An ice core of 15.5 m retrieved from Agassiz Ice Cap (Nunavut, Canada) in April 2009 was analyzed for perchlorate to obtain a temporal trend in the recent decades and to better understand the factors affecting High Arctic deposition. The continuous record dated from 1936 to 2007, covers the periods prior to and during the major atmospheric releases of organic chlorine species that affected the stratospheric ozone levels. Concentrations and yearly fluxes of perchlorate and chloride showed a significant correlation for the 1940-1959 period, suggesting a predominant tropospheric formation by lightning. While concentration of chloride remained unchanged from 1940s until 2009, elevated levels of perchlorate were observed after 1979. A lack of significant increases in either sulfate or chloride between 1980 and 2001 suggests that the effect of volcanic activities on the perchlorate at the study site during this period could be insignificant. Therefore, the elevated perchlorate in the ice could most likely be attributed to anthropogenic activities that influenced perchlorate sources and formation mechanisms after 1979. Our results show that anthropogenic contribution could be responsible for 66% of perchlorate found in the ice. Although with some differences in trends and amounts, deposition rate found in this study is similar to those observed at Devon Island (Nunavut, Canada), Eclipse Icefield (Yukon, Canada) and Summit Station (Greenland). Methyl chloroform, a chlorinated solvent largely used after 1976, peaked in the atmosphere in 1990 and has a much shorter atmospheric life than chlorofluorocarbons (CFCs). This study proposes methyl chloroform (CHCCl) as the significant anthropogenic source of perchlorate in the Canadian High Arctic between 1980 and 2000, with HCFC-141b (ClFC-CH), a relatively short-lived CFC probably responsible for a slower decrease in perchlorate deposition after the late 1990s. The presence of aerosols in the stratosphere appears to suppress perchlorate production after 1974. As both methyl chloroform and HCFC-141b had no new significant emissions after 2003, deposition of perchlorate in High Arctic is expected to remain at pre-1980 levels.

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.

Opioid 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 co-expression ofSst(gene encoding somatostatin) andOprm1(gene encoding MORs) mRNAs in brainstem regions involved in respiratory depression. Interestingly,Oprm1mRNA expression was found in the majority (> 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 knockout mice and found that the lack of MORs prevented respiratory rate depression from occurring. Next, using transgenic knockout mice lacking functional MORs specifically inSst-expressing cells, we compared respiratory responses to fentanyl between control and the conditional knockout mice. We found that respiratory rate depression by fentanyl was preserved when MORs were deleted only inSst-expressing cells. Our results show that despite co-expression 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.Significance statementOpioid drugs cause respiratory depression, a potentially lethal side-effect with overdose, by acting on µ-opioid receptors in brainstem regions regulating breathing, therefore limiting their effective use as analgesics. Somatostatin is a major neuropeptide found within these brainstem circuits, but it is unknown whether somatostatin circuits regulate respiratory depression by opioids. We found that somatostatin-expressing neurons co-express µ-opioid receptors in respiratory circuits but that respiratory rate depression by fentanyl was preserved despite genetic deletion of µ-opioid receptors in somatostatin-expressing cells. Our results suggest that somatostatin-expressing cells are resistant to the rate-depressive effects of opioids and that other cells contribute to the effects of fentanyl on breathing. Somatostatin-expressing cells may constitute a cell population that can be targeted to stimulate breathing when it fails with opioids.

Rhythmic breathing is generated by neural circuits located in the brainstem. At its core is the preBötzinger Complex (preBötC), a region of the medulla, necessary for the generation of rhythmic breathing in mammals. The preBötC is comprised of various neuronal populations expressing neurokinin-1 receptors, the cognate G-protein-coupled receptor of the neuropeptide substance P (encoded by the tachykinin precursor 1 orTac1). Neurokinin-1 receptors are highly expressed in the preBötC and destruction or deletion of neurokinin-1 receptor-expressing preBötC neurons severely impairs rhythmic breathing. Application of substance P to the preBötC stimulates breathing in rodents, however substance P is often associated with nociception and locomotion in various brain regions, suggesting thatTac1neurons found in the preBötC may have diverse functional roles. Here, we aim to characterize the role ofTac1-expressing preBötC neurons in the generation of rhythmic breathingin vivo, as well as motor behaviors. Using a cre-lox recombination approach, we injected adeno-associated virus containing the excitatory channelrhodopsin-2 ChETA in the preBötC region ofTac1-cre mice. Using a combination of histological, optogenetics, respiratory, and behavioral assays, we defined the identity and the role ofTac1preBötC neurons. These neurons are glutamatergic and their stimulation promotes rhythmic breathing in both anesthetized and freely moving/awake animals, but also triggers locomotion and overcomes respiratory depression by opioid drugs. Overall, our study identifies a new population of excitatory preBötC with major role in rhythmic breathing and behaviors.

Rhythmic breathing is generated by neural circuits located in the brainstem. At its core is the preBötzinger Complex (preBötC), a region of the medulla, necessary for the generation of rhythmic breathing in mammals. The preBötC is comprised of various neuronal populations expressing neurokinin-1 receptors, the cognate G-protein-coupled receptor of the neuropeptide substance P (encoded by the tachykinin precursor 1 or Tac1). Neurokinin-1 receptors are highly expressed in the preBötC and destruction or deletion of neurokinin-1 receptor-expressing preBötC neurons severely impairs rhythmic breathing. Although application of substance P to the preBötC stimulates breathing in rodents, substance P is also involved in nociception and locomotion in various brain regions, suggesting that Tac1 neurons found in the preBötC may have diverse functional roles. Here, we characterized the role of Tac1-expressing preBötC neurons in the generation of rhythmic breathing in vivo, as well as motor behaviors. Using a cre‑lox recombination approach, we injected adeno-associated virus containing the excitatory channelrhodopsin-2 ChETA in the preBötC region of Tac1-cre mice. Employing a combination of histological, optogenetics, respiratory, and behavioral assays, we showed that stimulation of glutamatergic or Tac1 preBötC neurons promoted rhythmic breathing in both anesthetized and freely moving animals, but also triggered locomotion and overcame respiratory depression by opioid drugs. Overall, our study identified a population of excitatory preBötC with major roles in rhythmic breathing and behaviors.