Subcellular pharmacokinetic measurements have informed the study of central nervous system (CNS)-acting drug mechanisms. Recent investigations have been enhanced by the use of genetically encoded fluorescent biosensors for drugs of interest at the plasma membrane and in organelles. We describe screening and validation protocols for identifying hit pairs comprising a drug and biosensor, with each screen including 13-18 candidate biosensors and 44-84 candidate drugs. After a favorable hit pair is identified and validated via these protocols, the biosensor is then optimized, as described in other papers, for sensitivity and selectivity to the drug. We also show sample hit pair data that may lead to future intensity-based drug-sensing fluorescent reporters (iDrugSnFRs). These protocols will assist scientists to use fluorescence responses as criteria in identifying favorable fluorescent biosensor variants for CNS-acting drugs that presently have no corresponding biosensor partner. eLife (2022), DOI: 10.7554/eLife.74648 Graphical abstract.

During development, regulatory factors appear in a precise order to determine cell fates over time. Consequently, to investigate complex tissue development, it is necessary to visualize and manipulate cell lineages with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here, we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labeling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation and inactivation of reporters and/or effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification.

Medial frontal cortical areas are thought to play a critical role in the brain's ability to flexibly deploy strategies that are effective in complex settings. Still, the specific circuit computations that underpin this foundational aspect of intelligence remain unclear. Here, by examining neural ensemble activity in rats that sample different strategies in a self-guided search for latent task structure, we demonstrate a robust tracking of individual strategy prevalence in the anterior cingulate cortex (ACC), especially in an area homologous to primate area 32D. Prevalence encoding in the ACC is wide-scale, independent of reward delivery, and persists through a substantial ensemble reorganization that tags ACC representations with contextual content. Our findings argue that ACC ensemble dynamics is structured by a summary statistic of recent behavioral choices, raising the possibility that ACC plays a role in estimating - through statistical learning - which actions promote the occurrence of events in the environment.

Fluorescent in-situ hybridization (FISH)-based methods are powerful tools to study molecular processes with subcellular resolution, relying on accurate identification and localization of diffraction-limited spots in microscopy images. We developed the Radial Symmetry-FISH (RS-FISH) software that accurately, robustly, and quickly detects single-molecule spots in two and three dimensions, making it applicable to several key assays, including single-molecule FISH (smFISH), spatial transcriptomics, and spatial genomics. RS-FISH allows interactive parameter tuning and scales to large sets of images as well as tera-byte sized image volumes such as entire brain scans using straight-forward distributed processing on workstations, clusters, and in the cloud.

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.

Cells regulate function by synthesizing and degrading proteins. This turnover ranges from minutes to weeks, as it varies across proteins, cellular compartments, cell types, and tissues. Current methods for tracking protein turnover lack the spatial and temporal resolution needed to investigate these processes, especially in the intact brain, which presents unique challenges. We describe a pulse-chase method (DELTA) for measuring protein turnover with high spatial and temporal resolution throughout the body, including the brain. DELTA relies on rapid covalent capture by HaloTag of fluorophores that were optimized for bioavailability in vivo. The nuclear protein MeCP2 showed brain region- and cell type-specific turnover. The synaptic protein PSD95 was destabilized in specific brain regions by behavioral enrichment. A novel variant of expansion microscopy further facilitated turnover measurements at individual synapses. DELTA enables studies of adaptive and maladaptive plasticity in brain-wide neural circuits.

Electrophysiology is one of the major experimental techniques used in neuroscience. The favorable spatial and temporal resolution as well as the increasingly larger site counts of brain recording electrodes contribute to the popularity and importance of electrophysiology in neuroscience. Such electrodes are typically mechanically placed in the brain to perform acute or chronic freely moving animal measurements. The micro positioners currently used for such tasks employ a single translator per independent probe being placed into the targeted brain region, leading to significant size and weight restrictions. To overcome this limitation, we have developed a miniature robotic multi-probe neural microdrive that utilizes novel phase-change-material-filled resistive heater micro-grippers. The microscopic dimensions, gentle gripping action, independent electronic actuation control, and high packing density of the grippers allow for micrometer-precision independent positioning of multiple arbitrarily shaped parallel neural electrodes with only a single piezo actuator in an inchworm motor configuration. This multi-probe-single-actuator design allows for significant size and weight reduction, as well as remote control and potential automation of the microdrive. We demonstrate accurate placement of multiple independent recording electrodes into the CA1 region of the rat hippocampus in vivo in acute and chronic settings. Thus, our robotic neural microdrive technology is applicable towards basic neuroscience and clinical studies, as well as other multi-probe or multi-sensor micro-positioning applications.

Mammalian development is characterized with transitions from homogeneous populations of precursor to heterogeneous population of specified cells. We review here the main dynamical mechanisms through which such transitions are conceptualized, and discuss that the differentiation timing, robust cell-type proportions and recovery upon perturbation are emergent property of proliferating and communicating cell populations. We argue that studying developmental systems using transitions in collective system states is necessary to describe observed experimental features, and propose additionally the basis of a novel analytical method to deduce the relationship between single-cell dynamics and the collective, symmetry-broken states in cellular populations.

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.

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.