned above, one mechanism that could explain changes in airflow in differently sized mice is if lung volume increased with body size. However once mice reach their adult size, although their body weight can increase, their lung volume does not. This has been clearly demonstrated with serial computed tomography imaging of lung size (Mitzner, Brown and Lee, 2001).

3) Another possible mechanism that could contribute to changes in airflow across different sized mice is if increased displacement of air in the plethysmography chamber distorts the airflow signal. However, in a toy experiment wherein the chamber is filled with objects that displace either 20, 25, or 30mL’s of air, the total volume of air injected by syringe (10ml) can be accurately calculated in all conditions. (See Author response image 1). This indicates that normalizing approximate airflow or tidal volume to weight, as a proxy for body volume, will negatively impact the accuracy of those estimates.

4) We can demonstrate the stability of airflow across recordings over time, even though the animal has gained some weight. When airflows from the same animal, after injection of saline, are compared at the beginning and end of our studies, the airflows are indistinguishable despite some mice increasing in weight (See Author response image 2 where specific breaths in hypercapnia are compared). In this case normalizing by weight would lead the experimenter to believe lung volume significantly decreases as animals increase in weight which would have no biological basis.

In summary, it is unlikely that either animal weight or size in adult mice is significantly biasing approximated respiratory airflow or tidal volume values, and therefore it would be inappropriate to normalize these values by weight.

Author response image 1

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Author response image 2

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c) One of the difficult of measuring breathing with plethysmography is to control for arousal levels. It is well know that sedation by opioids may affect the severity of respiratory depression so arousal levels before morphine may be an issue. Please discuss this important caveat.

We certainly appreciate that arousal levels impact the distribution of breaths that are taken by awake, behaving mice and in fact, this concern is exactly why we decided to measure breathing before and after morphine in hypercapnia in addition to normoxia (see Results, first paragraph). Although hypercapnia stimulates the respiratory system, it also normalizes the behavior and we presume the arousal state of mice. As a result, as we show in Figure 1, all of the changes in respiratory parameters are more reproducible between mice.

2) While using hypercapnia as a tool may simplify the approach, it will dramatically change the behavioral state of the animal. Under hypercapnia, arousal levels may be severely disrupted and it has been shown that arousal levels are tightly linked to respiratory depression. Hypercapnia will also affect the opiate sensitivity, and this effect will not be uniform in different areas. Carefully discuss this important caveat, and don't imply that this is an advantage, other than the fact that it regularizes the rhythm.

Although it is certain that hypercapnia alters arousal levels, breathing, and sensitivity to opioids, we use the regularization of breathing as a way to dissect the role of the preBötC and PBN/KF in OIRD. We acknowledge the benefit of hypercapnia (Results, first paragraph) is simply that it regularizes the respiratory rhythm which is in large part due to a reduction of behaviors performed, for example, far less sniffing and grooming. Although the changes to breathing in hypercapnia are quantitatively distinct from normoxia (Figure 1), the ability to eliminate the variability in breathing due to behavior enabled us to quantitively measure how breaths change in morphine and how these changes can be rescued after deletion of Oprm1 in the preBötC and/or PBN/KF sites.

Even if hypercapnia produces an altered arousal state that changes the OIRD response, we do not anticipate that it is due to the "masking" a third brain site that it critical for ORID in normoxia. This is because in all the animals studied, we also performed recordings in normoxia that demonstrate a rescue in respiratory rate and peak inspiratory flow after deletion of Oprm1 from the preBötC and then the PBN/KF which we now include in additional supplementary figures (Figure 2—figure supplement 2, Figure 3—figure supplement 3). This demonstrates that the rescues we observe are not unique to hypercapnia.

3) Figure 2 characterizes the necessity of PreBötC. Without quantitative immunohistochemical confirmation (Figure 2—figure supplement 1) it is indeed difficult to know whether the injections were restricted to the preBötC, same applies to the KF injections. (Figure 3—figure supplement 1).

We have now modified the supplementary figures that detail the preBötC and PBN/KF viral injections (Figure 2—figure supplement 1 and Figure 3—figure supplement 1). These figures now contain a schematic that details the brain anatomy from anterior to posterior brain sections and the extent of Cre-GFP expression from each animal. Note that the overlapping GFP-expressing region from all animals is the preBötC and PBN/KF.

4) Oprm1-/- animals were not compared with other mice that received injection of scrambled AAV, but were compared to themselves before surgery, microinjection, and recovery. It is possible that the inflammation induced by AAV, and the damage done by the microinjection may change the response to opioids. We recommend that this should be verified with proper controls.

We have now used a mixed repeated measures two-way ANOVA to compare our preBötC AAV-Cre injected animals with the control preBötC AAV-GFP or AAV-TdTomato injected animals (Figure 2 legend). Additionally, we have added 3 new control preBötC injected animals (new total is n=7) in order to match or exceed number of experimental animals (n=6). The addition of new mice has verified our original observations of no change in frequency, airflow and pause in control injected animals.

5) From the transcriptome profiling and neonatal in vitro results, the authors propose that opioids silence a small subset (~140) of glutamatergic neurons to depress preBötC activity, and that a molecularly defined subpopulation (~70 neurons) can be targeted to rescue these effects. It would be important to emphasize in the Discussion that tests need to be performed in the awake behaving mice in vivo with the transgenic lines utilized for the in vitro studies to determine if so few neurons are also responsible for OIRD in the adult system in vivo.

The limited expression of Oprm1 within the preBötC coupled with the in vivo OIRD rescue observed after deletion of Oprm1 from the preBötC makes (which is likely even incomplete, i.e. not all cells are expressing viral), makes the powerful prediction that just a small number of neurons is capable of dramatically changing the respiratory rhythm in vivo. Still, at the end of the Discussion we have now clarified that this is an important hypothesis that future experiment should test directly.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Please note, during the course of the revision we validated that our dose of morphine (20mg/kg) does not change respiration in mice completely lacking µOR-/-. This demonstrates that this dose of morphine is specific to µOR. As a result, we have removed a phrase in our Discussion suggesting this dose of morphine could activate kappa and δ opioid receptors (per the request of the reviewer in revision round 1).

Essential revisions:

There are still some outstanding issues that must be addressed.

Anatomy a) Regarding PBN/KF injection sites, it is clear that these encompassed a much larger area than the targeted region, which is not surprising for targeting these small areas in the mouse brainstem, although it looks like (at least from the examples shown) that the most concentrated fluorescent protein expression may typically be in and immediately around the targeted areas. So it is clear that the authors have included the targeted areas with their stereotaxic injection approach.

Ok.

b) Regarding preBötC targeting, the reconstructions shown in Figure 2—figure supplement 1, part A and the two anterior sites in part B are reasonable for demonstrating preBötC targeting in the mouse, but the labeling for the posterior site reconstructed extends too caudal as the authors have represented the anatomy, implying some receptor deletion in the rVRG. The authors thusly should modify statements in the manuscript regarding site-specificity and that injection sites were confirmed by the restricted expression of Cre-GFP (subsection “Stereotaxic injection”, and figure legends), and they need to discuss any caveats associated with this issue.

Subsection “Stereotaxic injection” now indicates that the site-specificity is indicated by the center of the Cre-GFP expression. We also have added additional discussion (subsection “Just two small brainstem sites mediate OIRD”) to note that we cannot exclude µOR deletion from neighboring regions.

c) Given the results showing functional rescue from depressed breathing frequency and peak inspiratory flow obtained with their Oprm1f/f transgenic and Cre vector construct, their results with control vector injections, and the injection site anatomical reconstructions, it is likely that the authors have deleted some opioid receptors in the regions of interest. We ask the authors to further clarify in the manuscript if/how they have validated deletion of opioid receptors with their experimental approach and to address any associated caveats. The authors could confirm the absence of MOR in knockout animals with Immunohistochemistry.

We clarified our methodology and describe that the Cre-GFP determined injection site-specificity indicates where the µ-opioid receptor deleted. Although we infer a deletion by the rescue of OIRD, we indicate that we do not explicitly confirm deletion by immunohistochemistry (Results, fourth paragraph and subsection “Stereotaxic injection”).

Hypercapnia

Measuring breathing in 5% CO2 likely limits the authors' conclusions. They need to mention that these data are valid in animals that are exposed to CO2, and discuss the issues related to high CO2, low pH, hyperventilation induced by CO2, and reduced arousal, which may limit the role of the preBotC in opioid-induced respiratory depression. The authors don't mention any papers in the discussion related to hypercapnia, medullary sites sensitive to CO2, arousal states, opioid receptor knockout animals etc.

We disagree with the reviewer that our conclusions are limited to 5% CO2 and a limited role of the preBötC for two simple reasons:

1) Much of the presented data are in under 5% CO2 conditions for uniformity and clarity of data presentation. However, as indicated in our experimental methods and Figure 2—figure supplement 2 and Figure 3—figure supplement 3, the OIRD rescues in peak inspiratory flow and frequency observed in 5% CO2 also occur in normoxia.

2) If 5% CO2 “[limits] the role of the preBötC in opioid induced respiratory depression” (as suggested above), then elimination of µOR from the preBötC would not result in rescued OIRD. This is simply not what we observe.

Pauses

Definition of pause versus expiration. The definition of a pause according to the authors is when volume goes below 0.5mL during the expiratory period. According to Figure 1G, the last part of the pause with morphine seems to be above this threshold. This arbitrary threshold is concerning. The threshold is likely detecting different pauses in saline versus morphine considering that volume is considerably reduced by morphine. For instance, in Figure 1G, peak flow with saline condition is -6 mL/sec versus -2ml/sec for morphine, with corresponding differences in volume. The use of an absolute value (0.5mL) as a threshold means that the detection of the "pause" will likely be different between saline and morphine conditions, so not due to different timings but different detections. It would be easier to use the simple definitions of inspiratory and expiratory times, which would be consistent with previous reports showing that expiratory duration is affected by opioids. Also, we are not sure what the physiological relevance of a pause is. Is it controlled by different brainstem circuits? The long expirations are likely due to a combination of hypercapnia and hypoxia with morphine.

We believe that by introducing the concept of a “pause” period is important for two reasons: 1) it is the most accurate way to describe our data and 2) reflective of the underlying physiology.

1) Perhaps the most obvious qualitative change in breathing under morphine are that many breaths have long periods (lasting several hundred milliseconds at 20mg/kg morphine) of little to no airflow (Figure 1A and D, Figure 1—figure supplement 1). We sought to rigorously and quantitatively describe this phenomenon. As we show in Figure 1—figure supplement 2, we do not “arbitrarily” choose the 0.5mL/sec. threshold, and instead chose this threshold because it identifies essentially no pauses in hypercapnia after saline injection (only 1.53% of breaths have a pause). Although pauses in length 50-100msec. could be considered false positives because they do not have a considerable apneic period (see Figure 1—figure supplement 1, panel 2), they occur at a low rate in saline (1.53%) and we see an increased distribution of pauses in morphine that last hundreds of milliseconds (Figure 1I, see Figure 1—figure supplement 1, panel 3 and 4). We now clarify this in the first paragraph of the subsection “Plethysmography, respiratory and behavioral analysis”.

2) We interpret the “pause” period as a delay to initiate the subsequent breath and this implicates the preBötC in OIRD. This falls in line with the fact that the autonomous respiratory rhythm generated by the preBötC slice slows after bath administration of opioid agonists, independent of any other brain areas. Further, after deletion of Oprm1 from the preBötC pause lengths are significantly decreased (Figure 2J). Since our recordings occur in hypercapnia, the pause terminates with a brief active expiration. However, active expiration depends upon preBötC activity (Huckstepp et al., 2016) and thus remains consistent with the pause reflecting a delay in preBötC inspiratory initiation.

Discussion

The authors do not discuss previous studies about MOR-/-, preBötC, etc. There are many studies investigating this topic that could be discussed in the Discussion. Dahan et al., 2001, Montandon et al., 2011, 2016, Varga et al., 2020, Stucke et al., Mustapic et al., Lalley et al. etc.

We appreciate your recommendations for citations. We have already discussed the importance of and cited Montandon et al., 2011, Varga et al., 2020 and Mustapic et al. in our manuscript.

Author details

1. Iris Bachmutsky

   1. Department of Physiology, University of California-San Francisco, San Francisco, United States
   2. Neuroscience Graduate Program, University of California-San Francisco, San Francisco, United States

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

2. Xin Paul Wei

   1. Department of Physiology, University of California-San Francisco, San Francisco, United States
   2. Biomedical Sciences Graduate Program, University of California-San Francisco, San Francisco, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

3. Eszter Kish

   1. Department of Physiology, University of California-San Francisco, San Francisco, United States
   2. Neuroscience Graduate Program, University of California-San Francisco, San Francisco, United States

   Contribution

   Conceptualization, Investigation, Methodology

   Competing interests

   No competing interests declared

4. Kevin Yackle

   Department of Physiology, University of California-San Francisco, San Francisco, United States

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   Kevin.Yackle@ucsf.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1870-2759

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