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The rates of opioid overdose in the United States quadrupled between 1999 and 2017, reaching a staggering 130 deaths per day. This health epidemic demands innovative solutions that require uncovering the key brain areas and cell types mediating the cause of overdose— opioid-induced respiratory depression. Here, we identify two primary changes to murine breathing after administering opioids. These changes implicate the brainstem’s breathing circuitry which we confirm by locally eliminating the µ-Opioid receptor. We find the critical brain site is the preBötzinger Complex, where the breathing rhythm originates, and use genetic tools to reveal that just 70–140 neurons in this region are responsible for its sensitivity to opioids. Future characterization of these neurons may lead to novel therapies that prevent respiratory depression while sparing analgesia. 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.

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. 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.

Opioids are perhaps the most effective analgesics in medicine. However, between 1999 and 2020, over 500,000 people in the United States died from opioid overdose. This health epidemic demands innovative solutions that will require uncovering the key brain areas and cell types mediating the cause of overdose— opioid-induced respiratory depression. Here, I identify two primary changes to breathing after administering opioids. These changes implicate the brainstem’s breathing circuitry which I confirm by locally eliminating the µ-Opioid receptor. I find the critical brain site is the preBötzinger Complex, where the breathing rhythm originates, and use genetic tools to reveal that just 70–140 neurons in this region are responsible for its sensitivity to opioids. Meanwhile, in the absence of this basic understanding of the mechanisms mediated opioid respiratory depression, some groups have already moved forward with development of therapeutics with novel biochemical properties they claim will dissociate opioid-like analgesia from effects on 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, I re-examine breathing in ß-arrestin 2-deficient mice and instead find no connection between ß-arrestin 2 and opioid respiratory depression. Put together, this work suggests a new approach to develop safer opioid-like drugs is needed, and that future characterization of the small group of neurons mediating respiratory depression may lead to novel therapies that prevent respiratory side effects while sparing analgesia.

The rates of opioid overdose in the United States quadrupled between 1999 and 2017, reaching a staggering 130 deaths per day. This health epidemic demands innovative solutions that require uncovering the key brain areas and cell types mediating the cause of overdose-opioid respiratory depression. Here, we identify two primary changes to breathing after administering opioids. These changes implicate the brainstem's breathing circuitry which we confirm by locally eliminating the μ-Opiate receptor. We find the critical brain site is the origin of the breathing rhythm, the preBötzinger Complex, and use genetic tools to reveal that just 70-140 neurons in this region are responsible for its sensitivity to opioids. Future characterization of these neurons may lead to novel therapies that prevent respiratory depression while sparing analgesia.

Opioids are perhaps the most effective analgesics in medicine. However, from 1999 to 2018, they also killed more than 400,000 people in the United States by suppressing breathing, a common side-effect known as opioid induced respiratory depression. This doubled-edged sword has inspired the dream of developing novel therapeutics that provide opioid-like analgesia without respiratory depression. One such approach has been to develop so-called ‘biased agonists’ that activate some, but not all pathways downstream of the µ-opioid receptor (MOR), the target of morphine and other opioid analgesics. This hypothesis stems from a study suggesting that MOR-mediated activation of ß2-Arrestin is the downstream signaling pathway responsible for respiratory depression, whereas inhibition of adenylyl cyclase produces analgesia. To further verify this model, which represents the motivation for the biased agonist approach, we examined respiratory behavior in mice lacking the gene for ß2-Arrestin. Contrary to previous findings, we find no correlation between ß2-Arrestin function and opioid-induced respiratory depression, suggesting that any effect of biased agonists must be mediated through an as-yet to be identified signaling mechanism.

Opioids are perhaps the most effective analgesics in medicine. However, from 1999 to 2018, they also killed more than 400,000 people in the United States by suppressing breathing, a common side-effect known as opioid induced respiratory depression. This doubled-edged sword has inspired the dream of developing novel therapeutics that provide opioid-like analgesia without respiratory depression. One such approach has been to develop so-called 'biased agonists' that activate some, but not all pathways downstream of the μ-opioid receptor (MOR), the target of morphine and other opioid analgesics. This hypothesis stems from a study suggesting that MOR-mediated activation of β2-Arrestin is the downstream signaling pathway responsible for respiratory depression, whereas inhibition of adenylyl cyclase produces analgesia. To further verify this model, which represents the motivation for the biased agonist approach, we examined respiratory behavior in mice lacking the gene for β2-Arrestin. Contrary to previous findings, we find no correlation between β2-Arrestin function and opioid-induced respiratory depression, suggesting that any effect of biased agonists must be mediated through an as-yet to be identified signaling mechanism. Competing Interest Statement The authors have declared no competing interest.

Loss of function mutations in CNTNAP2 cause a syndromic form of autism spectrum disorder (ASD) in humans and produce social deficits, repetitive behaviors, and seizures in mice. Yet, the functional effects of these mutations at the cellular and circuit level remain elusive. Using laser scanning photostimulation, whole-cell recordings, and electron microscopy, we found a dramatic decrease in functional excitatory and inhibitory synaptic inputs in L2/3 medial prefrontal cortex (mPFC) of Cntnap2 knock-out (KO) mice. In accordance with decreased synaptic input, KO mice displayed reduced spine and synapse densities, despite normal intrinsic excitability and dendritic complexity. To determine how this decrease in synaptic inputs alters coordination of neuronal firing patterns in vivo, we recorded mPFC local field potentials (LFP) and unit spiking in head-fixed mice during locomotion and rest. In KO mice, LFP power was not significantly altered at all tested frequencies, but inhibitory neurons showed delayed phase-firing and reduced phase-locking to delta and theta oscillations during locomotion. Excitatory neurons showed similar changes but only to delta oscillations. These findings suggest that profound ASD-related alterations in synaptic inputs can yield perturbed temporal coordination of cortical ensembles.