Opioid drugs are commonly prescribed due to their powerful painkilling properties. However, when misused, these compounds can cause breathing to become dangerously slow and shallow: between 1999 and 2018, over 400,000 people died from opioid drug overdoses in the United States alone.

Exactly how the drugs affect breathing remains unclear. What is known is that opioids work by binding to specific receptors at the surface of cells, an event which has a ripple effect on many biochemical pathways. Amongst these, research published in 2005 identified the β-arrestin 2 pathway as being responsible for altering breathing. This spurred efforts to find opioid-like drugs that would not interfere with the pathway, retaining their ability relieve pain but without affecting breathing. However, new evidence is now shedding doubt on the conclusions of this study.

In response, Bachmutsky, Wei et al. attempted to replicate the original 2005 findings. Mice with carefully controlled genetic background were used, in which the genes for the β-arrestin 2 pathway were either present or absent. Both groups of animals had similar breathing patterns under normal conditions and after receiving an opioid drug. The results suggest β-arrestin 2 is not involved in opioid-induced breathing suppression.

These findings demonstrate that research to develop opioid-like drugs that do not affect the β-arrestin 2 pathway are based on a false premise. Precisely targeting a drug’s molecular mechanisms to avoid suppressing breathing may still be a valid approach, but more research is needed to identify the right pathways.

More than 180 people died each day in 2018 from depressed breathing following an opioid overdose, a lethal side-effect termed opioid induced respiratory depression (OIRD) (Pattinson, 2008; Scholl et al., 2018). Nevertheless, opioids remain among the most effective and widely prescribed analgesics, as evidenced by the World Health Organization’s step ladder for pain management. These two contrasting characteristics have created the urgent desire to develop or discover novel µ-opioid receptor (MOR) agonists that provide analgesia without depressing breathing. Such a possibility emerged in 2005 when it was reported that mice with germline deletion of ß-arrestin 2 (Arrb2-/-) experience enhanced analgesia with attenuated respiratory depression when administered systemic morphine (Raehal et al., 2005). This finding inspired a new field of ‘biased agonist’ pharmacology with the goal of engaging MOR-dependent G-protein but not ß-arrestin 2 signaling. This approach remains a central strategy in drug discovery for analgesics (Turnaturi et al., 2019).

Germline deletion of the µ-opioid receptor gene (Oprm1) completely eliminates OIRD in murine models (Dahan et al., 2001) and selective deletion of Oprm1 within the brainstem medullary breathing rhythm generator, the preBötzinger Complex (preBötC) (Smith et al., 1991), largely attenuates OIRD (Bachmutsky et al., 2020; Varga et al., 2019). When engaged, MOR G-protein signaling activates inwardly rectifying potassium channels (Al-Hasani and Bruchas, 2011) and inhibits synaptic vesicle release (Zurawski et al., 2019), thereby depressing neural signaling. Additionally, the MOR signals intracellularly via a pathway dependent on MOR internalization by ß-arrestin 2 (Calebiro et al., 2010; Luttrell and Lefkowitz, 2002). Indeed, all three pathways have been implicated in OIRD (Montandon et al., 2016; Raehal et al., 2005; Wei and Ramirez, 2019) but a role for Arrb2 has been suggested to be important and selective for respiratory depression, relative to analgesia.

Since the original OIRD study of Arrb2-/- mice, multiple MOR agonists, dubbed ‘biased agonists’, have been created that signal through G-protein but not ß-arrestin 2 pathways. These agonists are reported to produce analgesia with reduced respiratory depression (Manglik et al., 2016; Schmid et al., 2017) and thereby provide pharmacological support for the proposed role of ß-arrestin 2 in OIRD. However, recently, these studies have been called into question (Hill et al., 2018; Kliewer et al., 2020), prompting us to independently investigate the underlying necessity of ß-arrestin 2 in mediating ORID. Here, in an experiment where we rigorously control for genetic background, we demonstrate that basal breathing and OIRD are similar in Arrb2+/+, Arrb2+/-, and Arrb2-/- littermates. Furthermore, the in vitro preBötC rhythm is similarly silenced by an MOR agonist in all three genotypes. Our data, together with another recent report, does not show a role of Arrb2 in opioid-induced respiratory depression and suggests that MOR biased agonists attenuate OIRD through a different mechanism.

Breathing behaviors and OIRD severity differ between strains of mice (Bubier et al., 2020). Therefore, we sought to compare basal and morphine depressed breathing in mice with the same genetic background. We bred F1 Arrb2+/- mice to generate littermates that were wildtype (+/+), heterozygous (+/-), and homozygous (-/-) for germline deletion of Arrb2 (Figure 1A). Breathing was assessed by whole-body plethysmography following intraperitoneal (IP) injection of saline (for control recordings) or after IP morphine (20 mg/kg) one day later (Figure 1B). This same breathing protocol was conducted in air with 21% O₂ and 0% CO₂ (hereby referred as normoxic) and air with 21% O₂ and 5% CO₂ (hereby referred as hypercapnic). The hypercapnic state minimizes potential confounding fluctuations in breathing rate and depth seen in normoxic conditions. We used our previously described analytical pipeline (Bachmutsky et al., 2020) to assay the two key parameters that define OIRD, slow and shallow breathing. Slow breathing is measured as the instantaneous frequency of each breath and shallow breathing is defined by the peak inspiratory flow since it strongly correlates with the volume of air inspired (PIF, Figure 1C; Bachmutsky et al., 2020).

Figure 1

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In the normoxic condition, the morphology of single breaths, respiratory rate, and peak inspiratory airflow after IP saline appeared similar in Abbr2+/+ and -/- mice (Figure 2A–B, Table 1 contains mean ± SEM and 95% CI). As expected for OIRD, IP morphine decreased the frequency and PIF, but the breathing characteristics remained indistinguishable in Abbr2+/+ versus -/- mice (Figure 2A–B, Table 1). Consistently, histograms of the instantaneous frequency and PIF for each breath after IP morphine showed overlapping distributions from Arrb2+/+ (combined from n = 5 mice), +/- (n = 6), and -/- (n = 7) animals (Figure 2C,E). We quantified OIRD as the ratio of the average instantaneous frequency or PIF after IP morphine normalized to IP saline. As expected from the raw data, OIRD was similar among the genotypes (rate decreased 60% and PIF by 40%, Figure 2D and F, Table 2 contains 95% CI for each mean and the comparisons). In fact, there was a small attenuation of respiratory rate depression in Arrb2+/+ compared to Arrb2-/- and +/- mice, inconsistent with the hypothesis that Arrb2 mutation attenuates OIRD. These data lead us to conclude that OIRD in normoxic conditions is not diminished in Arrb2-/- mice.

Figure 2

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Figure 2—source data 1

Raw respiratory data, OIRD ratio, and statistical tests for recordings performed in normoxic conditions.

This source data corresponds to Figure 2 and Table 1 and Table 2.

https://cdn.elifesciences.org/articles/62552/elife-62552-fig2-data1-v3.xlsx

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These same breathing assays and analysis were also performed in a hypercapnic state. Hypercapnia eliminates any changes in breathing rate and depth that are simply due to variation in behavioral state, like sniffing versus calm sitting. Thus, although breathing when hypercapnic is faster and deeper (like in Figure 3A, Table 3), the reduced variability minimizes any chance that the conclusions in the normoxic condition are due to additional effects of opioids on behaviors such as sedation and locomotion, or non-opioid-related differences in arousal. Importantly, OIRD is still robustly observed in the hypercapnic state (Figure 3A, Table 4). As anticipated, the opioid depression of instantaneous frequency (by ~30%) and PIF (by ~30%) were the same among all three genotypes (Figure 3C–F, Table 4). Therefore, OIRD is not diminished in Arrb2-deficient mice in two independent breathing assays.

Figure 3 with 1 supplement see all

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Figure 3—source data 1

Raw respiratory data, OIRD ratio, and statistical tests for recordings performed in hypercapnic conditions.

This source data corresponds to Figure 3 and Table 3 and Table 4.

https://cdn.elifesciences.org/articles/62552/elife-62552-fig3-data1-v3.xlsx

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To understand the confidence of these results, we determined the extent that Arrb2+/+ breathing parameters must be depressed (compared to Arrb2-/-) in order to produce a significant test statistic more than 80 % of the time, that is, power analysis. Given our cohort sizes and the observed variation in breathing parameters from Arrb2+/+ and -/- littermates, we plotted the relationship between power (0–1) and percent difference between the Arrb2-/- and hypothetical Arrb2+/+ means (see methods). When comparing the Arrb2-/- measured and Arrb2+/+ hypothetical means, we could confidently distinguish these mean breathing frequencies so long as they differed by at least ~12–20%, and mean PIFs by more than ~10%–22% (Figure 3—figure supplement 1). Thus, our experimental approach enabled us to only detect mild to large differences in OIRD between Arrb2+/+ and -/- littermates, if they had occurred. It remains possible that a small effect, much smaller than previously reported, was not identified in our study.

The most important site for OIRD is the preBötC (Bachmutsky et al., 2020). So, we directly measured the effects of the opioid peptide [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO) on the preBötC rhythm in all three Arrb2 genotypes. Importantly, DAMGO robustly stimulates both MOR signaling pathways, G-protein and ß-arrestin 2 dependent signaling. The preBötC slice was prepared from Arrb2 littermates and the genotype (+/+, +/-, -/-) was determined post hoc. The rhythm was monitored by measuring electrical activity in the hypoglossal nerve rootlet (Smith et al., 1991) for 20 minutes at baseline, and then with 20 nM then 50 nM DAMGO (Figure 4A–B). The preBötC rhythms of Arrb2+/+, +/-, and -/- littermates (n = 5, 20, 6) similarly slowed by ~70–80% at 20 nM DAMGO and nearly all were silenced at 50 nM (Figure 4C–D). If anything, it appears the Arrb2-/- slices showed a statistically significant increase in DAMGO sensitivity when compared to Arrb2+/- littermates (Figure 4C–D), although this likely stems from the differences in cohort sizes (6 vs 20). In conclusion, a MOR agonist that substantially activates ß-arrestin 2 signaling similarly slows the preBötC rhythm in mice lacking the Arrb2 gene and the littermate controls.

Figure 4

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Figure 4—source data 1

Raw data and statistical tests for in vitro OIRD recordings.

The data contains raw and normalized data and corresponding statistical tests for each Arrb2 genotype.

https://cdn.elifesciences.org/articles/62552/elife-62552-fig4-data1-v3.xlsx

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The proposed unique importance of MOR-dependent ß-arrestin 2 signaling in OIRD has motivated the development of biased agonists for analgesia. However, the recent failure to reproduce this result has called model into question (Kliewer et al., 2020). Therefore, the goal of our studies was to test the null hypothesis that germline deletion of Arrb2 does not attenuate OIRD. The results from our in vivo studies under normoxic and hypercapnic conditions, as well as our in vitro studies, failed to reject this null hypothesis. In order to directly compare to previous results, we sufficiently powered our cohort size to identify effect sizes reported in Raehal et al., 2005 (~50% less OIRD), and the statistical tests were performed conservatively by not correcting for multiple comparisons. Combined, these three independent assays demonstrate that the germline knockout of Arrb2 does not attenuate OIRD.

Unlike other studies, we designed ours with five important features to ensure a robust conclusion. First, the OIRD comparisons were made between littermates in an effort to control for any strain specific effects on breathing and opioid sensitivity (Bubier et al., 2020). Second, OIRD was defined as the comparison of breathing after IP injection of saline and morphine in the same animal to control for any within-animal specific breathing variation. Third, we analyzed the multiple breathing parameters with and without averaging across long stretches of breathing. Four, OIRD was measured under a hypercapnic state for a more precise quantification. And fifth, we validated our in vivo studies by directly measuring the impact of a MOR ligand on the preBötC rhythm, the key site for OIRD. Future studies should also confirm an unchanged sensitivity to opioids in other brain or peripheral sites that contribute to OIRD.

One difference between our study and the original Arrb2 study by Raehal et al. was our method of morphine delivery. Raehal et al. reported that at the maximal concentration of morphine delivered (150 mg/kg subcutaneous), the respiratory rate in Arrb2 knockouts was depressed to half that of wildtype controls (~20% vs 40% depression of breathing rate). In our study, although we delivered 20 mg/kg morphine by IP injection, we observed an even larger depression of breathing in all three Arrb2 genotypes (60% in normoxic and 30% in hypercapnic conditions), indicating our assay was sufficient to observe the reported attenuated OIRD response in Arrb2-/- mice. Beyond this, several important future studies to exhaust other methodological limitations include the measurement of respiratory depression in Arrb2-/- and littermates with other abused opioids like oxycodone and heroin, to temporally or spatially delete Arrb2 using Arrb2^flox/flox alleles to overcome possible compensatory mechanisms, and the study of biased agonists in models where potent opioids like fentanyl are lethal. Additional or other mechanisms may underlie the fatal apnea that was not studied here.

The core premise for the development of MOR biased agonists is that Arrb2-dependent signaling provides a molecular mechanism to dissociate analgesia from respiratory depression. Our findings do not support this claim. Consistently, a recent study demonstrated that an opioid receptor ligand that does not induce MOR ß-arrestin 2-dependent signaling still temporarily induces OIRD (Uprety et al., 2021). Given all of this, how then do some biased agonists show analgesia while minimizing respiratory depression? Perhaps biased agonists are just partial MOR agonists for activation of G-protein signaling. In this case, we imagine analgesia is more sensitive than respiratory depression, and therefore certain concentrations of MOR ligand enable these two effects to be separated. This would be akin to providing a lower dose of standard opioid-like drugs. Regardless, our results, along with similar data from another recent study (Kliewer et al., 2020), refute the foundational model that Arrb2 selectively mediates OIRD and suggest that now we must reconsider and in the future reinvestigate the mechanism of biased agonism in vivo.

The data generated in Figures 2-4 are provided in the source files.

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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, formal-analysis, i.b.-and-a.d.-conducted-all-in-vivo-experiments.-p.w.-and-i.b.-conducted-in-vitro-experiments., investigation, methodology, software, validation, visualization, writing-original-draft, writing-review-and-editing

   Contributed equally with

   Xin Paul Wei

   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

   conceptualization, data-curation, investigation, methodology, p.w.-and-i.b.-conducted-in-vitro-experiments.-i.b.-analyzed-all-data., validation, writing-review-and-editing

   Contributed equally with

   Iris Bachmutsky

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6621-3143

3. Adelae Durand

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

   Contribution

   data-curation, investigation, writing-review-and-editing

   Competing interests

   No competing interests declared

4. Kevin Yackle

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

   Contribution

   conceptualization, funding-acquisition, project-administration, supervision, visualization, writing-original-draft, 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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