After finding food, a foraging animal must decide whether to continue feeding or to explore the environment for potentially better options. One strategy to negotiate this tradeoff is to perform local searches around the food while repeatedly returning to feed. We studied this behavior in flies and used genetic tools to uncover the underlying behavioral strategies. Over time, flies gradually expand their search, shifting from primarily exploiting food sources to exploring the environment, a change likely driven by increased satiety. We found that flies' search patterns preserve these dynamics even as the overall range of the search is modulated by starvation. In contrast, search induced by optogenetic activation of sugar-sensing neurons does not show these dynamics. We asked what navigational strategies underlie local search. Using a generative model, we found that a change in locomotor pattern after food consumption could account for repeated returns to the food, but not the relatively direct return trajectories that flies make even from far away. Such trajectories likely rely on alternative strategies, such as path integration or sensory taxis. We tested this by individually silencing their likely neural components, the compass system, olfaction, and hygrosensation. The only substantial effect was from perturbing hygrosensation, which reduced the number of long exploratory trips with subsequent return to the food. Our study illustrates that local search comprises multiple behavioral features that evolve over time based on both internal and external factors, providing a path toward uncovering the underlying neural mechanisms.

Mammalian development takes place inside the maternal uterus, creating technological constraints that make difficult the study of embryogenesis in live developing embryos. A central challenge for understanding the role of metabolism in mammalian development is discriminating placental and uterine-regulated signals from embryo-intrinsic processes independent of maternal influence, a process that until now has remained inseparable during gastrulation and organogenesis1–3. Ex utero culture systems allowing continuous growth of embryos during pre-gastrulation to organogenesis4,5 offer a promising solution to this challenge. Here, we present optimized ex utero culture platforms that support faithful development of mouse embryos from gastrulation (embryonic day 6.5/7.5) through the fetal period (embryonic day \~12.5) and harnessed these platforms for dissecting metabolic transitions in vivo during embryogenesis independently of uterus and placenta. We characterized the metabolome of in utero and ex utero whole embryos, fetal organs and culture medium between embryonic days E6.5 and E12.5 by liquid chromatography mass-spectrometry (LC-MS) metabolomics, isotope tracing, and single cell transcriptomics. These datasets present a comprehensive overview of the dynamic embryonic metabolism during gastrulation and organogenesis in utero and ex utero. This analysis revealed that the midgestational metabolic switch occurring at E10.5-E11.5 is faithfully recapitulated ex utero, indicating that this transition is intrinsically programmed in embryonic tissues and does not require direct maternal or placental cues. Notably, oxygen availability modulated the extent of this transition, but elevated oxygen was insufficient to induce it prematurely, demonstrating that the switch is developmentally timed and only partially environmental-responsive. We further harnessed the ex utero platform for identifying and perturbing a mitochondrial redox shift at E7.5-E8.5 that is critical for developmental progress after gastrulation. These findings uncover the remarkable metabolic plasticity of the mammalian embryo, demonstrating its capacity to sustain growth independently of maternal inputs from the establishment of the body plan through the onset of the fetal period. Moreover, they highlight the use of long-term ex utero culture as a unique framework for dissecting the mechanisms that shape embryogenesis under physiological and experimentally perturbed conditions, while functionally uncoupling embryonic programs from maternal and placental influences.

Electron Microscopy (EM) is widely used in many scientific fields, particularly in life sciences, offering high-resolution information on the ultrastructure of biological organisms. Accurate characterization of EM image quality is important for assessing the EM tool performance, in addition to sample preparation protocol, imaging conditions, etc.This paper provides an overview of tools we developed as plugins for the popular image processing package Fiji (ImageJ) (1). These tools include signal-to-noise ratio analysis, contrast evaluation, and resolution analysis, as well as the capability to import images acquired on custom FIB-SEM instruments (2). We have also made these tools available in Python, with both versions available on GitHub.

Motion is an essential component of any living system. It is rich with information, but it is often challenging to quantitatively extract biologically informative results from the motion apparent in microscopy images. This challenge is exacerbated by the wide variety in biological movement, which often takes the form of difficult-to-segment amorphous structures undergoing complex motion. An image processing technique known as optical flow can capture motion at each pixel in an image, thus bypassing the need for object segmentation or a priori definition of motion types. This makes it a powerful tool for quantitative assessment of biological systems from the protein to organism scale. However, despite its flexibility and strengths for analyzing fluorescence microscopy images, its adoption in the bioimaging community has been limited by the availability of easy-to-use tools and guidance in results interpretation. Here we describe an optical flow tool, OpticalFlow3D, that can be run in Python or MATLAB and is compatible with three-dimensional microscopy images. Using biological examples across length scales, we illustrate how OpticalFlow3D can enable new biological insight.

On evolutionary timescales, brain circuits adapt to support survival in each species’ ecological niche. While some anatomical aspects of neural circuitry are conserved across species with distant evolutionary origins, each species also exhibits specific circuit adaptations that enable its behavioral repertoire. It remains unclear whether homologous brain regions leverage analogous neural computations as different species perform common behaviors such as reaching and manipulating objects. Here, we directly assessed conservation of neural computations using intracortical recordings from mouse, monkey, and human motor cortex—a homologous region across many mammals—during motor behaviors crucial for survival. We hypothesized that, despite their phylogenetic distance, rodents and primates produce movements through conserved neural computations implemented by motor cortical population dynamics. Remarkably, we found that movement-related neural dynamics were highly conserved across species, while variations in behavioral output were uniquely captured in neural trajectory geometries. Strikingly, neural dynamics during movement across species were more conserved than those across brain regions in the same human and between motor preparation and execution in the same monkeys. Lastly, through manipulation of neural network models trained to perform reaching movements, we reinforce that conservation of neural dynamics across species likely stems from shared circuit constraints. We thus assert that evolution maintains neural computations across phylogeny even as behavioral repertoires expand.

Acoustic communication is widespread among vertebrates and central to social behavior. Yet how brain-wide circuits identify conspecific signals and distinguish acoustic elements with different, often sex-specific social valence remains poorly understood. Here we present the first whole-brain analysis of neuronal responses to conspecific vocalisations in vertebrates, using the transparent fish Danionella cerebrum. Combining volumetric calcium imaging with playbacks probing the stimulus space of the natural sound repertoire, we uncover an unexpectedly early and specialized processing hierarchy: hindbrain nuclei already segregate vocalization-like pulse trains from tones, midbrain regions sharpen these representations and extract temporal features that define vocalization type, and the central posterior thalamic nucleus responds selectively to conspecific vocalization rates and thus acts as a gate for social sounds. Male and female brains share this early feature code but diverge in diencephalic and telencephalic regions, where identical acoustic features evoke sex-specific population activity patterns that parallel dimorphic behavior. Together, our results provide the first cellular-resolution, brain-wide account of social sound processing in a vertebrate, from early categorical segregation to thalamic gating and sex-specific population responses in social circuits.

Cfr methylates C8 of adenosine 2503 (A2503) in 23S ribosomal RNA (rRNA) and will also methylate C2 of A2503 after methylating C8. C8methylation confers resistance to more than five classes of clinically used antibiotics, highlighting it as a worrisome mechanism of antibiotic resistance. Here, we report the structure of Cfr, determined by cryogenic electron microscopy (Cryo-EM). Despite its small size (∼36 kDa), we exploit a transient protein–RNA crosslink that forms during catalysis, which requires Cys105 to resolve. Using a Cfr Cys105Ala variant and an 87-nucleotide strand of rRNA, we isolate the crosslinked species and determine its structure to 3.0 Å resolution. Notably, the 87-mer rRNA adopts an L-shaped conformation characteristic of tRNAs, rather than the conformation it assumes in the ribosome.

Connectomics has become essential for the study of brain function, yet for most research groups it remains prohibitively costly in imaging time, data storage, and analysis. Here, we present an imaging, processing, and analysis pipeline for multi-resolution image acquisition and circuit reconstruction. Applied to the central complex of six insect species, we were able to obtain global projectomes at cellular resolution (40-50 nm) with embedded local connectomes describing key computational compartments at synaptic resolution (8-12 nm). We provide standardized protocols for volume EM sample preparation, image acquisition and image alignment, combined with existing methods for µCT block trimming, automatic segmentation, synapse detection, collaborative skeleton tracing with CATMAID, and segmentation proofreading via CAVE. We validated our workflow by reconstructing head direction cells across all six insect species, which revealed deep conservation at the level of cell types, cell numbers and projection patterns, while also revealing circuit level specializations. Overall, our pipeline democratizes comparative connectomics by making this method accessible for small research groups with modest resources.

Monitoring GABAergic inhibition in the nervous system has been enabled by development of an intensiometric molecular sensor that directly detects GABA. However, the first generation iGABASnFR exhibits low signal-to-noise and suboptimal kinetics, making in vivo experiments challenging. To improve sensor performance, we targeted several sites in the protein for near-saturation mutagenesis and evaluated the resulting sensor variants in a high throughput screening system using evoked synaptic release in primary cultured neurons. This identified a sensor variant, iGABASnFR2, with 4.2-fold improved sensitivity and 20% faster kinetics, and binding affinity that remained in a range sensitive to changes in GABA concentration at synapses. We also identified sensors with an inverted response, decreasing fluorescence intensity upon GABA binding. We termed the best such negative-going sensor iGABASnFR2n, which can be used to corroborate observations with the positive-going sensor. These improvements yielded a qualitative enhancement of in vivo performance when compared directly to the original sensor. iGABASnFR2 enabled the first measurements of direction-selective GABA release in the retina. In vivo imaging in somatosensory cortex revealed that iGABASnFR2 can report volume-transmitted GABA release following whisker stimulation. Overall, the improved sensitivity and kinetics of iGABASnFR2 make it a more effective tool for imaging GABAergic transmission in intact neural circuits.

Mitochondria utilize calcium to increase ATP synthesis. However, excessive matrix calcium activates the mitochondrial permeability transition (mPT), a process that permeabilizes the mitochondrial inner membrane and leads to cell death. While initially characterized 50 y ago, the proteins underlying the process are unclear, although integral membrane proteins were expected to be the porous entities during calcium overload. Here, we designed two assays to study the mPT using high-throughput methodologies. By surveying 19,113 proteins in human cells, we identified four proteins that sensitize the human mPT, but only one that was essential for mPT activation, mitochondrial-localized NRLX1. Surprisingly, NLRX1 is not an integral membrane protein, and our work did not identify any essential integral membrane proteins for the human mPT. The mitochondrial permeability transition (mPT) is an evolutionarily conserved destructive process that permeabilizes the inner mitochondrial membrane in response to calcium overload. The molecular mechanism underlying the mPT is not established. To unambiguously identify essential proteins, we designed two phenotypic assays for mitochondrial calcium overload and applied them to FACS-based CRISPR screening in human cells, ultimately evaluating 19,113 genes. The first screen studied mitochondrial membrane potential (MMP) collapse in response to calcium overload. Top-ranked genes were the essential proteins of the mitochondrial calcium uniporter complex, MCU and EMRE, reflecting that the calcium-induced MMP collapse results from mitochondrial calcium entry and not the mPT. The second screen measured the permeability of the inner mitochondrial membrane. Here, the fluorescent interaction of a membrane impermeant 600 Da dye and a mitochondrial-targeted HaloTag protein was studied under mPT activating conditions; calcium overload and the thiol-reactive molecule phenylarsine oxide. With secondary validation, we identified four protein-encoding genes that delayed or prevented the mPT under knockout: NF2, REST, BPTF, and NRLX1. Knockout of the nonmitochondrial proteins BPTF, NF2, or REST increased mitochondrial calcium retention capacity (CRC). However, calcium release or sensitivity to cyclosporin A (CsA) persisted, indicative of mPT sensitizers. Only knockout of the mitochondrial matrix protein, NLRX1, increased CRC, abolished calcium release, and was CsA-insensitive. This top-ranked hit of the mitochondrial permeability screen meets the definition of an essential mPT activator. Integral membrane proteins, including all previously proposed mPT candidates, were not essential activators.