Pretrained neural network models for biological segmentation can provide good out-of-the-box results for many image types. However, such models do not allow users to adapt the segmentation style to their specific needs and can perform suboptimally for test images that are very different from the training images. Here we introduce Cellpose 2.0, a new package that includes an ensemble of diverse pretrained models as well as a human-in-the-loop pipeline for rapid prototyping of new custom models. We show that models pretrained on the Cellpose dataset can be fine-tuned with only 500-1,000 user-annotated regions of interest (ROI) to perform nearly as well as models trained on entire datasets with up to 200,000 ROI. A human-in-the-loop approach further reduced the required user annotation to 100-200 ROI, while maintaining high-quality segmentations. We provide software tools such as an annotation graphical user interface, a model zoo and a human-in-the-loop pipeline to facilitate the adoption of Cellpose 2.0.

Precise synaptic connection of neurons with their targets is essential for the proper functioning of the nervous system. A plethora of signaling pathways act in concert to mediate the precise spatial arrangement of synaptic connections. Here we show a novel role for a gap junction protein in controlling tiled synaptic arrangement in the GABAergic motor neurons in , in which their axons and synapses overlap minimally with their neighboring neurons within the same class. We found that while EGL-20/Wnt controls axonal tiling, their presynaptic tiling is mediated by a gap junction protein UNC-9/Innexin, that is localized at the presynaptic tiling border between neighboring dorsal D-type GABAergic motor neurons. Strikingly, the gap junction channel activity of UNC-9 is dispensable for its function in controlling tiled presynaptic patterning. While gap junctions are crucial for the proper functioning of the nervous system as channels, our finding uncovered the novel channel-independent role of UNC-9 in synapse patterning.

Chemogenetics is a technique for obtaining selective pharmacological control over a cell population by expressing an engineered receptor that is selectively activated by an exogenously administered ligand. A promising approach for neuronal modulation involves the use of “Pharmacologically Selective Actuator Modules” (PSAMs); these chemogenetic receptors are selectively activated by ultrapotent “Pharmacologically Selective Effector Molecules” (uPSEMs). To extend the use of PSAM/PSEMs to studies in nonhuman primates it is necessary to thoroughly characterize the efficacy and safety of these tools. We describe the time course and brain penetrance in rhesus monkeys of two compounds with promising binding specificity and efficacy profiles in in vitro studies, uPSEM792 and uPSEM817, after systemic administration. Rhesus macaques received subcutaneous (s.c.) or intravenous (i.v.) administration of uPSEM817(0.064 mg/kg) or uPSEM792 (0.87 mg/kg) and plasma and CSF samples were collected over the course of 48 hours. Both compounds exhibited good brain penetrance, relatively slow washout and negligible conversion to potential metabolites - varenicline or hydroxyvarenicline. In addition, we found that neither of these uPSEMs significantly altered heart rate or sleep. Our results indicate that both compounds are suitable candidates for neuroscience studies using PSAMs in nonhuman primates.

Chemogenetics is a technique for obtaining selective pharmacological control over a cell population by expressing an engineered receptor that is selectively activated by an exogenously administered ligand. A promising approach for neuronal modulation involves the use of "Pharmacologically Selective Actuator Modules" (PSAMs); these chemogenetic receptors are selectively activated by ultrapotent "Pharmacologically Selective Effector Molecules" (uPSEMs). To extend the use of PSAM/PSEMs to studies in nonhuman primates, it is necessary to thoroughly characterize the efficacy and safety of these tools. We describe the time course and brain penetrance in rhesus monkeys of two compounds with promising binding specificity and efficacy profiles in studies, uPSEM792 and uPSEM817, after systemic administration. Rhesus monkeys received subcutaneous (s.c.) or intravenous (i.v.) administration of uPSEM817 (0.064 mg/kg) or uPSEM792 (0.87 mg/kg), and plasma and cerebrospinal fluid samples were collected over 48 h. Both compounds exhibited good brain penetrance, relatively slow washout, and negligible conversion to potential metabolites─varenicline or hydroxyvarenicline. In addition, we found that neither of these uPSEMs significantly altered the heart rate or sleep. Our results indicate that both compounds are suitable candidates for neuroscience studies using PSAMs in nonhuman primates.

We report the rational engineering of a remarkably stable yellow fluorescent protein (YFP), 'hyperfolder YFP' (hfYFP), that withstands chaotropic conditions that denature most biological structures within seconds, including superfolder green fluorescent protein (GFP). hfYFP contains no cysteines, is chloride insensitive and tolerates aldehyde and osmium tetroxide fixation better than common fluorescent proteins, enabling its use in expansion and electron microscopies. We solved crystal structures of hfYFP (to 1.7-Å resolution), a monomeric variant, monomeric hyperfolder YFP (1.6 Å) and an mGreenLantern mutant (1.2 Å), and then rationally engineered highly stable 405-nm-excitable GFPs, large Stokes shift (LSS) monomeric GFP (LSSmGFP) and LSSA12 from these structures. Lastly, we directly exploited the chemical stability of hfYFP and LSSmGFP by devising a fluorescence-assisted protein purification strategy enabling all steps of denaturing affinity chromatography to be visualized using ultraviolet or blue light. hfYFP and LSSmGFP represent a new generation of robustly stable fluorescent proteins developed for advanced biotechnological applications.

While fluorescence microscopy has proven to be an exceedingly useful tool in bioscience, it is difficult to offer simultaneous high resolution, fast speed, large volume, and good biocompatibility in a single imaging technique. Thus, when determining the image data required to quantitatively test a complex biological hypothesis, it often becomes evident that multiple imaging techniques are necessary. Recent years have seen an explosion in development of novel fluorescence microscopy techniques, each of which features a unique suite of capabilities. In this Technical Perspective, we highlight recent studies to illustrate the benefits, and often the necessity, of combining multiple fluorescence microscopy modalities. We provide guidance in choosing optimal technique combinations to effectively address a biological question. Ultimately, we aim to promote a more well-rounded approach in designing fluorescence microscopy experiments, leading to more robust quantitative insight.

The availability of both anatomical connectivity and brain-wide neural activity measurements in C. elegans make the worm a promising system for learning detailed, mechanistic models of an entire nervous system in a data-driven way. However, one faces several challenges when constructing such a model. We often do not have direct experimental access to important modeling details such as single-neuron dynamics and the signs and strengths of the synaptic connectivity. Further, neural activity can only be measured in a subset of neurons, often indirectly via calcium imaging, and significant trial-to-trial variability has been observed. To address these challenges, we introduce a connectome-constrained latent variable model (CC-LVM) of the unobserved voltage dynamics of the entire C. elegans nervous system and the observed calcium signals. We used the framework of variational autoencoders to fit parameters of the mechanistic simulation constituting the generative model of the LVM to calcium imaging observations. A variational approximate posterior distribution over latent voltage traces for all neurons is efficiently inferred using an inference network, and constrained by a prior distribution given by the biophysical simulation of neural dynamics. We applied this model to an experimental whole-brain dataset, and found that connectomic constraints enable our LVM to predict the activity of neurons whose activity were withheld significantly better than models unconstrained by a connectome. We explored models with different degrees of biophysical detail, and found that models with realistic conductance-based synapses provide markedly better predictions than current-based synapses for this system.

The representation of contextual information peripheral to a salient stimulus is central to an animal's ability to correctly interpret and flexibly respond to that stimulus. While the computations and circuits underlying the context-dependent modulation of stimulus-response pairings have typically been studied in vertebrates, the genetic tractability, numeric simplification, and well-characterized connectivity patterns of the Drosophila melanogaster brain have facilitated circuit-level insights into contextual processing. Recent studies in flies reveal the neuronal mechanisms that create flexible context-dependent behavioral responses to sensory events in conditions of predation threat, feeding regulation, and social interaction.

Viruses use a plethora of mechanisms to evade immune responses. A recent example is neutralization of the nuclear DNA cytosine deaminase APOBEC3B by the Epstein-Barr virus (EBV) ribonucleotide reductase subunit BORF2. Cryo-EM studies of APOBEC3B-BORF2 complexes reveal a large >1000-Å binding surface composed of multiple structural elements from each protein, which effectively blocks the APOBEC3B active site from accessing single-stranded DNA substrates. Evolutionary optimization is suggested by unique insertions in BORF2 absent from other ribonucleotide reductases and preferential binding to APOBEC3B relative to the highly related APOBEC3A and APOBEC3G enzymes. A molecular understanding of this pathogen-host interaction has potential to inform the development of drugs that block the interaction and liberate the natural antiviral activity of APOBEC3B. In addition, given a role for APOBEC3B in cancer mutagenesis, it may also be possible for information from the interaction to be used to develop DNA deaminase inhibitors.

mTORC1 controls cellular metabolic processes in response to nutrient availability. Amino acid signals are transmitted to mTORC1 through the Rag GTPases, which are localized on the lysosomal surface by the Ragulator complex. The Rag GTPases receive amino acid signals from multiple upstream regulators. One negative regulator, GATOR1, is a GTPase activating protein (GAP) for RagA. GATOR1 binds to the Rag GTPases via two modes: an inhibitory mode and a GAP mode. How these two binding interactions coordinate to process amino acid signals is unknown. Here, we resolved three cryo-EM structural models of the GATOR1-Rag-Ragulator complex, with the Rag-Ragulator subcomplex occupying the inhibitory site, the GAP site, and both binding sites simultaneously. When the Rag GTPases bind to GATOR1 at the GAP site, both Rag subunits contact GATOR1 to coordinate their nucleotide loading states. These results reveal a potential GAP mechanism of GATOR1 during the mTORC1 inactivation process.