Evaluation and comparison of pharmacokinetic profiles and safety of two extended-release buprenorphine formulations in common marmosets (Callithrix jacchus).

While sustained-release buprenorphine (BSR) is used as a long-lasting opioid analgesic in common marmosets (Callithrix jacchus), there are no published studies on pharmaceutical-grade extended-release buprenorphine options such as Ethiqa XR (EXR) for this species. However, BSR is a compounded product and has been reported to cause injection site reactions in multiple species, including marmosets. Additionally, now with the availability of EXR, a pharmaceutical-grade veterinary product, the use of BSR in laboratory animals is not compliant with the Guide for the Care and Use of Laboratory Animals (Guide) unless scientifically justified and approved by the IACUC. We compared pharmacokinetic and safety profiles of BSR (0.15 mg/kg) and EXR (0.1-0.2 mg/kg) administered subcutaneously to adult marmosets. Blood was collected by venipuncture of the saphenous vein at multiple time points (0.25-72 h) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). EXR between 0.1 and 0.2 mg/kg resulted in a dose-dependent increase in Cmax (1.43-2.51 ng/mL) and were not statistically different from BSR (1.82 ng/mL). Tmax, lambdaz, and t1/2 were not statistically different between formulations. Mean plasma buprenorphine concentrations for BSR and EXR exceeded the therapeutic threshold (0.1 ng/mL) within 0.25 h and lasted for > 72 h. Mild sedation, but neither respiratory depression nor ataxia, was observed for both formulations. BSR injection sites had significantly higher histopathological scores compared to EXR. Video recordings for monitoring drug-induced behavioral changes showed increased animal activity levels after BSR and EXR versus saline controls. Norbuprenorphine, a buprenorphine metabolite associated with respiratory depression, was detected in the plasma after BSR and EXR administration as well as by in vitro liver microsome assays. In conclusion, we recommend using EXR over BSR as a long-lasting buprenorphine analgesic in marmosets because EXR is a pharmaceutical-grade formulation that is compliant with FDA guidelines and the Guide as well as exhibits comparable PK and safety profiles as BSR.

Multi-animal pose estimation, identification and tracking with DeepLabCut.

Estimating the pose of multiple animals is a challenging computer vision problem: frequent interactions cause occlusions and complicate the association of detected keypoints to the correct individuals, as well as having highly similar looking animals that interact more closely than in typical multi-human scenarios. To take up this challenge, we build on DeepLabCut, an open-source pose estimation toolbox, and provide high-performance animal assembly and tracking-features required for multi-animal scenarios. Furthermore, we integrate the ability to predict an animal's identity to assist tracking (in case of occlusions). We illustrate the power of this framework with four datasets varying in complexity, which we release to serve as a benchmark for future algorithm development.

Atypical behaviour and connectivity in SHANK3-mutant macaques.

Mutation or disruption of the SH3 and ankyrin repeat domains 3 (SHANK3) gene represents a highly penetrant, monogenic risk factor for autism spectrum disorder, and is a cause of Phelan-McDermid syndrome. Recent advances in gene editing have enabled the creation of genetically engineered non-human-primate models, which might better approximate the behavioural and neural phenotypes of autism spectrum disorder than do rodent models, and may lead to more effective treatments. Here we report CRISPR-Cas9-mediated generation of germline-transmissible mutations of SHANK3 in cynomolgus macaques (Macaca fascicularis) and their F1 offspring. Genotyping of somatic cells as well as brain biopsies confirmed mutations in the SHANK3 gene and reduced levels of SHANK3 protein in these macaques. Analysis of data from functional magnetic resonance imaging revealed altered local and global connectivity patterns that were indicative of circuit abnormalities. The founder mutants exhibited sleep disturbances, motor deficits and increased repetitive behaviours, as well as social and learning impairments. Together, these results parallel some aspects of the dysfunctions in the SHANK3 gene and circuits, as well as the behavioural phenotypes, that characterize autism spectrum disorder and Phelan-McDermid syndrome.

Planar-Polarized Semaphorin-5c and Plexin A Promote the Collective Migration of Epithelial Cells in Drosophila.

Collective migration of epithelial cells is essential for morphogenesis, wound repair, and the spread of many cancers, yet how individual cells signal to one another to coordinate their movements is largely unknown. Here, we introduce a tissue-autonomous paradigm for semaphorin-based regulation of collective cell migration. Semaphorins typically regulate the motility of neuronal growth cones and other migrating cell types by acting as repulsive cues within the migratory environment. Studying the follicular epithelial cells of Drosophila, we discovered that the transmembrane semaphorin, Sema-5c, promotes collective cell migration by acting within the migrating cells themselves, not the surrounding environment. Sema-5c is planar polarized at the basal epithelial surface such that it is enriched at the leading edge of each cell. This location places it in a prime position to send a repulsive signal to the trailing edge of the cell ahead to communicate directional information between neighboring cells. Our data show that Sema-5c can signal across cell-cell boundaries to suppress protrusions in neighboring cells and that Plexin A is the receptor that transduces this signal. Finally, we present evidence that Sema-5c antagonizes the activity of Lar, another transmembrane guidance cue that operates along leading-trailing cell-cell interfaces in this tissue, via a mechanism that appears to be independent of Plexin A. Together, our results suggest that multiple transmembrane guidance cues can be deployed in a planar-polarized manner across an epithelium and work in concert to coordinate individual cell movements for collective migration.

Dopamine neurons projecting to the posterior striatum reinforce avoidance of threatening stimuli.

Midbrain dopamine neurons are well known for their role in reward-based reinforcement learning. We found that the activity of dopamine axons in the posterior tail of the striatum (TS) scaled with the novelty and intensity of external stimuli, but did not encode reward value. We demonstrated that the ablation of TS-projecting dopamine neurons specifically inhibited avoidance of novel or high-intensity stimuli without affecting animals' initial avoidance responses, suggesting a role in reinforcement rather than simply in avoidance itself. Furthermore, we found that animals avoided optogenetic activation of dopamine axons in TS during a choice task and that this stimulation could partially reinstate avoidance of a familiar object. These results suggest that TS-projecting dopamine neurons reinforce avoidance of threatening stimuli. More generally, our results indicate that there are at least two axes of reinforcement learning using dopamine in the striatum: one based on value and one based on external threat.

A Self-Killing Rabies Virus That Leaves a Trace on the DNA.

Although modified rabies viruses have emerged as a powerful tool for tracing the inputs to genetically defined populations of neurons, the toxicity of the virus has limited its utility. A recent study employed a self-inactivating rabies (SiR) virus that enables recording or manipulation of targeted neurons for months.

Opposite initialization to novel cues in dopamine signaling in ventral and posterior striatum in mice.

Dopamine neurons are thought to encode novelty in addition to reward prediction error (the discrepancy between actual and predicted values). In this study, we compared dopamine activity across the striatum using fiber fluorometry in mice. During classical conditioning, we observed opposite dynamics in dopamine axon signals in the ventral striatum ('VS dopamine') and the posterior tail of the striatum ('TS dopamine'). TS dopamine showed strong excitation to novel cues, whereas VS dopamine showed no responses to novel cues until they had been paired with a reward. TS dopamine cue responses decreased over time, depending on what the cue predicted. Additionally, TS dopamine showed excitation to several types of stimuli including rewarding, aversive, and neutral stimuli whereas VS dopamine showed excitation only to reward or reward-predicting cues. Together, these results demonstrate that dopamine novelty signals are localized in TS along with general salience signals, while VS dopamine reliably encodes reward prediction error.

Dopamine neurons projecting to the posterior striatum form an anatomically distinct subclass.

Combining rabies-virus tracing, optical clearing (CLARITY), and whole-brain light-sheet imaging, we mapped the monosynaptic inputs to midbrain dopamine neurons projecting to different targets (different parts of the striatum, cortex, amygdala, etc) in mice. We found that most populations of dopamine neurons receive a similar set of inputs rather than forming strong reciprocal connections with their target areas. A common feature among most populations of dopamine neurons was the existence of dense 'clusters' of inputs within the ventral striatum. However, we found that dopamine neurons projecting to the posterior striatum were outliers, receiving relatively few inputs from the ventral striatum and instead receiving more inputs from the globus pallidus, subthalamic nucleus, and zona incerta. These results lay a foundation for understanding the input/output structure of the midbrain dopamine circuit and demonstrate that dopamine neurons projecting to the posterior striatum constitute a unique class of dopamine neurons regulated by different inputs.

A screen for round egg mutants in Drosophila identifies tricornered, furry, and misshapen as regulators of egg chamber elongation.

The elongation of tissues and organs during embryonic development results from the coordinate polarization of cell behaviors with respect to the elongation axis. Within the Drosophila melanogaster ovary, initially spherical egg chambers lengthen dramatically as they develop to create the elliptical shape of the mature egg. This morphogenesis depends on an unusual form of planar polarity within the egg chamber's outer epithelial cell layer known as the follicle cells. Disruption of follicle cell planar polarity leads to the production of round rather than elongated eggs; however, the molecular mechanisms that control this tissue organization are poorly understood. Starting from a broadly based forward genetic screen, we have isolated 12 new round egg complementation groups, and have identified four of the mutated genes. In mapping the largest complementation group to the fat2 locus, we unexpectedly discovered a high incidence of cryptic fat2 mutations in the backgrounds of publicly available stocks. Three other complementation groups correspond to the genes encoding the cytoplasmic signaling proteins Tricornered (Trc), Furry (Fry), and Misshapen (Msn). Trc and Fry are known members of an NDR kinase signaling pathway, and as a Ste20-like kinase, Msn may function upstream of Trc. We show that all three proteins are required for follicle cell planar polarity at early stages of egg chamber elongation and that Trc shows a planar polarized distribution at the basal follicle cell surface. These results indicate that this new mutant collection is likely to provide novel insight into the molecular mechanisms controlling follicle cell planar polarity and egg chamber elongation.