Calcium imagining and deep behavior analysis in fentanyl vapor self-administration and relapse

The prevalence of opioid use disorder (OUD) and overdose deaths have reached epidemic proportions and constitute a global crisis. In 2019 synthetic opioids, including fentanyl, were being used by 1.2% of the worldwide population and contributed to more than 70% of the record-breaking number of overdose deaths. Fentanyl, which is often used clinically for anesthesia and analgesia, is commonly administered intravenously or by inhalation (smoking/vaping), which results in rapid drug bioavailability in the brain. Technical challenges have contributed greatly to our lack of understanding of the neurobiology of OUD, including limitations of behavioral models, difficulty tracking individual neurons longitudinally in freely behaving animals, and inadequate behavioral analysis tools. Intravenous drug self-administration is considered the “gold standard” model to investigate the neurobiology of OUD preclinically, but it remains difficult to perform in vivo electrophysiology or calcium imaging during drug self-administration due to the tangling of drug catheter and recording cable. This technical challenge was overcome with the development of a noninvasive mouse model of opioid self-administration using vaporized fentanyl that recapitulates key features of OUD. Imaging freely behaving animals is difficult, and conventional single-unit recordings can neither distinguish neuron subtypes nor track individual neurons longitudinally. In contrast, in vivo imaging using miniaturized fluorescence microscope (miniscope) systems allows for examining spatially and temporally coordinated activity in hundreds of individual neurons longitudinally in freely behaving animals. Complex behavioral analysis is infrequently incorporated in preclinical models, which likely contributes to limited translational impact. Recent computational advances in convolutional neural networks, pose estimation, and machine learning analysis has overcome these challenges to provide tools for computational neuroethology. We are leveraging these cutting-edge imaging technologies and behavioral analysis tools to gain a deeper insight into the neuronal ensembles that encode opioid-related behaviors during fentanyl self-administration and relapse

Chandra X-Ray Observatory Observations of the Globular Cluster M71

We observed the nearby, low-density globular cluster M71 (NGC 6838) with the Chandra X-ray Observatory to study its faint X-ray populations. Five X-ray sources were found inside the cluster core radius, including the known eclipsing binary millisecond pulsar (MSP) PSR J1953+1846A. The X-ray light curve of the source coincident with this MSP shows marginal evidence for periodicity at the binary period of 4.2 h. Its hard X-ray spectrum and luminosity resemble those of other eclipsing binary MSPs in 47 Tuc, suggesting a similar shock origin of the X-ray emission. A further 24 X-ray sources were found within the half-mass radius, reaching to a limiting luminosity of 1.5 10^30 erg/s (0.3-8 keV). From a radial distribution analysis, we find that 18+/-6 of these 29 sources are associated with M71, somewhat more than predicted, and that 11+/-6 are background sources, both galactic and extragalactic. M71 appears to have more X-ray sources between L_X=10^30--10^31 erg/s than expected by extrapolating from other studied clusters using either mass or collision frequency. We explore the spectra and variability of these sources, and describe the results of ground-based optical counterpart searches.Comment: 36 pages including 7 figures and 8 tables, accepted by The Astrophysical Journa

Coronaridine congeners attenuate fentanyl seeking during prolonged abstinence

Background: The prevalence of opioid use disorder (OUD) has reached epidemic proportions with a record-breaking number of overdose deaths. Over 70% of the record-breaking number of overdose deaths are caused by synthetic opioids, including fentanyl. Fentanyl is commonly administered intravenously or by inhalation (smoking/vaping), which results in rapid drug bioavailability in the brain. There is a current need to identify a novel pharmacologic therapy to treat OUD, and there is increasing evidence to support the use of novel compounds referred to as coronaridine congeners to treat OUD and other psychiatric illnesses. In preclinical models, coronaridine congeners have been shown to decrease self-administration of drugs of abuse and induce antidepressant and anxiolytic effects. Here we used a preclinical fentanyl vapor self-administration model and fast-scan cyclic voltammetry (FSCV) to study the anti-addictive effects of two coronaridine congeners, 18-methoxycoronaridine (18-MC) and catharanthine (Cath).Methods: C57BL/6J mice were trained to self-administer vaporized fentanyl (5 mg/mL) or vehicle in airtight operant chambers. Mice self-administered vapor for 1 hour per day for 10 days (sessions were conducted for 5 consecutive days, followed by 2 days off). Chambers were equipped with two nosepokes, one active and one inactive. A successful response in the active nosepoke resulted in a vapor delivery that coincided with the presentation of a cue light, followed by a 1-minute timeout period. Mice learned to self-administer vapor with 3-second vapor deliveries for the first 3 days of training, which was then reduced to 1.5-second vapor deliveries the remaining 7 days. After training, mice were returned to their home cages for a forced abstinence period. Cue-induced drug seeking tests were conducted on abstinence days (AD) 20 and 25. During cue-induced seeking tests, successful responses in the active nosepoke resulted in presentation of the drug-associated cue, but no vapor was delivered (i.e. extinction conditions). Cue-induced drug seeking tests were conducted using a crossover design where half of subjects received coronaridine treatment (18-MC or Cath), while the other half received vehicle (ddH2O), on AD20. On AD25, subjects received the opposite treatment compared to AD20. Mice were injected (i.p.) with either vehicle or coronaridine treatment 1 hour before seeking tests. To examine the molecular mechanism of coronaridine congeners, FSCV was conducted on dopaminergic pre-synaptic terminals in the nucleus accumbens (NAc) neurons to measure dopamine (DA) release in the presence of 18-MC and Cath with or without nicotinic acetylcholine receptor antagonists.Results: We found that both 18-MC and Cath significantly reduced fentanylseeking during prolonged abstinence with no effect on mice that had previously self-administered vehicle. Furthermore, FSCV revealed that 18-MC and Cath significantly reduced DA release onto NAc neurons.Conclusion: In this study, we report that both 18-MC and Cath decrease fentanyl seeking during prolonged abstinence. DA release is important for opioid-related behaviors, and we found that 18-MC and Cath reduce DA release in the NAc, a mechanism that may underlie the effect of coronaridine congeners on fentanyl seeking. Together, these results provide evidence that coronaridine congeners may be promising novel compounds for the pharmacotherapeutic treatment of OUD

The XMM Cluster Survey: X-ray analysis methodology

The XMM Cluster Survey (XCS) is a serendipitous search for galaxy clusters using all publicly available data in the XMM-Newton Science Archive. Its main aims are to measure cosmological parameters and trace the evolution of X-ray scaling relations. In this paper we describe the data processing methodology applied to the 5,776 XMM observations used to construct the current XCS source catalogue. A total of 3,675 > 4-sigma cluster candidates with > 50 background-subtracted X-ray counts are extracted from a total non-overlapping area suitable for cluster searching of 410 deg^2. Of these, 993 candidates are detected with > 300 background-subtracted X-ray photon counts, and we demonstrate that robust temperature measurements can be obtained down to this count limit. We describe in detail the automated pipelines used to perform the spectral and surface brightness fitting for these candidates, as well as to estimate redshifts from the X-ray data alone. A total of 587 (122) X-ray temperatures to a typical accuracy of < 40 (< 10) per cent have been measured to date. We also present the methodology adopted for determining the selection function of the survey, and show that the extended source detection algorithm is robust to a range of cluster morphologies by inserting mock clusters derived from hydrodynamical simulations into real XMM images. These tests show that the simple isothermal beta-profiles is sufficient to capture the essential details of the cluster population detected in the archival XMM observations. The redshift follow-up of the XCS cluster sample is presented in a companion paper, together with a first data release of 503 optically-confirmed clusters.Comment: MNRAS accepted, 45 pages, 38 figures. Our companion paper describing our optical analysis methodology and presenting a first set of confirmed clusters has now been submitted to MNRA

Defensive properties of pyrrolizidine alkaloids against microorganisms

The understanding of the selection factors that drive chemical diversification of secondary metabolites of constitutive defence systems in plants, such as pyrrolizidine alkaloids (PAs), is still incomplete. Historically, plants always have been confronted with microorganisms. Long before herbivores existed on this planet, plants had to cope with microbial pathogens. Therefore, plant pathogenic microorganisms may have played an important role in the early evolution of the secondary metabolite diversity. In this review, we discuss the impact that plant-produced PAs have on plant-associated microorganisms. The objective of the review is to present the current knowledge on PAs with respect to anti-microbial activities, adaptation and detoxification by microorganisms, pathogenic fungi, root protection and PA induction. Many in vitro experiments showed effects of PAs on microorganisms. These results point to the potential of microorganisms to be important for the evolution of PAs. However, only a few in vivo studies have been published and support the results of the in vitro studies. In conclusion, the topics pointed out in this review need further exploration by carrying out ecological experiments and field studies

Metal A and Metal B Sites of Nuclear RNA Polymerases Pol IV and Pol V Are Required for siRNA-Dependent DNA Methylation and Gene Silencing

Plants are unique among eukaryotes in having five multi-subunit nuclear RNA polymerases: the ubiquitous RNA polymerases I, II and III plus two plant-specific activities, nuclear RNA polymerases IV and V (previously known as Polymerases IVa and IVb). Pol IV and Pol V are not required for viability but play non-redundant roles in small interfering RNA (siRNA)-mediated pathways, including a pathway that silences retrotransposons and endogenous repeats via siRNA-directed DNA methylation. RNA polymerase activity has not been demonstrated for Polymerases IV or V in vitro, making it unclear whether they are catalytically active enzymes. Their largest and second-largest subunit sequences have diverged considerably from Pol I, II and III in the vicinity of the catalytic center, yet retain the invariant Metal A and Metal B amino acid motifs that bind magnesium ions essential for RNA polymerization. By using site-directed mutagenesis in conjunction with in vivo functional assays, we show that the Metal A and Metal B motifs of Polymerases IV and V are essential for siRNA production, siRNA-directed DNA methylation, retrotransposon silencing, and the punctate nuclear localization patterns typical of both polymerases. Collectively, these data show that the minimal core sequences of polymerase active sites, the Metal A and B sites, are essential for Pol IV and Pol V biological functions, implying that both are catalytically active