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ß-arrestin 2 germline knockout does not attenuate opioid respiratory depression

  1. Iris Bachmutsky
  2. Xin Paul Wei
  3. Adelae Durand
  4. Kevin Yackle  Is a corresponding author
  1. Department of Physiology, University of California, San Francisco, United States
  2. Neuroscience Graduate Program, University of California, San Francisco, United States
  3. Biomedical Sciences Graduate Program, University of California, San Francisco, United States
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Cite this article as: eLife 2021;10:e62552 doi: 10.7554/eLife.62552

Abstract

Opioids are perhaps the most effective analgesics in medicine. However, between 1999 and 2018, over 400,000 people in the United States died from opioid overdose. Excessive opioids make breathing lethally slow and shallow, a side-effect called opioid-induced respiratory depression. This doubled-edged sword has sparked the desire to develop novel therapeutics that provide opioid-like analgesia without depressing breathing. One such approach has been the design of so-called ‘biased agonists’ that signal through some, but not all pathways downstream of the µ-opioid receptor (MOR), the target of morphine and other opioid analgesics. This rationale stems from a study suggesting that MOR-induced ß-arrestin 2 dependent signaling is responsible for opioid respiratory depression, whereas adenylyl cyclase inhibition produces analgesia. To verify this important result that motivated the ‘biased agonist’ approach, we re-examined breathing in ß-arrestin 2-deficient mice and instead find no connection between ß-arrestin 2 and opioid respiratory depression. This result suggests that any attenuated effect of ‘biased agonists’ on breathing is through an as-yet defined mechanism.

eLife digest

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.

Introduction

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.

Results

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).

Experimental approach to measure OIRD in each Arrb2 genotype.

(A) Breeding scheme to generate F2 Arrb2+/+, +/-, and -/- littermates. (B), Whole body plethysmography experimental scheme. On Day 1, recordings were performed 15 min after IP saline injection. Day 2, recordings 15 min after IP morphine (20 mg/kg). Recordings were first conducted under normoxic conditions (21% O2, 0% CO2) and then at least one week later under hypercapnic conditions (21% O2, 5% CO2). (C), Example analysis of a single breath. The approximated airflow (mL/s) was used to identify inspiration (insp. <0 mL/s) and expiration (expir. >0 mL/s). Instantaneous frequency (Hz, s–1) defined as the interval between inspiration onset and expiration offset. PIF, peak inspiratory airflow. These two parameters were used to define OIRD.

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.

Basal respiration and OIRD in Arrb2 littermates in normoxic conditions.

(A) Example breathing trace in normoxic conditions (21% O2, 0% CO2) following IP saline (left) and morphine (right) for Arrb2+/+. (B), Example breathing traces from Arrb2-/-. (C), Histogram of instantaneous respiratory frequency (Hz) for all breaths in morphine from Arrb2-/- (red, combined from n = 7 animals), Arrb2+/- (blue, n = 6), and Arrb2+/+ (black, n = 5). PDF, probability density function. Top, mean (circle)± standard deviation (bars). Values of respiratory measurements are reported in Table 1. (D), OIRD defined as the ratio of average respiratory frequency in morphine to saline for Arrb2-/-, Arrb2+/-, and Arrb2+/+. Mean (circle)± standard deviation (bars). Data values included in Table 2. Note, Arrb2+/+ mice have less OIRD when compared to Arrb2+/- and -/-. (E–F), Analysis of peak inspiratory airflow (PIF) displayed as in C–D. Note, PIF in Arrb2±mice shows more OIRD when compared to Arrb2+/+ and -/-. There is no statistically significant difference between Arrb2+/+ and -/-. Single and Two Factor ANOVA and unpaired t-test statistics reported in Table 2. *, indicates the post-hoc single factor ANOVA comparisons with p-value < 0.05. Statistics were not corrected for multiple comparisons to maximize the possibility of identifying differences between Arrb2 genotypes.

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
Table 1
Mean and confidence interval for normoxic condition raw respiratory frequency and peak inspiratory airflow after saline and morphine intraperitoneal injection.
Arrb2 -/- mean ± SEMArrb2 -/-95% CIArrb2+/- mean ± SEMArrb2 +/-95% CIArrb2 +/+ mean ± SEMArrb2 +/+95% CI
Freq. saline (Hz)7.05 ± 0.346.38 → 7.727.51 ± 0.416.71 → 8.316.93 ± 0.326.30 → 7.56
Freq. morphine2.67 ± 0.162.36 → 2.982.83 ± 0.142.56 → 3.103.27 ± 0.162.96 → 3.58
PIF saline(mL/s)–3.17 ± 0.15–2.88 → –3.46–4.03 ± 0.25–3.54 → –4.52–3.5 ± 0.23–3.04 → –3.95
PIF morphine–2.09 ± 0.09–1.91 → –2.23–2.19 ± 0.10–1.99 → –2.39–2.33 ± 0.10–2.13 → –2.53
Table 2
OIRD values of respiratory frequency and peak inspiratory airflow in normoxic conditions and the several types of statistical tests.

The respiratory frequency OIRD in Arrb2+/+ is larger than Arrb2+/- and -/-. The PIF OIRD for Arrb2+/- is smaller than Arrb2+/+ and -/-.

Arrb2 -/-OIRD mean (95% CI)Arrb2+/- mean (95% CI)Arrb2 +/+ mean (95% CI)Arrb2 -/- vs. Arrb2 +/+ (t-test)Arrb2 -/- vs. Arrb2 +/+(t-test 95% CI)One-way anovaTukey HSD/Kramer Arrb2 -/- vs. +/+Two-way anova regression(interaction)
Freq.0.38 (0.35→0.41)0.38 (0.35→0.41)0.47 (0.44→0.50)P = 0.0010.05→0.140.0020.0030.22
PIF0.66 (0.61→0.71)0.55 (0.48→0.56)0.68 (0.63→0.71)P = 0.83–0.13→0.160.030.960.05

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
Basal respiration and OIRD in Arrb2 littermates in hypercapnic conditions.

(A) Example breathing trace in hypercapnic conditions (21% O2, 5% CO2) following IP saline (left) and morphine (right) for Arrb2+/+. (B), Example breathing traces from Arrb2-/-. (C), Histogram of instantaneous respiratory frequency (Hz) for all breaths in morphine from Arrb2-/- (red, combined from n = 7 animals), Arrb2+/- (blue, n = 6), and Arrb2+/+ (black, n = 5). PDF, probability density function. Top, mean (circle)± standard deviation (bars). Respiratory measurements for saline and morphine are reported in Table 3. (D), OIRD defined as the ratio of average respiratory frequency in morphine to saline for Arrb2-/-, Arrb2+/-, and Arrb2+/+. Mean (circle)± standard deviation (bars). Data included in Table 4. (E–F), Analysis of peak inspiratory airflow (PIF) as in C–D. but for peak inspiratory airflow. Single and Two Factor ANOVA and unpaired t-test statistics reported in Table 4. Statistics were not corrected for multiple comparisons to maximize the possibility of identifying differences between Arrb2 genotypes.

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
Table 3
Mean and confidence interval for hypercapnic condition raw respiratory frequency and peak inspiratory airflow after saline and morphine intraperitoneal injection.
Arrb2 -/- mean ± SEMArrb2 -/-95% CIArrb2+/- mean ± SEMArrb2 +/-95% CIArrb2 +/+ mean ± SEMArrb2 +/+95% CI
Freq. saline (Hz)7.68 ± 0.197.31 → 8.057.69 ± 0.167.38 → 8.007.77 ± 0.397.01 → 8.53
Freq. morphine5.20 ± 0.105.00 → 5.404.79 ± 0.194.42 → 5.165.38 ± 0.185.02 → 5.73
PIF saline (mL/s)–5.34 ± 0.27–4.81 → –5.87–5.75 ± 0.25–5.26 → –6.24–5.71 ± 0.20–5.32 → –6.10
PIF morphine–3.54 ± 0.16–3.22 → –3.85–3.86 ± 0.15–3.57 → –4.15–3.93 ± 0.24–3.46 → –4.40
Table 4
OIRD values of respiratory frequency and peak inspiratory airflow in hypercapnic conditions and the several types of statistical tests.
Arrb2 -/-OIRD median or mean (95% CI)Arrb2+/- mean (95% CI)Arrb2 +/+ mean (95% CI)Arrb2 -/- vs. Arrb2 +/+ (Mann-Whitney or unpaired t-test, two tail)Arrb2 -/- vs. Arrb2 +/+(95% CI)Kruskal-Wallis orOne-way anovaTwo-way Anova - regression(interaction)
Freq.0.660.63 (0.55→0.71)0.70 (0.60→0.80)P = 0.52 (MW)–0.11→0.150.16 (KW)0.45
PIF0.66 (0.64→0.68)0.68 (0.61→0.77)0.69 (0.64→0.72)P = 0.46 (t)–0.04→0.080.86 (anova)0.96

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.

Slowing of preBötC rhythmicity with a MOR agonist from Arrb2 littermates.

A, Neonatal medullary preBötC slice preparation. PreBötC inspiratory activity is measured via the hypoglossal nerve rootlet (∫CNXII, arbitrary units). Example twenty minute recordings from an Arrb2+/+ slice where the rhythm is recorded at baseline (0 nM DAMGO), then 20 nM and 50 nM DAMGO, and then with the addition of 100 nM Naloxone. (B), As in A, but example recording from an Arrb2-/- littermate. (C), The preBötC frequency (Hz) for each slice from Arrb2+/+ (black, n = 5), +/- (blue, n = 20), and -/- (red, n = 6) littermates at each DAMGO dose. Average frequency (bursts per second) is measured during the last five minutes of each recording. (D), Mean ± SEM for normalized frequency for each genotype at each DAMGO dose. *, p-value < 0.05 for Kruskal-Wallis test. p-Values for pairwise Mann-Whitney: 0.10 for +/+ vs -/-, 0.45 for +/+ vs +/-, 0.02 for±vs -/-.

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

Discussion

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 Arrb2flox/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.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus, male and female)Arrb2-/-The Jackson Laboratory011130
Strain, strain background (Mus musculus, male and female)C57Bl/6 JThe Jackson Laboratory000664
Peptide, recombinant proteinDAMGOAbcamAb12067
Chemical compound, drugMorphine sulfateHenry Schein057202
Chemical compound, drugNaloxoneSigma AldrichN7758
Software, algorithmMatlabMathworkshttps://github.com/YackleLab/Opioids-depress-breathing-through-two-small-brainstem-sites

Animals

Arrb2 -/- mice (Bohn et al., 1999) were bred to C57BL/6 to generate heterozygous F1. The F1 littermates were then crossed to make Arrb2-/-, Arrb2-/+, and Arrb2+/+ (F2). Mice were housed in a 12 hour light/dark cycle with unrestricted food and water. Mice were given anonymized identities for experimentation and data collection. All animal experiments were performed in accordance with national and institutional guidelines with standard precautions to minimize animal stress and the number of animals used in each experiment. Institutional Animal Care and Use Committee approval number AN181239.

Plethysmography and respiratory analysis

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Plethysmography and respiratory analysis were performed as in Bachmutsky et al., 2020. Briefly, on the first recording day, adult (6–12 weeks) Arrb2-/-, Arrb2-/+, and Arrb2+/+ mice were administered IP 100 µL of saline and placed in an isolated recovery cage for 15 min. After, individual mice were then monitored in a 450 mL whole animal plethysmography chamber at room temperature (22°C) in 21% O2 balanced with N2 (normoxic condition) or 21% O2, 5% CO2 balanced with N2 (hypercapnic condition). After 1 day, the same protocol was used to monitor breathing after IP injection of morphine (20 mg/kg, Henry Schein 057202). The morphine recordings under normoxic and hypercapnic conditions were separated from saline recordings by at least 3 days. Each breath was automatically segmented based on airflow crossing zero as well as quality control metrics. Respiratory parameters (e.g. peak inspiratory flow, instantaneous frequency) for each breath, as well as averages, were then calculated. Reported airflow in mL/sec. is an approximate of true volumes. The analysis was performed with custom Matlab code available on Github with a sample dataset (https://github.com/YackleLab/Opioids-depress-breathing-through-two-small-brainstem-sites). All animals in the study were included in the analysis and cohorts including all these genotypes were run together.

Statistics

A power analysis was performed using the reported effect size from Raehal et al. In this case, 1–4 mice were necessary to observe a statistically significant result. Each cohort (Arrb2+/+, +/-, -/-) exceeded 4. Statistical tests were performed on the ratio of IP morphine to IP saline for instantaneous respiratory frequency and peak inspiratory flow separately for normoxic and hypercapnic conditions. A Shapiro Wilks test was first done to determine if the data was normally distributed (Figure 2—source data 1, Figure 3—source data 1). If normal, a single factor ANOVA was performed to determine any differences among the three genotypes (alpha <0.05). In the instance the p-value was <0.05, the Tukey HSD post-hoc test was done to determine which of the pairwise comparisons were statistically different (alpha <0.05). Additionally, one-way unpaired parametric T-tests were used to compare Arrb2 +/+ and -/- genotypes (alpha <0.05). If the data failed to pass the Shapiro Wilks test, then the non-parametric Kruskal-Wallis test was used to determine if any differences (alpha <0.05). And the Mann-Whitney U test was used to compare Arrb2+/+ and -/- genotypes (alpha <0.05). A two-way ANOVA with regression was used to determine interactions between each of the genotypes IP saline and IP morphine values. To determine the power of our data, we compared hypothetical Arrb2+/+ and measured Arrb2-/- means. The power calculation included our cohort size and the measured standard deviation of the two genotypes. A similar statistical approach was used to analyze the in vitro data (Figure 4—source data 1). All the above statistics were performed using the publicly available Excel package ‘Real Statistics Functions’ SPSS and Matlab.

Slice electrophysiology

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Rhythmic 550–650 μm-thick transverse medullary slices which contain the preBötC and cranial nerve XII (XIIn) from neonatal Arrb2 -/-, +/-, +/+ mice (P0-5) were prepared as described (Bachmutsky et al., 2020). Slices were cut in ACSF containing (in mM): 124 NaCl, 3 KCl, 1.5 CaCl2, 1 MgSO4, 25 NaHCO3, 0.5 NaH2PO4, and 30 D-glucose, equilibrated with 95% O2 and 5% CO2 (4 °C, pH = 7.4). Recordings were performed in 9 mM at a temperature of 27°C. Slices equilibrated for 20 min before experiments were started. The preBötC neural activity was recorded from CNXII rootlet. Activity was recorded with a MultiClamp700A or B using pClamp9 at 10,000 Hz and low/high pass filtered at 3/400 Hz. After equilibration, baseline activity and then increasing concentrations of DAMGO (ab120674) were bath applied (20 nM, 50 nM). After the rhythm was eliminated, 100 nM Naloxone (Sigma Aldrich N7758) was bath applied to demonstrate slice viability. The rate was determined from the last 5 min of each 20-min recording and rhythmic activity was normalized to the first control recording for dose response curves.

Data availability

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

References

    1. Luttrell LM
    2. Lefkowitz RJ
    (2002)
    The role of beta-arrestins in the termination and transduction of G-protein-coupled receptor signals
    Journal of Cell Science 115:455–465.

Decision letter

  1. Allan Basbaum
    Reviewing Editor; University of California San Francisco, United States
  2. Christian Büchel
    Senior Editor; University Medical Center Hamburg-Eppendorf, Germany
  3. Jack L Feldman
    Reviewer; University of California, Los Angeles, United States
  4. Bryan L Roth
    Reviewer; University of North Carolina, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

There is definitely great interest in the mechanisms that underlie the adverse side effects that occur when opioids are used to relieve pain. Your study has very convincingly demonstrated that a prevailing hypothesis, namely that Mu opioid receptor-induced ß-arrestin 2-dependent signaling is responsible for opioid respiratory depression, cannot be supported. Alternative mechanisms must be investigated.

Decision letter after peer review:

Thank you for submitting your article "ß-arrestin 2 germline deletion does not attenuate opioid respiratory depression" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Christian Büchel as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Jack L Feldman (Reviewer #1); Bryan Roth (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.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.

Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in eLife, either of which would be linked to the original paper.

Essential revisions:

There is considerable support for your manuscript. We appreciate that your submission was intended as a Short Report, however, there is consensus among the reviewers that were you to provide the new experiments indicated, eLife should definitely consider publishing as a regular manuscript. We hope that you are able to complete these experiments as they will greatly increase the impact of your manuscript. Key points of the Reviewers comments are noted below.

Since the in vivo experiments were germline knockouts, some comment should be made as to the possibility that the results are impacted by effects in sites other than the preBotizinger Complex or the parabrachial nuclear complex. The in vitro experiments address this implicitly, but an explicit sentence or two would be helpful.

Deaths in OIRD are typically the result of asphyxiation, the result of a long lasting apnea. In this study, as well as all of their references, the depression of breathing in mice was far from apnea. How does this impact the significance of these findings to OIRD in humans?

One methodological concern is the use of morphine as the µOR agonist. Most of the prescribed and/or abused µOR agonists (e.g., oxycodone, fentanyl, heroin) are much more potent than morphine (https://www.ncbi.nlm.nih.gov/books/NBK537482/table/appannex6.tab2/ ), and account for most of the OIRD deaths. While not necessary for this paper, the authors might consider using more potent agonists in follow up studies.

One reviewer suggests that the writing could be a bit more precise in distinguishing between gas mixtures and presumptive blood gas concentrations of CO2 or O2. For example, Line 79: "hypercapnia" is not a "gas". Do you mean "hypocapnic gas mixture"? Note: same issue elsewhere with using "normoxia" to refer to a different gas mixture.

Statistics: The proper way to analyze the data is a two-way repeated measures ANOVA with the factors genotype and treatment.

The authors need to define the β-error and confidence intervals to state that there was no difference between genotypes (within given confidence intervals).

The DAMGO slice experiments refer to a "historical control". In order to be conclusive, the authors must repeat these experiments in wild-type mice (in a blinded manner!) and do a proper side-by-side comparison.

In order to convincingly contradict previous results, the experimental design should be comprehensive. In this specific case, testing more than a single dose of morphine would make the results more convincing.

One reviewer believes that the electrophysiological studies are incomplete. At a minimum they should provide dose-response findings in WT and KO slices to an endogenous peptide and morphine. Additionally, they would need to provide some estimates of experimental variability and, at the least, biological and technical replicates. Finally, they would need to provide statistical analysis of the findings. Simply exposing N=1 slice to DAMGO is not persuasive.

The authors may wish to consider that since these mice have had bArr2 deleted from conception, if this molecule is involved in the actions of opioids it is possible that compensatory changes have occurred during development to abrogate the effect in adults. Of course, this does not explain the difference between these results and prior results but does provide additional context for the findings.

https://doi.org/10.7554/eLife.62552.sa1

Author response

Essential revisions:

There is considerable support for your manuscript. We appreciate that your submission was intended as a Short Report, however, there is consensus among the reviewers that were you to provide the new experiments indicated, eLife should definitely consider publishing as a regular manuscript. We hope that you are able to complete these experiments as they will greatly increase the impact of your manuscript. Key points of the Reviewers comments are noted below.

Since the in vivo experiments were germline knockouts, some comment should be made as to the possibility that the results are impacted by effects in sites other than the preBotizinger Complex or the parabrachial nuclear complex. The in vitro experiments address this implicitly, but an explicit sentence or two would be helpful.

Our study is inconsistent with the original description of OIRD in Arrb2 germline knockouts (Raehal et al. 2005). in vitro, we demonstrate the preBötC is similarly impacted by opioids in all three genotypes. However, as the reviewer notes, the similar impact upon the preBötC may only partially explain the in vivo results. We now note on Page 8, line 156-157 that future studies should explore the sensitivity of other important central and peripheral OIRD sites in all three genotypes.

Deaths in OIRD are typically the result of asphyxiation, the result of a long lasting apnea. In this study, as well as all of their references, the depression of breathing in mice was far from apnea. How does this impact the significance of these findings to OIRD in humans?

The focus of our work was to reproduce the results from Raehal et al. 2005 which led to the conclusion that Arrb2 dependent signaling is a key mediator of OIRD. This provided the rationale for developing MOR ‘biased agonists’. Despite not assaying lethality or other abused opioids, our preclincal results remain significant as they inform the groups and companies currently developing ‘biased agonists’ as well as the NIH programs currently funding or planning to fund such approaches. We have added such comments into our discussion on Page 8, lines 165-167.

One methodological concern is the use of morphine as the µOR agonist. Most of the prescribed and/or abused µOR agonists (e.g., oxycodone, fentanyl, heroin) are much more potent than morphine (https://www.ncbi.nlm.nih.gov/books/NBK537482/table/appannex6.tab2/ ), and account for most of the OIRD deaths. While not necessary for this paper, the authors might consider using more potent agonists in follow up studies.

This is an important point and future experiment that must be conducted. We had added this comment into our discussion on Page 8, lines 165-167.

One reviewer suggests that the writing could be a bit more precise in distinguishing between gas mixtures and presumptive blood gas concentrations of CO2 or O2. For example, Line 79: "hypercapnia" is not a "gas". Do you mean "hypocapnic gas mixture"? Note: same issue elsewhere with using "normoxia" to refer to a different gas mixture.

Thank you for this feedback. We have corrected our terminology throughout the text.

Statistics: The proper way to analyze the data is a two-way repeated measures ANOVA with the factors genotype and treatment.

The results of a Two-Way ANOVA with regression are now included in Table 2 and 4. A regression was used since the number of mice for each Arrb2 genotype is unbalanced. The only interaction observed was the PIF in the normoxic condition. Upon visualization of the means, it appears the interaction is due to the increased saline PIF in the Arrb2+/- mice.

Author response image 1

The authors need to define the β-error and confidence intervals to state that there was no difference between genotypes (within given confidence intervals).

The confidence intervals for the respiratory parameters, OIRD, and t-test/Mann-Whitney U are now included in Tables 1-4. We now comprehensively describe the power of our study in Figure 3-Supplement 1 and text Page 6, Lines 111-121.

The DAMGO slice experiments refer to a "historical control". In order to be conclusive, the authors must repeat these experiments in wild-type mice (in a blinded manner!) and do a proper side-by-side comparison.

This experiment has been conducted in litters containing Arrb2+/+, +/-, and -/- littermates in a blinded fashion. The data is displayed in Figure 4 and described on Page 6-7, Lines 121-135.

In order to convincingly contradict previous results, the experimental design should be comprehensive. In this specific case, testing more than a single dose of morphine would make the results more convincing.

We agree with the reviewer that a dose response curve would more comprehensively describe OIRD. However, the single dose used produced a profound respiratory depression equivalent to or even larger than what is displayed in Figure 5 of Raehal et al. 2005. Given this, we believe the single chosen dose is sufficient to refute the prior claim, and this is the primary message of the manuscript.

Please note the impact of COVID-19 on our revision experiments. The shutdown necessitated a culling of our mouse colony. This required us to re-breed all F1 and then F2 animals from the very few Arrb2-/- we retained. This breeding alone has taken more than 8 months. Furthermore, the Arrb2-/- stocks at Jax were culled and these mice are backordered due to breeding to re-establish the colony. So far, the F2 mice we have been generated have been dedicated to our in vitro studies.

One reviewer believes that the electrophysiological studies are incomplete. At a minimum they should provide dose-response findings in WT and KO slices to an endogenous peptide and morphine. Additionally, they would need to provide some estimates of experimental variability and, at the least, biological and technical replicates. Finally, they would need to provide statistical analysis of the findings. Simply exposing N=1 slice to DAMGO is not persuasive.

As described in Point 7, we have repeated our in vitro study with a dose response curve in all three Arrb2 genotypes (Figure 4). We now display the variability of our data and performed the appropriate statistical tests.

We chose to conduct these in vitro studies with DAMGO since this peptide is the archetype MOR agonist used in biochemical studies to demonstrate if MOR ligands have signaling bias (for example, see Schmid et al. 2017). Although the pharmacokinetics of morphine make it more suitable for the in vivo studies, DAMGO is the most appropriate MOR ligand to investigate activation of both Gαi and ß-arrestin 2 dependent MOR signaling in vitro.

The authors may wish to consider that since these mice have had bArr2 deleted from conception, if this molecule is involved in the actions of opioids it is possible that compensatory changes have occurred during development to abrogate the effect in adults. Of course, this does not explain the difference between these results and prior results but does provide additional context for the findings.

As described in Point 7, we have repeated our in vitro study with a dose response curve in all three Arrb2 genotypes (Figure 4). We now display the variability of our data and performed the appropriate statistical tests.

We chose to conduct these in vitro studies with DAMGO since this peptide is the archetype MOR agonist used in biochemical studies to demonstrate if MOR ligands have signaling bias (for example, see Schmid et al. 2017). Although the pharmacokinetics of morphine make it more suitable for the in vivo studies, DAMGO is the most appropriate MOR ligand to investigate activation of both Gαi and ß-arrestin 2 dependent MOR signaling in vitro.

https://doi.org/10.7554/eLife.62552.sa2

Article and author information

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

Funding

NIH Office of the Director (DP5-OD023116)

  • Kevin Yackle

Program for Breakthrough Biomedical Research

  • Kevin Yackle

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

All animal experiments were performed in accordance with national and institutional guidelines with standard precautions to minimize animal stress and the number of animals used in each experiment. All animal protocols have been approved by the UCSF 'Office of Research'.approval number AN181239.

Senior Editor

  1. Christian Büchel, University Medical Center Hamburg-Eppendorf, Germany

Reviewing Editor

  1. Allan Basbaum, University of California San Francisco, United States

Reviewers

  1. Jack L Feldman, University of California, Los Angeles, United States
  2. Bryan L Roth, University of North Carolina, United States

Publication history

  1. Received: October 1, 2020
  2. Accepted: May 17, 2021
  3. Accepted Manuscript published: May 18, 2021 (version 1)
  4. Accepted Manuscript updated: May 24, 2021 (version 2)
  5. Version of Record published: June 18, 2021 (version 3)

Copyright

© 2021, Bachmutsky et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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