Respiratory Rate Depression Research Articles

Establishing short-term occupational exposure limits (STELs) for sensory irritants using predictive and in silico respiratory rate depression (RD50) models

Sensory irritation is a health endpoint that serves as the critical effect basis for many occupational exposure limits (OELs). Schaper 1993 described a significant relationship with high correlation between the measured exposure concentration producing a 50% respiratory rate decrease (RD50) in a standard rodent assay and the American Conference of Governmental Industrial Hygienists (ACGIH®) Threshold Limit Values (TLVs®) as time-weighted averages (TWAs) for airborne chemical irritants. The results demonstrated the potential use of the RD50 values for deriving full-shift TWA OELs protective of irritant responses. However, there remains a need to develop a similar predictive model for deriving workplace short-term exposure limits (STELs) for sensory irritants. The aim of our study was to establish a model capable of correlating the relationship between RD50 values and published STELs to prospectively derive short-term exposure OELs for sensory irritants. A National Toxicology Program (NTP) database that included chemicals with both an RD50 and established STELs was used to fit several linear regression models. A strong correlation between RD50s and STELs was identified, with a predictive equation of ln (STEL) (ppm) = 0.86 * ln (RD50) (ppm) − 2.42 and an R2 value of 0.75. This model supports the use of RD50s to derive STELs for chemicals without existing exposure recommendations. Further, for data-poor sensory irritants, predicted RD50 values from in silico quantitative structure activity relationship (QSAR) models can be used to derive STELs. Hence, in silico methods and statistical modeling can present a path forward for establishing reliable OELs and improving worker safety and health.

Opioid Overdose Detection in a Murine Model Using a Custom-Designed Photoplethysmography Device

Background and ObjectiveOver 68,000 opioid-overdose related deaths occurred within the United States in 2020 alone, indicating a need to develop technologies to help curb this growing epidemic. The ability to detect respiratory rate (RR) depression in real-time has the potential to decrease adverse outcomes by alerting emergency medical services or willing bystanders to an overdose event. The aim of this investigation was to design, build, and test a novel photoplethysmography (PPG)-based measurement device capable of monitoring RR and identifying respiratory depression. Materials and MethodsWe developed a novel murine model for opioid-induced respiratory depression (OIRD) to demonstrate the PPG device's capabilities. We induced respiratory depression in mice using both isoflurane and opioid-overdose and initiated recovery events with injections of naloxone while monitoring respiration via PPG and a laboratory reference system. Results and DiscussionThe device accurately identified all anesthesia-induced respiratory depression (n = 5) and OIRD events (n = 3). Our PPG-based monitor showed significant correlation with a reference respiratory measurement system (p<0.01). The bias measured across the isoflurane trials was 0.6 breaths per minute (BrPM), while the bias measured across the oxycodone trials was −1.0 BrPM, with mean absolute errors of 1.5 and 3.6 BrPM, respectively, indicating that our device was able to accurately measure RR in a murine model. ConclusionsThese preliminary experiments suggest that our device could detect OIRD and could potentially be adaptable to humans with modifications to firmware and more extensive validation in human subjects. Our present study is a proof-of-concept for detecting OIRD and alerting bystanders and health professionals in real-time.

Fentanyl-Induced Respiratory Depression and Locomotor Hyperactivity Are Mediated by μ-Opioid Receptors Expressed in Somatostatin-Negative Neurons

AbstractOpioid drugs are widely used as analgesics but cause respiratory depression, a potentially lethal side effect with overdose, by acting on μ-opioid receptors (MORs) expressed in brainstem regions involved in the control of breathing. Although many brainstem regions have been shown to regulate opioid-induced respiratory depression, the types of neurons involved have not been identified. Somatostatin is a major neuropeptide found in brainstem circuits regulating breathing, but it is unknown whether somatostatin-expressing circuits regulate respiratory depression by opioids. We examined the coexpression ofSst(gene encoding somatostatin) andOprm1(gene encoding MORs) mRNAs in brainstem regions involved in respiratory depression. Interestingly,Oprm1mRNA expression was found in the majority (&gt;50%) ofSst-expressing cells in the preBötzinger Complex, the nucleus tractus solitarius, the nucleus ambiguus, and the Kölliker–Fuse nucleus. We then compared respiratory responses to fentanyl between wild-type andOprm1full knock-out mice and found that the lack of MORs prevented respiratory rate depression from occurring. Next, using transgenic knock-out mice lacking functional MORs specifically inSst-expressing cells, we compared respiratory responses to fentanyl between control and the conditional knock-out mice. We found that respiratory rate depression by fentanyl was preserved when MORs were deleted only inSst-expressing cells. Our results show that despite coexpression ofSstandOprm1in respiratory circuits and the importance of somatostatin-expressing cells in the regulation of breathing, these cells do not mediate opioid-induced respiratory rate depression. Instead, MORs found in respiratory cell populations other thanSst-expressing cells likely contribute to the respiratory effects of fentanyl.

Tolerability and pharmacokinetics of intravenous allopregnanolone with and without midazolam pretreatment in two healthy dogs.

The neurosteroid allopregnanolone (ALLO) is under investigation as a treatment for benzodiazepine-refractory status epilepticus (SE). Here we assess the cardiopulmonary safety of intravenous ALLO by itself and after a clinically recommended dose of midazolam (MDZ) in two healthy adult beagles. Each dog received ALLO (1 mg/kg, IV), and after a washout period of 2 weeks, the dog was dosed with MDZ (0.2 mg/kg, IV) followed 10 min later by ALLO. Behavioral state, vital signs, arterial blood gasses, blood chemistries, and plasma ALLO concentrations were monitored for up to 6 h after dosing. The dogs appeared sleepy but were fully responsive after both treatments. No depression of mean arterial pressure or respiratory rate was noted. Blood gas measurements failed to show evidence of drug-induced acute respiratory acidosis. Estimated maximum plasma ALLO concentrations were in the range of 1500 to 3000 ng/mL. The results indicate that intravenous ALLO can be used safely to treat benzodiazepine-refractory SE, even when administered shortly after a benzodiazepine.

βγ G-proteins, but not regulators of G-protein signaling 4, modulate opioid-induced respiratory rate depression.

Opioid medications are the mainstay of pain management but present substantial side-effects such as respiratory depression which can be lethal with overdose. Most opioid drugs, such as fentanyl, act on opioid receptors such as the G-protein-coupled µ-opioid receptors (MOR). G-protein-coupled receptors activate pertussis toxin-sensitive G-proteins to inhibit neuronal activity. Binding of opioid ligands to MOR and subsequent activation G proteins βγ is modulated by regulator of G-protein signaling (RGS). The roles of G-proteins βγ and RGS in MOR-mediated inhibition of the respiratory network are not known. Using rodent models to pharmacologically modulate G-protein signaling, we aim to determine the roles of βγ G-proteins and RGS4. We showed that inhibition of βγ G-proteins using gallein perfused in the brainstem circuits regulating respiratory depression by opioid drugs results in complete reversal of respiratory depression. Blocking of RGS4 using CCG55014 did not change the respiratory depression induced by MOR activation despite co-expression of RGS4 and MORs in the brainstem. Our results suggest that neuronal inhibition by opioid drugs is mediated by G-proteins, but not by RGS4, which supports the concept that βγ G-proteins could be molecular targets to develop opioid overdose antidotes without the risks of re-narcotization often found with highly potent opioid drugs. On the other hand, RGS4 mediates opioid analgesia, but not respiratory depression, and RGS4 may be molecular targets to develop pain therapies without respiratory liability.

Heroin- and Fentanyl-Induced Respiratory Depression in a Rat Plethysmography Model: Potency, Tolerance, and Sex Differences.

The opioid overdose death toll in the United States is an ongoing public health crisis. We characterized the magnitude and duration of respiratory depression, the leading cause of death in opioid overdose cases, induced by heroin or fentanyl and the development of tolerance in male and female rats. We used whole-body plethysmography to first establish dose-response curves by recording breathing for 60 minutes post-intravenous opioid injection. We then tested the development of respiratory tolerance to acute heroin or fentanyl over several weeks and to chronic fentanyl with acute fentanyl or heroin challenge. Heroin and fentanyl each provoked dose-dependent respiratory depression. Heroin caused prolonged (45-60 minute) respiratory depression in female and male rats, characterized by decreased frequency, tidal volume, and minute ventilation and increased inspiratory time and apneic pause. Fentanyl produced similar changes with a shorter duration (10-15 minutes). High-dose heroin or fentanyl produced robust respiratory depression that was slightly more severe in females and, when given intermittently (acute doses 2 to 3 weeks apart), did not lead to tolerance. In contrast, chronic fentanyl delivered with an osmotic minipump resulted in tolerance to acute fentanyl and heroin, characterized by a shorter duration of respiratory depression. This effect persisted during withdrawal in males only. Our model and experimental design will allow for investigation of the neurobiology of opioid-induced respiratory depression and for testing potential therapeutics to reverse respiratory depression or stimulate breathing. SIGNIFICANCE STATEMENT: Fentanyl was more potent and had shorter duration in producing respiratory depression than heroin in both sexes, whereas female rats were more sensitive than males to heroin-induced respiratory depression. Tolerance/cross-tolerance develops in chronic fentanyl administration but is minimized with long interadministration intervals.

A Neuromodulatory System that Links Opioid‐Induced Sedation and Respiratory Depression

The main cause of death from opioid overdose is respiratory depression, mediated by mu‐opioid‐receptors located in the respiratory brainstem. Opioids also cause sedation, which further reduces respiratory drive through largely unknown mechanisms. The Kölliker‐Fuse nucleus (KF) in the pons is highly susceptible to inhibition by opioids and significantly contributes to opioid‐induced respiratory rate depression and associated changes in breathing pattern. Taking advantage of cell‐type specific retrograde neural tracing, we characterized anatomical projections between the main noradrenergic arousal center of the brain, the locus coeruleus (LC) and the KF, which represent a potential link between arousal/sedation and breathing. Our results show that 30% of noradrenergic LC neurons send projections to the KF. To begin to understand the functional role and to define the neurochemical identity of LC to KF projections, we used whole‐cell patch clamp recordings from the KF combined with optogenetic stimulation of LC neurons. Optical stimulation of LC axon terminals induces light‐evoked glutamatergic excitatory postsynaptic currents in KF neurons. Pharmacological isolation of LC to KF synapses suggests that this pathway is monosynaptic. The glutamatergic excitatory postsynaptic currents are dependent on vesicular glutamate transporter 2 expression in noradrenergic LC neurons. Additionally, prolonged optical stimulations lead to noradrenergic excitatory postsynaptic currents that are blocked by alpha1 receptor antagonist. Thus, LC may drive KF neuron activity via glutamate and/or noradrenaline release. This direct excitatory drive in the LC‐KF circuit is sensitive to opioids, which can directly inhibit KF neuron somas, as well as inhibit neurotransmitter release from presynaptic LC terminals. This work provides exciting evidence for an excitatory pathway between the LC and the KF, that may play an important link between opioid‐induced sedation and respiratory depression.

Nitazenes are potent mu‐opioid receptor agonists with profound respiratory depression

BackgroundNitazene opioids (2‐benzylbenzimidazole opioids) were first identified in multiple overdose fatalities a few years ago. It has been shown that nitazene drugs are highly active at the mu‐opioid receptor, with potency and efficacy exceeding fentanyl. Because of this, isotonitazene was added to Schedule I of the controlled substance list in August 2020 by the Drug Enforcement Agency. Further studies have shown the high potency of the N‐desethyl‐isotonitazene metabolite, which was similar in potency of isotonitazene itself. The objective of this study was to compare the pharmacokinetics with regards to respiratory depression between N‐desethyl‐isotonitazene and fentanyl in vivo rabbit model.MethodsThe study was approved by the local Animal Care Committee and conformed to NIH standards. Adult New Zealand White rabbits (3‐4kg) were anesthetized, tracheotomized, ventilated, decerebrated and vagotomized. Phrenic nerve activity was recorded from the c5 rootlet. Phrenic neural activity, respiratory rate, arterial blood pressure, and airway carbon dioxide concentration were recorded on a computerized chart recorder (Powerlab/16SP; ADInstrumentes, Australia). We determined the magnitude and time course of respiratory rate depression from 1mcg/kg N‐desethyl‐isotonitazene and fentanyl and subsequently the dose required to achieve apnea. Data were collected using LabChart data and exported to SigmaPlot 11 (Systat Software, USA) for data processing, data plotting and statistical analysis. Using the phrenic neurogram, we determined the respiratory rate and peak phrenic activity. We plotted the average respiratory rate for each minute normalized to control frequency, removed artefacts, and determined the best fitting curve for each plot. Plots were averaged for each dose and displayed as mean± standard error.ResultsFentanyl (1mcg/kg) depressed respiratory rate by 25±5% from baseline (n=3) while N‐desethyl‐isotonitazene caused a 38±2% depression (n=6). Recovery to baseline was 150±15 minutes for N‐desethyl‐isotonitazene and 46±8 minutes for fentanyl. N‐desethyl‐isotonitazene caused apnea at 4mcg/kg (n=6), and complete recovery to baseline respiratory rate was 201±33 min minutes. Fentanyl caused apnea at 8mcg/kg (n=4), and 64±7 minutes were needed to recovery.ConclusionsNitazenes are potent synthetic opioids. At equal doses, they cause more profound and longer lasting respiratory rate depression than fentanyl.

Chemogenetic activation of glutamatergic preBötzinger Complex neurons increases breathing in mice

ObjectiveRespiratory rhythm is generated by complex neural circuits in the medulla. At the core of these circuits is the preBötzinger Complex (preBötC), a collection of cells generating respiratory rhythm in the ventral part of the medulla. In addition to its role in generating respiratory rhythm, the preBötC also plays a significant role in opioid‐induced respiratory depression. The preBötC is made up of a heterogenous group of neurons, with a large portion being glutamatergic and expressing µ‐opioid receptors. A marker for these glutamatergic neurons is vesicular glutamate transporter 2 (vGLUT2). Here we propose that targeting vGLUT2‐expressing preBötC neurons using chemogenetics can stimulate respiratory rhythm and potentially alleviate opioid‐induced respiratory depression.HypothesisActivation of vGLUT2 preBötC neurons stimulates breathing and alleviates respiratory depression by fentanyl in mice.MethodsUsing a cre‐lox recombination approach, we expressed the excitatory hM3Dq (a modified version of the excitatory M3 muscarinic receptor) into vGLUT2 preBötC neurons. To this aim, we injected into the preBötC recombinant adeno‐associated virus transcriptionally activated by Cre‐recombinase containing hM3Dq and mCherry DNA flanked by lox sites in vglut2‐IRES‐Cre male mice (Jackson Labs). Four weeks after adeno‐associated virus, mice were anesthetized with isoflurane and respiratory activity was measured through electrodes implanted into the diaphragm and the genioglossus muscle. Mice were given consecutive intramuscular injections of saline and the hM3Dq ligand, clozapine‐N‐oxide (CNO; 0.5 and 1 mg/kg), while respiratory responses were monitored. Brains were collected for histological analysis with expression of hM3Dq determined by expression of mCherry.ResultsInjection of 0.5mg/kg CNO elicited a 24.55% increase in respiratory rate 8 minutes following CNO injection. Injection of 1mg/kg CNO destabilized breathing within 6‐25 minutes following injection. Histology confirmed expression of mCherry in the region of the preBötC. Further studies will determine whether stimulation of vGLUT2 preBötC neurons can prevent or reverse respiratory rate depression elicited by fentanyl.ConclusionsOur preliminary data showed that stimulation of glutamatergic preBötC neurons can increase respiratory rate in anesthetized conditions. Further studies will determine whether stimulation of glutamatergic preBötC cells also increases respiratory rate in freely‐behaving mice and whether the glutamatergic preBötC neurons are viable targets for the development of preventive drug therapies for respiratory depression during opioid overdose.

Roles of GABAergic and Glutamatergic Prebötzinger Complex Circuits in Opioid‐Induced Respiratory Depression

Introduction. Opioids are widely prescribed analgesic medication but are highly addictive and can be lethal with overdose. Respiratory depression is the primary cause of death by opioid overdose, and it limits the ability to use opioid analgesics safely and effectively. Respiratory depression by opioids is mediated by the inhibition of neural circuits in the brainstem, such as the preBötzinger Complex (preBötC), a small region of the medulla involved in respiratory rhythm generation. The generation of respiratory rhythm requires the activation of excitatory and inhibitory neurons in the preBötC. Glutamate is a main excitatory neurotransmitter, and preBötC glutamatergic neurons are involved in generating respiratory rhythm. γ‐aminobutyric acid (GABA) is a main inhibitory neurotransmitter, and GABAergic neurons are found in the preBötC. Interestingly, GABAergic neurons often co‐express µ‐opioid receptors, and disinhibition of GABAergic neurons by opioid drugs is involved in opioid addiction and euphoria. Here, we aim to determine the roles of excitatory glutamatergic and inhibitory GABAergic neurons in opioid‐induced respiratory depression.MethodsTo control glutamatergic and GABAergic neurons, we expressed opsin channels in cells expressing the vesicular glutamate transporter 2 (vGLUT2) and the vesicular GABA transporter (vGAT), respectively. Using a cre‐lox recombination approach, we injected into the preBötC cre‐dependent adeno‐associated viruses to locally express the inhibitory archaerhodopsin or the excitatory channelrhodopsin in vGLUT2 or vGAT‐expressing cells. Following a 4‐week incubation period, we applied 554 nm or 470 nm lights through optical fibres focused onto the preBötC while recording diaphragm and genioglossal muscle activities in anesthetized mice. Following systemic injection of fentanyl (5µg/kg), we inhibited or activated glutamatergic or GABAergic cells, respectively.ResultsOur preliminary data indicate that inhibition of vGLUT2 cells severely depressed respiratory rate, mimicking the respiratory depression observed with the opioid fentanyl. We are currently performing optical inhibition of vGAT preBötC cells in anesthetized mice and determining their role in respiratory depression by fentanyl.DiscussionWe will identify the role of GABAergic and glutamatergic preBötC cells in coordinating respiratory rhythm in preBötC cells and whether GABAergic cells contribute to respiratory depression by opioid drugs. Understanding the role of the populations of cells involved in respiratory depression by opioid drugs is critical to identify new molecular targets to reduce respiratory depression and provide safe and reliable pain killers without the risks associated with opioid pain killers.

Contribution of the caudal medullary raphe to opioid induced respiratory depression

BackgroundOpioid-induced respiratory depression can be partially antagonized in the preBötzinger Complex and Parabrachial Nucleus/Kölliker-Fuse Complex. We hypothesized that additional opioid antagonism in the caudal medullary raphe completely reverses the opioid effect. MethodsIn adult ventilated, vagotomized, decerebrate rabbits, we administrated remifentanil intravenously at “analgesic”, “apneic”, and “very high” doses and determined the reversal with sequential naloxone microinjections into the bilateral Parabrachial Nucleus/Kölliker-Fuse Complex, preBötzinger Complex, and caudal medullary raphe. In separate animals, we injected opioid antagonists into the raphe without intravenous remifentanil. ResultsSequential naloxone microinjections completely reversed respiratory rate depression from “analgesic” and “apneic” remifentanil, but not “very high” remifentanil concentrations. Antagonist injection into the caudal medullary raphe without remifentanil independently increased respiratory rate. ConclusionsOpioid-induced respiratory depression results from a combined effect on the respiratory rhythm generator and respiratory drive. The effect in the caudal medullary raphe is complex as we also observed local antagonism of endogenous opioid receptor activation, which has not been described before.

Pre-treatment with Tandospirone attenuates fentanyl-induced respiratory depression without affecting the analgesic effects of fentanyl in rodents

Opioid analgesics are widely used to treat acute, postoperative, and chronic pain. However, opioid receptor activation can result in severe respiratory depression. In this study, we demonstrated that Tandospirone (TS), a selective serotonin-1A receptor partial agonist, is effective against opioid-induced respiratory depression. Fentanyl was used to establish a respiratory depression model in rodents. We observed the effects of TS on respiratory depression in rats by using plethysmographic recordings and arterial oxygen saturation. In addition, we evaluated the effects of TS on fentanyl-induced sedation and analgesia by using the loss of righting reflex (LORR) and hot-plate tests, respectively. Rats (n = 5) were treated with TS or saline 5 min prior to fentanyl administration. TS [2 mg/kg, intravenous (i.v.)] dose-dependently attenuated fentanyl-induced respiratory depression versus saline + fentanyl group. Furthermore, pre-treatment with TS (2 mg/kg, i.v.) increased arterial oxygen saturation to 76.5 ± 2.0% at 5 min after fentanyl injection, compared with 35.9 ± 2.5% in saline pre-treated rats (P < 0.001), whereas the time to induction of LORR (P > 0.99) and duration of LORR (P = 0.95) did not differ between the “TS + fentanyl” and “saline + fentanyl” group. The antinociceptive effect of fentanyl was not affected by the administration of TS (P = 0.99) in mice (n = 10). In conclusion, we found that TS, a novel non-benzodiazepine anxiolytic/antidepressant drug, could attenuate severe fentanyl-induced respiratory depression and did not affect the analgesic/sedative effect of fentanyl. The clinical application of TS could significantly improve pain management.

Opioids and obstructive sleep apnea.

Freire C, Sennes LU, Polotsky VY. Opioids and obstructive sleep apnea. J Clin Sleep Med. 2022;18(2):647-652.

The use of hypercapnic conditions to assess opioid-induced respiratory depression in rats

IntroductionWhole-body plethysmography (WBP) in unrestrained, non-anesthetized rodents is a preclinical method to assess the respiratory depressant effects of opioids, the leading cause of opioid overdose death in humans. However, low baseline respiration rates under normocapnic conditions (i.e., “floor” effect) can render the measurement of respiratory decreases challenging. We assessed hypercapnia-induced increases in respiration as a strategy to assess opioid-induced decreases in respiration in rats. MethodsWBP was used to assess respiration frequency, tidal volume and minute volume in the presence of normocapnic and hypercapnic (8% CO2) conditions in rats during the rat diurnal period of the light cycle. The mu-opioid receptor agonist fentanyl was administered intravenously, and the hot plate test was used to assess acute antinociception. Results and discussionHypercapnia-induced increases in respiratory parameters (frequency, minute volume, and tidal volume) were decreased by fentanyl at doses that did not decrease the same parameters under the normocapnic conditions. These findings show that hypercapnia increases sensitivity to respiratory depressant effects of fentanyl, as compared with assessments during the rat diurnal period when activity and breathing rate are generally low, i.e., there is a floor effect. The current approach is highly sensitive to opioid-induced respiratory depression, and therefore provides a useful method for assessment in a pre-clinical setting.

Naloxone Injections into the Parabrachial Nucleus/ Kölliker‐Fuse Complex, the preBötzinger Complex and the Caudal Medullary Raphe Reverse Remifentanil‐Induced Respiratory Depression

Background We have shown that naloxone microinjections into the Parabrachial Nucleus/ Kölliker-Fuse (PBN/KF) complex and the preBötzinger Complex (preBC) partially reversed remifentanil-induced respiratory rate depression (1). The magnitude of reversal was dose dependent. The goal of this study was to determine whether additional naloxone injection into the caudal medullary raphe (CMR (2)) could completely reverse the opioid effect. Methods The study was approved by the local Animal Care Committee and conformed to NIH standards. Adult New Zealand White rabbits (3-4kg) were anesthetized, tracheotomized, ventilated, decerebrated and vagotomized. Phrenic nerve activity was recorded from the c5 rootlet, time averaged and used to calculate inspiratory and expiratory duration and peak phrenic activity (PPA). Gridwise localized pressure microinjections of AMPA (50 μM, 70 nl) with a multibarrel glass pipette were used to functionally identify the PBN/KF and preBC. The CMR was defined as the area between 2.0mm caudal and 1.5mm rostral to preBC in the midline. We then injected an “apneic” remifentanil IV bolus (12±2mcg), which interrupted inspiratory activity for at least 60 sec. Next, a remifentanil infusion was started at an “analgesic” dose-rate, defined to achieve respiratory rate depression of 50% of control. At steady-state, we sequentially injected naloxone (1mM, 700nl) into the bilateral PBN/KF, the preBC, and the CMR. We repeated the “apneic” remifentanil bolus after naloxone injection into each area. To determine whether injection order affected the naloxone reversal, in separate sets of animals we microinjected naloxone in the reverse order, or solely into the preBC and CMR. Drug effects were compared with Paired t-tests. Results Figure 1 illustrates the effects of sequential naloxone microinjection into the bilateral PBN/KF, preBC, and CMR. At the end of the naloxone injection sequence, respiratory rate depression was completely reversed (A+B) or even increased above baseline (C). Depression of PPA was completely reversed (p=0.756). Conclusion Naloxone injection into the CMR reversed remifentanil-induced respiratory depression in addition to the reversal achieved in the PBN/KF and preBC at “analgesic” and “apneic” remifentanil doses. Further research is needed to determine whether the naloxone effect in the CMR was partially due to reversal of endogenous opioid-receptor activation (see Palkovic B, Endogenous Opioid-Receptor Activation in the Caudal Medullary Raphe Depresses Respiratory Rate in Decerebrate Rabbits, EB 2021). (1) Palkovic et al., Anesthesiology 2020, A3038 (2) Zhang et al., Anesthesiology 2007, 107: 288-97

The ability of fentanyls and other opioids to increase EMG amplitude in respiratory muscles correlates with their agonist efficacy

Acute overdose deaths involving fentanyls have been dramatically increasing in the USA since 2013. These potent opioids depress respiratory drive and produce rigidity in the intercostal muscles and diaphragm (resulting in what is referred to as Wooden Chest Syndrome) as well as in glottic and supraglottic muscles which further reduces the ability to breathe. In this study we have sought to characterise the ability of various opioid agonists to induce respiratory muscle rigidity using electromyographic (EMG) recording. EMG amplitude was recorded simultaneously from diaphragm, external and internal intercostal muscles of P21 male Wistar rats, using an in situ decerebrated and arterially perfused preparation. First, the effects of fentanyl and morphine on respiratory muscle EMG were studied using a dose of each agonist that depressed respiratory rate by approximately 40%. Fentanyl produced a marked increase in EMG amplitude during expiration in all three respiratory muscles whereas the effect of morphine was very slight. In order to understand whether the difference between fentanyl and morphine related to differences in chemical structure, lipid solubility or agonist intrinsic efficacy we studied the effects of a range of opioids that differed in these characteristics – carfentanil and fentanyl (piperidine derivatives, high lipid solubility, high efficacy), alfentanil (piperidine derivative, moderate lipid solubility, high efficacy) norbuprenorphine (thebaine derivative, high lipid solubility, high efficacy), levorphanol (morphinan, high lipid solubility, low efficacy), morphine and hydromorphone (morphine derivatives, low lipid solubility, low efficacy). With doses of each that produced around 40% depression of respiratory rate we observed that the rank order of ability to increase internal and external intercostal muscle EMG amplitude was carfentanil = fentanyl = norbuprenorphine > alfentanil = hydromorphone > morphine = levorphanol but for the diaphragm the rank order was fentanyl > alfentanil = norbuprenorphine ≥ hydromorphone > carfentanil ≥ morphine = levorphanol. These results suggest that the ability of opioid agonists to increase respiratory muscle EMG amplitude and therefore respiratory muscle rigidity does not depend solely on either chemical structure or lipid solubility of the agonist. For the intercostal muscles there is a better correlation with agonist efficacy. Why the effect of carfentanil on the diaphragm is weak remains to be elucidated.

Optogenetic activation of preBötzinger Complex cells alleviates respiratory depression by opioids

Rationale Opioids are extensively used for their analgesic properties but present a variety of unwanted side effects, including tolerance, dependence and respiratory depression. The analgesic effect of opioids is due to activation of µ-opioid receptors (MOR) in the central nervous system and no treatments are currently available to prevent respiratory depression without reducing their analgesic properties. The neural circuits and mechanisms regulating respiratory depression, sedation, and analgesia by opioids often overlap, therefore making challenging the identification of the mechanisms regulating respiratory depression. Neurons expressing tachykinin precursor 1 peptide (TAC1) located in the preBötzinger Complex (preBötC) also co-express neurokinin-1 receptors (NK1R) and MORs. NK1R neurons are preferentially inhibited by opioids and play an essential role in mediating opioid-induced respiratory depression. Objective Here, we tested the hypothesis that optogenetic activation of TAC1-expressing preBötC cells will prevent or reverse respiratory rate depression by opioids. Methods Using a Cre-loxP recombination approach, we injected, in TAC1 Cre recombinase mice the adeno-associated virus containing the gene cassette of the excitatory channelrhodopsin-2 ChETA flanked between loxP sites. After a four weeks period to allow ChETA expression in targeted cells, a 200 µm optical fiber was positioned above the preBötC for laser stimulation with blue light (wavelength: 480 nm). Respiratory rate was measured in anesthetized mice with electromyographic recordings of thediaphragm and genioglossus muscles activity. Once breathing was stable (>20 min), the clinically-relevant µ-opioid drug fentanyl (1mg/kg) was administered by intramuscular injection. About 5 minutes after fentanyl injection, targeted preBötC cells were stimulated using laser stimulations (frequency: 30Hz, duration of stimulation: 330ms). Results Data show that fentanyl depressed respiratory rate by about 40% in 5 minutes and stimulation of TAC1-expressing preBötC cells increased breathing rate back to baseline level when the laser was on; an effect that was reversible. Conclusion TAC1-expressing preBötC may constitute cell targets to prevent respiratory rate depression by opioids. These results may help identify the cells mediating respiratory depression by opioids, an essential step toward the development of therapeutic targets to reduce the risk of opioids overdose and associated mortality.

Endogenous Opioid Receptor Activation in the Caudal Medullary Raphe Depresses Respiratory Rate in Decerebrate Rabbits

Background The caudal medullary raphe (CMR) is an essential structure in controlling pain, cardiovascular, and respiratory function (1). Partial reversal of systemic opioid-induced respiratory depression was achieved with naloxone injection into the CMR (2). This study investigated the presence of endogenous opioid activity in the CMR. Methods The study was approved by the local Animal Care Committee and conformed to NIH standards. Adult New Zealand White rabbits (3-4kg) were anesthetized, tracheotomized, ventilated (FiO2 0.6, PCO2 45-55 mmHg), decerebrated and vagotomized. Phrenic nerve activity was recorded from the c5 rootlet, time averaged and used to calculate inspiratory (TI) and expiratory (TE) duration, breaths per minute (BPM), and peak phrenic activity (PPA). Using a multibarrel glass micropipette, we performed the following injection protocols into the CMR in the midline between 2 mm caudal and 1.5 mm rostral to the functionally identified preBötzinger Complex (preBC, (3)) and into the middle medullary raphe (middle MR (2), 2.5-3.5mm rostral to the preBC) in separate sets of animals: i) α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 50 μM, 70 nl), stepsize 0.5mm rostro-caudal, 0.5mm dorso-ventral. ii) The non-selective opioid antagonist naloxone (1-5 mM, 700 nl), stepsize 0.5mm rostro-caudal, 1st injection at the ventral edge of neuronal activity, 2nd injection 1.5-2mm dorsal; and iii) the µ-opioid antagonist CTAP and δ-opioid antagonist naltrindole (each 1 mM, 350-700 nl), stepsize same as ii). Statistical comparison with Wilcoxon signed rank test. Median (25-75% range). Results i) In 6 animals, AMPA injection into the CMR caudal to and at the level of the preBC increased respiratory rate by up to 59% and PPA by up to 38% at multiple dorso-ventral levels. This was due to a decrease in TI and TE. In the middle MR, AMPA injection depressed respiratory rate, at times to apnea, through an increase in TI and TE, and also depressed PPA. ii) Naloxone injection into the CMR increased respiratory rate from 30 (23-34) to 37 (30-6) BPM (p=0.031, n=6) through a decrease in TI from 0.9 (0.8-1.0) to 0.7 (0.6-0.8) sec (p=0.031) and TE from 1.2 (1.0-1.6) to 0.9 (0.7-1.2) sec (p=0.031). We observed effects with injections caudal to, at the level of and rostral to the preBC. iii) CTAP injection increased respiratory rate from 26 (22-27) to 34 (29-34) BPM, and subsequent naltrindole injection increased respiratory rate to 43 (37-44) BPM (n=3). In the middle MR, CTAP + naltrindole increased respiratory rate between 2-5 BPM. Conclusion Excitation of the CMR increased respiratory rate over an area of several millimeters. During quiet breathing under moderate hyperoxic hypercapnia in our decerebrate rabbit preparation, respiratory raphe neurons were depressed by endogenous opioid activity. It will be important to further investigate the role of the CMR in opioid-induced respiratory depression. (1) Holtman et al., J Neurosci 1986, 6: 1185-93 (2) Zhang et al., Anesthesiology 2007, 107: 288-97 (3) Cook-Snyder et al., Resp Physiol Neurobiol 2019, 260: 37-52

The Role of Somatostatin‐Expressing Cells in Opioid‐Induced Respiratory Depression

Introduction Opioids have the ability to cause respiratory depression, a severe decline in respiratory rate that can be lethal with overdose. While key brainstem respiratory regions have been shown to play a role in respiratory depression by opioids, the cells involved remain unknown. One region of interest is the preBötzinger Complex, a site that mediates respiratory depression and contains a subgroup of somatostatin (SST)-expressing cells that are required to maintain normal breathing. We aimed to determine the role of SST-expressing cells in respiratory depression by opioids. We hypothesized that deletion of mu-opioid receptors (MORs) in SST-expressing cells will prevent opioid-induced respiratory depression. Methods We developed transgenic knockout mice that lack MORs in SST-expressing cells (SST-MOR-/-) using Cre-lox recombination. SST-Cre+/+ mice (Ssttm2.1(cre)Zjh/J) were bred with Oprm1fl/fl mice (Oprm1tm1.1Cgrf/KffJ) to produce SST-MOR-/- mice. We used in situ hybridization to determine whether MOR mRNAs are co-expressed with SST mRNA in the brainstem of control (wild-type, SST-MOR+/+) and SST-MOR-/- mice. To determine the effect of removing MORs from SST-expressing cells, respiratory depression by intraperitoneal injection of fentanyl (0.3mg/kg) was quantified in control (wild-type, SST-MOR+/+) and SST-MOR-/- mice. Respiratory rate was recorded using whole-body plethysmography. Results MORs were expressed in several brainstem regions regulating breathing such as the preBötzinger Complex, the Bötzinger Complex, the nucleus ambiguus, the Kölliker-Fuse nucleus, the locus coeruleus and the nucleus tractus solitarius, while SST and MORs were co-expressed primarily in the preBötzinger Complex. Knockout of MORs in SST-expressing cells was confirmed by microscopy. Intraperitoneal injection of fentanyl (0.3mg/kg) induced a similar, significant respiratory rate depression in both wild-type (p<0.001, n=14) and SST-MOR-/- (p<0.001, n=12) mice. Conclusions SST and MORs are co-expressed in the preBötzinger Complex while MORs are expressed in other key brainstem respiratory sites. Preliminary data suggests that MORs in SST-expressing cells are not required for fentanyl-induced respiratory depression. Our previous work showed that arousal state can affect the severity of respiratory depression by opioids. For this reason, we are currently investigating the impact of arousal state (active versus inactive) on opioid-induced respiratory depression in control and SST-MOR-/- mice. We are also looking at the role of SST-expressing cells in respiratory depression by other opioid drugs such as morphine.

Investigating Opioid‐induced Respiratory Depression and Analgesia Using Larval Zebrafish

Introduction Opioid drugs produce both analgesia and severe respiratory depression which can be lethal. In 2019, over 50,000 North Americans died of an opioid overdose. Our understanding on how to differentiate between these two effects is limited due to a lack of simple animal models. Therefore, we aim to understand respiratory depression and analgesia by opioids using novel zebrafish models, allowing for drug screening. Using these models, we tested a variety of drugs including serotoninergic agonists, glutamatergic modulators, and calcium channel activators, for their ability to reverse opioid-induced respiratory depression. Methods We have established phenotype-based approaches using in-vivo zebrafish models of respiratory depression and analgesia by fentanyl. To quantify respiratory depression, we measured respiratory activity by quantifying mandible movements in 12-14 day post-fertilization larvae. To quantify analgesia, we measured the escape swimming response to nociceptive stimuli such as formalin or allyl isothiocyanate (AITC) combined with fentanyl to induce analgesia. We then introduced various candidate drugs to measure their effects on respiratory depression and analgesia. Results Respiratory rate was normalized to each fish's baseline (baseline=100%). Zebrafish strains were found to be differentially sensitive to opioids, where Tübingen (TU) fish did not respond to fentanyl (1µM, p=0.4240, n=9) and AB fish showed significant respiratory rate depression (p<0.001, n=11). AB and TU crosses did not show respiratory depression (n=7). In AB zebrafish, a dose-dependent decrease in respiratory rate was observed with 1μM and 3µM fentanyl (p=0.01, n=11 and p<0.001, n=27, respectively). The opioid antagonist naloxone (20µM) and selective antagonist CTAP (4μM) reversed respiratory depression by fentanyl (p<0.001, n=9 and p=0.049, n=11, respectively). Respiratory depression by 1μM fentanyl was reversed by the AMPA receptor positive allosteric modulator CX614 (5μM, p<0.001, n=9) but not the serotoninergic 5-HT4 agonist BIMU8 (10μM, n=21). Calcium channel activators nefiracetam (1µM) and FLP-64176 (1µM) reversed respiratory depression by fentanyl (1µM, p=0.005, n=11 and3µM, p<0.001, n=15, respectively). Formalin (0.075%) and AITC (100µM) significantly increased swimming velocity (p=0.005, n=21 and p<0.001, n=23, respectively), which was reduced by fentanyl (4μM, p<0.001, n=12and 6µM, p=0.004, n=14, respectively). FLP-64176 reversed fentanyl analgesia when administered with formalin (p<0.001, n=10). Discussion: Our models show that respiratory depression and analgesia by opioids can be measured in zebrafish larvae. The strain differences point to a potential genetic protection which we are investigating using quantitative real-time polymerase chain reaction (qPCR). Our novel zebrafish models can be used to investigate the effects of opioids, as well as for drug screening to find ways to treat respiratory depression while preserving analgesia.