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In the mouse, each class of olfactory receptor neurons expressing a given odorant receptor has convergent axonal projections to two specific glomeruli in the olfactory bulb, thereby creating an odour map. However, it is unclear how this map is represented in the olfactory cortex. Here we combine rabies-virus-dependent retrograde mono-trans-synaptic labelling with genetics to control the location, number and type of ‘starter’ cortical neurons, from which we trace their presynaptic neurons. We find that individual cortical neurons receive input from multiple mitral cells representing broadly distributed glomeruli. Different cortical areas represent the olfactory bulb input differently. For example, the cortical amygdala preferentially receives dorsal olfactory bulb input, whereas the piriform cortex samples the whole olfactory bulb without obvious bias. These differences probably reflect different functions of these cortical areas in mediating innate odour preference or associative memory. The trans-synaptic labelling method described here should be widely applicable to mapping connections throughout the mouse nervous system. Scent tracking In the mouse, glomeruli in the olfactory bulb receive projections from single classes of olfactory neurons, thereby forming an odour map. Information from the glomeruli is then relayed to the cortex but the projection patterns from individual glomeruli are not known. Three papers now examine the details of this projection. Luo and colleagues use a combination of genetics and retrograde mono-trans-synaptic rabies virus labelling. They trace the presynaptic connections of individual cortical neurons and find no evidence of connections supporting a stereotyped odour map in the cortex, but see systematic topographical differences in amygdala connectivity. The lack of stereotypical cortical projection is corroborated, both at the level of bulk axonal patterning and in projections of individually labelled neurons, by two papers — one from the Axel laboratory, and one from the Baldwin laboratory — that examine the anterograde projections from individual glomeruli. Together, these findings provide anatomical evidence for combinatorial processing of information from diverse glomeruli by cortical neurons and may also reflect different functions of various areas in mediating innate or learned odour preferences.

Previous studies have demonstrated that aneuploidy in human embryos is surprisingly frequent with 50–80% of cleavage-stage human embryos carrying an abnormal chromosome number. Here we combine non-invasive time-lapse imaging with karyotypic reconstruction of all blastomeres in four-cell human embryos to address the hypothesis that blastomere behaviour may reflect ploidy during the first two cleavage divisions. We demonstrate that precise cell cycle parameter timing is observed in all euploid embryos to the four-cell stage, whereas only 30% of aneuploid embryos exhibit parameter values within normal timing windows. Further, we observe that the generation of human embryonic aneuploidy is complex with contribution from chromosome-containing fragments/micronuclei that frequently emerge and may persist or become reabsorbed during interphase. These findings suggest that cell cycle and fragmentation parameters of individual blastomeres are diagnostic of ploidy, amenable to automated tracking algorithms, and likely of clinical relevance in reducing transfer of embryos prone to miscarriage. Abnormal human embryo development is implicated in the embryo arrest observed during in vitro fertilization. Chavez and colleagues perform time-lapse imaging on human embryos and find that chromosomally abnormal embryos exhibit diverse cell cycle parameters that may contribute to arrest.

Acute respiratory distress syndrome (ARDS) is associated with significant morbidity and mortality, and neutrophils are critical to its pathogenesis. Neutrophil activation is closely regulated by inhibitory tyrosine phosphatases including Src homology region 2 domain-containing phosphatase-1 (Shp1). Here, we report that loss of neutrophil Shp1 in mice produced hyperinflammation and lethal pulmonary hemorrhage in sterile inflammation and pathogen-induced models of acute lung injury (ALI) through a Syk kinase-dependent mechanism. We observed large intravascular neutrophil clusters, perivascular inflammation, and excessive neutrophil extracellular traps in neutrophil-specific Shp1-KO mice, suggesting an underlying mechanism for the observed pulmonary hemorrhage. Targeted immunomodulation through the administration of a Shp1 activator (SC43) reduced agonist-induced reactive oxygen species in vitro and ameliorated ALI-induced alveolar neutrophilia and NETs in vivo. We propose that the pharmacologic activation of Shp1 has the potential to fine tune neutrophil hyperinflammation that is central to the pathogenesis of ARDS.

Acute respiratory distress syndrome (ARDS) is associated with significant morbidity and mortality, and neutrophils are critical to its pathogenesis. Neutrophil activation is closely regulated by inhibitory tyrosine phosphatases including Src homology region 2 domain-containing phosphatase-1 (Shp1). Here, we report that loss of neutrophil Shp1 in mice produced hyperinflammation and lethal pulmonary hemorrhage in sterile inflammation and pathogen-induced models of acute lung injury (ALI) through a Syk kinase-dependent mechanism. We observed large intravascular neutrophil clusters, perivascular inflammation, and excessive neutrophil extracellular traps in neutrophil-specific Shp1-KO mice, suggesting an underlying mechanism for the observed pulmonary hemorrhage. Targeted immunomodulation through the administration of a Shp1 activator (5C43) reduced agonist-induced reactive oxygen species in vitro and ameliorated ALI-induced alveolar neutrophilia and NETs in vivo. We propose that the pharmacologic activation of Shp1 has the potential to fine tune neutrophil hyperinflammation that is central to the pathogenesis of ARDS.

In the mouse, each class of olfactory receptor neurons expressing a given odorant receptor converges their axons onto two specific glomeruli in the olfactory bulb (OB), thereby creating an odor map. How is this map represented in the olfactory cortex? Here we combine rabies virus-dependent retrograde mono-transsynaptic labeling with genetics to control the location, number and type of ‘starter’ cortical neurons, from which we trace their presynaptic neurons. We find that individual cortical neurons receive input from multiple mitral cells representing broadly distributed glomeruli. Different cortical areas represent the OB input differently. For example, the cortical amygdala preferentially receives dorsal OB input, whereas the piriform cortex samples the whole OB without obvious bias. These differences likely reflect different functions of these cortical areas in mediating innate odor preference or associative memory. The transsynaptic labeling method described here should be widely applicable to mapping connections throughout the mouse nervous system.

Cryogenic Electron Tomography (Cryo-ET) has gained increasing interest in recent years due to its ability to image whole cells and subcellular structures in 3D at nanometer resolution in their native environment. However, due to dose restrictions and the inability to acquire high tilt angle images, the reconstructed volumes are noisy and have missing information. In order to overcome these limitations and fulfill the promise of this method, it is necessary to image numerous instances of the same underlying object and average them, requiring a high throughput pipeline. Furthermore, recent advances in microscope automation have increased the data generation capacity of this method by many times, placing additional strain on the postprocessing portion of the electron tomography pipeline. Currently, the bottlenecks in this pipeline are a set of image inference tasks which require manual intervention by an expert due to weak and unreliable local image features. In this thesis we propose the use of geometric context in a structured probabilistic models framework to overcome the low reliability of local features and achieve automation and high throughput for two of the bottleneck tasks---precision registration of 2D images and 3D segmentation of whole cells. The central idea in our approach is to overcome the uncertainty from unreliable features by exploiting their mutual geometric and spatial relationships in varying degrees of locality to classify them more accurately. Structured probabilistic models provide a framework for encoding a diverse set of geometric relationships, as well as a substantial body of efficient yet effective approximate inference algorithms.In the first problem of precision registration of 2D images, the features are a set of gold markers which can be difficult to distinguish at high tilt angles. Precision alignment of the images requires the successful tracking of these markers throughout the series of images. We track markers jointly as a group, using their geometric relationships. Therefore the geometric relationship of interest for overcoming the unreliable features in this case is the pattern formed by the gold markers. We encode the relative geometric arrangement of pairs of markers as pairwise factors in a Conditional random field (CRF) framework, and use loopy belief propagation to find the most likely correspondence of markers between images. This approach, called RAPTOR (Robust Alignment and Projection estimation for TOmographic Reconstruction) has resulted in successful automatic full precision alignment of electron tomography tilt series.The second problem of 3D segmentation of whole cells is challenging due to uncertain boundary characteristics. Intensity and intensity gradients based methods easily confuse many non boundary pixels as boundaries, and therefore precision extraction of the cell boundary is difficult, manual and time intensive. We present an efficient recursive algorithm called BLASTED (Boundary Localization using Adaptive Shape and TExture Discovery) to automatically extract the cell boundary using another CRF framework in which boundary points and shape are jointly inferred with the help of a learned boundary feature detector and shape evolution model. The algorithm learns the texture of the boundary region progressively, and uses a global shape model and shape-dependent features to propose candidate boundary points on a slice of the membrane. It then updates the shape of that slice by accepting the appropriate candidate points using local spatial clustering, the global shape model, and trained boosted texture classifiers. This method has successfully segmented numerous datasets starting from one hand labelled slice each, reducing the processing time from days to hours.

Microbes, toxins, therapeutics and cells are often instilled into lungs of mice to model diseases and test experimental interventions. Consistent pulmonary delivery is critical for experimental power and reproducibility, but we observed variation in outcomes between handlers using different anesthetic approaches for intranasal dosing into mice. We therefore used a radiotracer to quantify lung delivery after intranasal dosing under inhalational (isoflurane) versus injectable (ketamine/xylazine) anesthesia in C57BL/6 mice. We found that ketamine/xylazine anesthesia resulted in delivery of a greater proportion (52±9%) of an intranasal dose to lungs relative to isoflurane anesthesia (30±15%). This difference in pulmonary dose delivery altered key outcomes in a model of viral pneumonia, with mice anesthetized with ketamine/xylazine for intranasal infection with influenza A virus developing worse lung pathology and more consistently losing body weight relative to control animals randomized to isoflurane anesthesia. Pulmonary dosing efficiency through oropharyngeal aspiration was not affected by anesthetic method and resulted in delivery of 63±8% of dose to lungs, and a non-surgical intratracheal dosing approach further increased lung delivery to 92±6% of dose. We conclude that anesthetic approach and dosing route can impact pulmonary dosing efficiency. These factors should be considered when planning and reporting studies involving delivery of fluids to lungs of mice.Competing Interest StatementThe authors have declared no competing interest.

Tracking objects in three-dimensional space is critical for autonomous driving. To ensure safety while driving, the tracker must be able to reliably track objects across frames and accurately estimate their states such as velocity and acceleration in the present. Existing works frequently focus on the association task while either neglecting the model performance on state estimation or deploying complex heuristics to predict the states. In this paper, we propose STT, a Stateful Tracking model built with Transformers, that can consistently track objects in the scenes while also predicting their states accurately. STT consumes rich appearance, geometry, and motion signals through long term history of detections and is jointly optimized for both data association and state estimation tasks. Since the standard tracking metrics like MOTA and MOTP do not capture the combined performance of the two tasks in the wider spectrum of object states, we extend them with new metrics called S-MOTA and MOTPS that address this limitation. STT achieves competitive real-time performance on the Waymo Open Dataset.