You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: Opioids depress breathing through two small brainstem sites}
Article:
Abstract:
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.
Introduction:
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.
Discussion:
We find that although multiple breathing parameters are impacted by opioids, decreased inspiratory airflow and delayed breath initiation, which we term pause, represent the primary changes that result in OIRD. Pauses occur during expiration and account for tens to hundreds of milliseconds of low airflow per breath. In hypercapnia, these pause periods terminate with an active expiration. The force, timing of inspiration, and expression of active expiration are ultimately determined by the inspiratory rhythm generator, the preBötC, and focused our initial studies to this site( Smith et al., 1991; Feldman et al., 2013; Cui et al., 2016; Huckstepp et al., 2016). Additionally, correlates of these two changes manifest as decreased burst size and frequency in the activity of the preBötC slice in vitro. These two key changes in the breath can guide future OIRD studies and efforts to characterize and test novel opioid drugs. Additionally, this workflow can be applied to the analysis of other respiratory depressants. Our experimental design allowed us to determine that both the preBötC and PBN/KF have independent and additive rescue of OIRD. Of these two, the preBötC has the larger magnitude rescue of our two core breathing parameters. The combined deletion of µ-Opioid receptor from both sites essentially eliminates OIRD, even to extremely high doses of the potent opioid fentanyl. This suggests that targeting just these two sites is sufficient to rescue opioid respiratory depression. We interpret the small remaining effect of opioids we observe to be due to incomplete transduction of these brain areas but cannot rule out other minor contributing sites. It is also possible, given the challenge of restricting viral transduction, that some of the demonstrated effects are already due to spillover deletion of µ-Opioid receptor in neighboring brain areas, such as the bulbospinal rostral ventral respiratory group. Our studies were limited to two opioids( morphine and fentanyl) at a specific dose and it will be important in future work to determine if these two brain sites are also critical for OIRD caused by these opioids at different doses or other opioids altogether. The two hallmark changes during OIRD, decreased inspiratory airflow and delayed initiation, perfectly match the opioid induced depression of amplitude and frequency in the preBötC slice. We show that ~140 Oprm1 glutamatergic preBötC neurons mediate this effect. And surprisingly, half this number, just ~70 glutamatergic neurons are sufficient to rescue opioid depression of the preBötC rhythm( 50 Dbx1/FOXP2 and 20 FOXP2 in Foxp2-Cre;Oprm1f/f, Figure 4—figure supplement 3). Further, given the importance of Dbx1 neurons in respiratory rhythm generation( Gray et al., 2010; Bouvier et al., 2010), rescuing Oprm1 in just ~50/140 Dbx1 neurons( the Foxp2+ subset) may be sufficient to prevent preBötC depression, the smallest number of neurons we propose. This small number is remarkably consistent with the number of Dbx1 neurons that must be lesioned to arrest preBötC activity( Wang et al., 2014). Given the similarity of these effects, we hypothesize that opioids are primarily acting by silencing presynaptic release, effectively removing these neurons from the network. Alternatively, it is proposed that just a small subset of preBötC excitatory neurons may generate the inspiratory rhythm( Phillips et al., 2019) and perhaps these key Oprm1 neurons are enriched within this subgroup. In this instance, since hyperpolarization of preBötC rhythmogenic neurons slows and silences breathing( Koizumi et al., 2016), opioids may act postsynaptically as proposed by others( Montandon et al., 2011; Johnson et al., 1996). Regardless, it is profound that such a small number can abruptly halt the respiratory rhythm in a network of more than 1000 neurons and suggests that either these neurons act as a key population for rhythmogenesis, or that recurrent excitatory networks are exquisitely sensitive to the number of participating cells. Important future work will need to use the Dbx1/Oprm1 and Foxp2/Oprm1 molecular codes to selectively eliminate Oprm1 from these neurons to test if truly so few neurons profoundly control or modulate breathing in vivo.
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 .