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One of the many goals of neuroscience is to understand how the brain encodes and transforms sensory information into behavior. These animal behaviors can be studied at the level of multi-limb poses or through the focused analysis of individual body parts. Techniques for tracking animal pose, such as DeepLabCut and SLEAP, enable detailed studies of large-scale multi-limb behaviors but show reduced accuracy when used for single-keypoint tracking, where insufficient spatial context leads to increased drift and instability in tracking (Arent I, Schmidt FP, Botsch M et al. Marker-less motion capture of insect locomotion with deep neural networks pre-trained on synthetic videos. Frontiers in Behavioral Neuroscience. Vol. 15. 2021. Tang G, Han Y, Sun X, et al. Anti-drift pose tracker (ADPT), a transformer-based network for robust animal pose estimation cross-species. eLife. Vol. 13. 2025). More general techniques, such as Faster Region-based Convolutional Neural Network (Faster R-CNN) and You Only Look Once (YOLO), have also been used to track location-based behaviors such as center-of-mass position and velocity. However, behaviors localized to a single body structure, such as the pharyngeal pumping (i.e., feeding) in the microscopic roundworm Caenorhabditis elegans (C. elegans), are particularly sensitive to noise from moving non-target body parts. This limitation cannot be resolved by simply adding more training data, as doing so often leads to overfitting rather than improved robustness, and instead requires additional processing beyond existing object tracking packages. To address these challenges, we present a fast, automated method that reliably measures pumping in freely moving C. elegans by combining a state-of-the-art object detector (Faster R-CNN) with a tunable noise filter in a technique we call PumpKin. To validate its performance, we demonstrate both its speed (average of 0.4 seconds/frame) and its robust estimation capabilities through application to eight different experimental conditions that encompass both satiety and genetically-driven changes to feeding. PumpKin accurately estimates average pumping rates under eight different experimental conditions, which are positively correlated with the estimates of two expert annotators. Furthermore, PumpKin provides reliable estimates of the instantaneous pumping rate dynamics, achieving an average overlap that exceeds the human-human agreement measured via leave-one-out analysis. Applying PumpKin to conditions differing in satiety revealed a shared basal pumping rate of 0.5 Hz across all worm groups recorded off food, regardless of genetic background or satiety state. Together, these findings highlight PumpKin's ability to accurately isolate and estimate the motion of a single body part during locomotion. Although we present results specific to C. elegans, we anticipate that PumpKin will generalize to behaviors localized to a single body structure in other systems.

The unfolded protein response (UPR) is a crucial signaling network that preserves endoplasmic reticulum (ER) homeostasis, impacting both health and disease. When ER stress occurs, often due to an accumulation of unfolded proteins in the ER lumen, the UPR initiates a broad cellular program to counteract cytotoxic effects. Inositol-requiring enzyme 1 (IRE1), a conserved ER-bound protein, is a key sensor of ER stress and activator of the UPR. While biochemical studies confirm IRE1's role in recognizing unfolded polypeptides, high-resolution structures showing direct interactions remain elusive. Consequently, the precise structural mechanism by which IRE1 senses unfolded proteins is debated. In this study, we employed advanced molecular modeling and 137 µs of atomistic molecular dynamics simulations to clarify how IRE1 detects unfolded proteins. Our results demonstrate that IRE1's luminal domain directly interacts with unfolded peptides and reveal how these interactions can stabilize higher-order oligomers. We provide a detailed molecular characterization of unfolded peptide binding, identifying two distinct binding pockets at the dimer's center, separate from its central groove. Furthermore, we present high-resolution structures illustrating how BiP associates with IRE1's oligomerization interface, thus preventing the formation of larger complexes. Our structural model reconciles seemingly contradictory experimental findings, offering a unified perspective on the diverse sensing models proposed. We elucidate the structural dynamics of unfolded protein sensing by IRE1, providing key insights into the initial activation of the UPR.

PKMζ is a persistently active atypical PKC (aPKC) isoform thought to maintain late-phase long-term potentiation (late-LTP) and long-term memory. PKMζ-knockout mice, however, still exhibit hippocampal LTP and spatial memory while lacking neocortical LTP, questioning whether this kinase is fundamental to enduring synaptic potentiation and memory. Tsokas et al. (2016) suggested that the other aPKC, PKCι/λ, may compensate for PKMζ during maintenance in the hippocampus of PKMζ-null mice. In wild-type mice, PKCι/λ drives early-LTP and short-term memory, whereas in PKCι/λ-knockout mice, PKMζ compensates by supporting both early- and late-phase processes. Here, we show that PKCι/λ is persistently upregulated during maintenance in two mouse models: PKMζ-conditional knockout mice, and double-knockout mice carrying both conditional deletion of PKCι/λ and constitutive loss of PKMζ. Because PKCι/λ-gene excision is inducible in the double-knockout line, we could characterize the persistent increase of PKCι/λ in late-LTP prior to its deletion. To examine PKCι/λ function, we induced its deletion in the hippocampus. Whereas mutual compensation preserves LTP when either PKCι/λ or PKMζ alone is knocked out, double-knockout of both PKCι/λ and PKMζ eliminates late-LTP. Double-knockout also abolishes spatial long-term memory without affecting short-term memory. Thus, when PKMζ is absent, PKCι/λ persists to maintain hippocampal late-LTP and long-term memory.

Effective decision making in dynamic environments requires flexible evidence accumulation. Although models often express this flexibility as a property of the accumulator, its implementation in the brain may involve adaptive mechanisms operating at other stages of the decision process. We examined two such mechanisms: (1) stimulus-specific sensory adaptation at the level of evidence encoding, and (2) arousal-related neuromodulation, which could, in principle, affect both evidence encoding and accumulation. We measured single-unit activity in the middle temporal (MT) area and pupil-linked arousal while monkeys performed a modified random-dot motion direction-discrimination task in which an adapting stimulus with varied temporal stability preceded a behaviorally relevant test stimulus. The monkeys' decisions reflected adaptive evidence accumulation that depended on temporal-context stability and corresponded to context-dependent changes in both stimulus-specific sensory adaptation in MT and task-evoked pupil responses. However, adaptation and pupil adjustments were not related to each other. Together, these findings suggest that multiple mechanisms contribute to flexible, context-dependent evidence accumulation, including changes in sensory adaptation that shape evidence encoding and changes in arousal that may shape the accumulation process itself.

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We generated time-series data for 105 morphine- and naloxone-related traits across ~700 BXD mice (64 diverse strains for both sexes) for 3 hr after a single morphine injection. Variations in responses were mapped using genome sequencing-based genotypes. The locomotor responses to morphine mapped to the µ opioid receptor gene (Oprm1) on chromosome (Chr) 10 with a peak linkage of 12.4 (-logp). The B allele inherited from C57BL/6J was associated with up to 60% higher activity. This effect climaxed at 75 min but was exhausted by 160 min. A second major modulator of locomotion emerged after approximately 100 min. This locus was located on Chr 16 with peak linkage of 10.6 in females and included one compelling candidate, fibroblast growth factor 12 (Fgf12). A strong and transient epistatic interaction existed between the Oprm1 and Fgf12 loci during a short time window (45-75 min). In heterogeneous stock rats, we demonstrated that Oprm1 and Fgf12 were co-expressed in one subtype of Drd1+ medium spiny neuron. A Bayesian network analysis supported an Oprm1-to-Fgf12 network that involves a MAP kinase cascade that modulates FGF12 phosphorylation and locomotor activation. OPRM1 and FGF12 networks in human genome-wide association study (GWAS) data highlight enrichment of signals associated with substance use disorder. This study represents the first demonstration of a time-dependent epistatic interaction modulating drug response in mammals and the first linkage of Fgf12 to opioid-induced behavior.

The dorsal raphe nucleus (DRN) serotonergic (5-HT) system has been implicated in regulating sleep and motor control; however, its specific role remains controversial. In this study, we found that optogenetic activation of DRN 5-HT neurons in larval zebrafish induced a quiescent state and a reduced response to acoustic stimuli. Unlike sleep, the induced quiescent state was not accompanied by a loss of postural control, and nighttime activation of DRN 5-HT neurons led to a subsequent sleep rebound. Whole brain light field imaging combined with demixed principal component analysis (dPCA) revealed distinct neural subspaces related to DRN activation, sound responses, and motor activity. DRN 5-HT activation selectively modulated the motor-related subspace while leaving the sound-evoked subspace unaffected. Unlike DRN activation, sleep induced by mepyramine significantly altered sound-evoked neuronal activity patterns. Further analysis demonstrated that serotonin had a graded effect on the motor subspace, wherein downstream neurons responsible for particular bout types were more significantly influenced. Embedding motor population activity in a curved geometric space revealed that the degree of curvature scales with behavioral suppression across animals, providing a quantitative signature of the quiescent state. Together, these results elucidate that serotonergic modulation promotes behavioral quiescence through selective regulation of motor populations.

Color discrimination thresholds-the smallest detectable color differences-provide a benchmark for models of color vision, enable quantitative evaluation of eye diseases, and inform the design of display technologies. Despite their importance, a comprehensive characterization of these thresholds has long been considered intractable due to the psychophysical curse of dimensionality. Here, we address this challenge using a novel semiparametric Wishart process psychophysical model (WPPM), which leverages the feature that the internal noise limiting color discrimination varies smoothly across stimulus space. The model was fit to data collected with a nonparametric adaptive trial-placement procedure, enabling efficient stimulus selection. Together, through the combination of adaptive trial placement and post hoc WPPM fitting, we achieved a comprehensive characterization of color discrimination in the isoluminant plane with only ∼6000 trials per participant (N = 8). Once fit, the WPPM allows readouts of discrimination performance for any stimulus pair. We validated these readouts against 25 probe psychometric functions, measured with an additional 6000 trials per participant held out from model fitting. In conclusion, our study provides a foundational dataset for color vision, and our approach generalizes beyond color to any domain in which the internal noise limiting performance varies smoothly across stimulus space, offering a powerful and efficient method for comprehensively characterizing various perceptual discrimination thresholds.

Local field potentials (LFPs) are compound signals that represent the dynamic flow of information across the brain, which have been historically associated with chemical synaptic inputs. How do gap junctional inputs onto active compartments shape LFPs? We developed a methodology to record extracellular potentials associated with different patterns of gap junctional inputs onto conductance-based models. We found that synchronous inputs through chemical synapses yielded a negative deflection in proximal extracellular electrodes whereas those onto gap junctions manifested a positive deflection. Importantly, we observed extracellular dipoles only when inputs arrived through chemical synapses but not with gap junctions. Remarkably, hyperpolarization-activation cyclic nucleotide-gated channels, which typically conduct inward currents, mediated outward currents triggered by the fast voltage transition caused by synchronous inputs. With rhythmic inputs at different frequencies arriving through gap junctions, we found strong suppression of LFP power at higher frequencies as well as frequency-dependent differences in the spike phase associated with the LFP when compared to respective chemical synaptic counterparts. All observed differences in LFP were mediated by the relative dominance of synaptic currents vs. voltage-driven transmembrane currents with chemical synapses vs. gap junctions, respectively. Our analyses unveil a hitherto unknown role for active dendritic gap junctions in shaping extracellular potentials.

Chronic malaria exposure has been proposed to modulate immune function, but its long-term effects on antibody-mediated responses to unrelated pathogens remain poorly defined. Whether these effects persist beyond periods of active infection and how early-life exposure shapes humoral immunity over time is not well understood. We leveraged a natural experiment in coastal Kenya - where two regions (Junju and Ngerenya) diverged sharply in malaria transmission from around 2004 - to evaluate the long-term immunological consequences of malaria exposure in childhood. Using a protein microarray platform, we measured IgG responses to vaccine and pathogen antigens in 123 children sampled longitudinally over a 15-year period. Active weekly malaria surveillance enabled precise reconstruction of individual exposure histories. IgG responses to Plasmodium falciparum apical membrane antigen 1 (AMA1) tracked closely with clinical malaria episodes, confirming the ability of the microarray platform to detect biologically meaningful variation in antigen-specific immunity. Despite comparable vaccination histories, children from the high malaria transmission setting (Junju) exhibited persistently lower measles-specific IgG levels than children from the low-transmission setting (Ngerenya), a pattern validated by ELISA. In longitudinal analyses, children from Junju exhibited lower antibody responses to a range of unrelated antigens, including Bordetella pertussis, CMV, rubella, and measles, with similar differences evident in cross-sectional analyses at 10 years of age. Within the Ngerenya cohort, children with documented early-life malaria had broadly lower IgG responses at age 10 compared to malaria-naive peers, despite identical geography, vaccines, and follow-up duration. These findings suggest that malaria exposure during early childhood is linked with durable suppression of antibody responses to unrelated pathogens and vaccines. This effect persists long after infection and may partially explain the overall diminished long-term vaccine effectiveness in malaria-endemic settings. This study was supported by fellowship funding to C.J.S. from the Wellcome Trust (WT105882MA). The funder played no role in the conceptualization, design, data collection, analysis, decision to publish, or preparation of the manuscript. M.S.S was funded in whole by Science for Africa Foundation to the Developing Excellence in Leadership, Training, and Science in Africa (DELTAS Africa) program [DEL-22-012] with support from Wellcome Trust and the UK Foreign, Commonwealth & Development Office and is part of the EDCPT2 programme supported by the European Union. For purposes of open access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. Malaria alters the immune system during infection, but it remains unclear whether it leaves lasting effects. Previous studies have suggested that children living in malaria-endemic areas sometimes develop weaker responses to vaccines and other infections, indicating that early childhood exposure may influence immune responses many years later. To explore the effect of malaria infection during early childhood, Safari et al. studied 123 children from two neighbouring communities on the Kenyan coast with different malaria histories. Over 15 years, children in both locations were closely followed with weekly visits to identify malaria cases; blood samples were collected annually. Based on these data, Safari et al. reconstructed individual malaria exposure histories over time and measured antibody levels to different infections and vaccines over more than a decade. They analysed more than 1,200 serum samples and measured antibodies against a range of pathogens and vaccine antigens. In one community, infection rates fell to negligible levels in 2004, while the other community experienced consistent malaria cases over the 15 years. Children who grew up in the area with a persistent malaria burden had lower antibody levels against several unrelated pathogens and vaccine antigens than children from the area where malaria had disappeared. These differences persisted throughout childhood. Safari et al. also found that children who experienced malaria early in life in the malaria-low community had lower antibody levels at 10 years of age than children in the same community who had never had malaria. The more malaria episodes a child experienced, the lower their antibody responses tended to be. The findings of Safari et al. suggest that malaria may leave a long-lasting imprint on the immune system. If confirmed in other settings, this could help explain why vaccines are sometimes less effective in malaria-endemic regions and why the benefits of malaria control may extend beyond preventing malaria itself. Understanding how malaria influences long-term immune development could help improve vaccination strategies and child health in areas where malaria remains common.

Although lifespan has long been the focus of ageing research, preventing functional decline late in life is a more pressing societal need. Here, we investigate the basis of senescence and declining fitness during replicative ageing in budding yeast, and describe a metabolic perturbation that preserves late-life fitness even on an unrestricted glucose diet. We show that senescence can be prevented by constitutive activation of AMPK, though only for approximately half the ageing population, and use genetic and functional assays to link this heterogeneous response with differences in cytosolic acetyl coenzyme A (Acetyl-CoA) metabolism. In one class of ageing cell, AMPK activity maintains fitness late in life through pathways that transport cytosolic Acetyl-CoA into mitochondria, but AMPK also inhibits fatty acid synthesis which leads to lipid starvation in the other class of ageing cell. Therefore, AMPK activity has both positive and negative effects, but we show that constitutive AMPK activity uncoupled from fatty acid synthesis inhibition (the A2A mutant) suppresses senescence and maintains fitness in both classes of ageing cell. Our findings support a model in which lipid starvation and excess Acetyl-CoA availability are major drivers of senescence in replicatively aged wild-type yeast. This work shows that ageing is not intrinsically associated with declining fitness, at least in yeast, and that re-engineering highly conserved metabolic pathways allows fitness to be preserved very late in life.

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Nucleic acid aptamers hold promise for clinical applications, yet understanding their molecular binding mechanisms to target proteins, and efficiently optimizing their binding affinities, remain challenging. Here, we present CAAMO (Computer-Aided Aptamer Modeling and Optimization), which integrates in silico aptamer design with experimental validation to accelerate the development of aptamer-based RNA therapeutics. Starting from the sequence information of a reported RNA aptamer, Ta, for the SARS-CoV-2 spike protein, our CAAMO method first determines its binding mode with the spike protein's receptor binding domain (RBD) through a multi-strategy computational approach. We then optimize its binding affinity via structure-based rational design. Among the six designed candidates, five were experimentally verified and exhibited enhanced binding affinities compared to the original Ta sequence. Furthermore, we directly compared the binding properties of the RNA aptamers to neutralizing antibodies and found that the designed aptamer TaG34C demonstrated a comparable binding affinity to the RBD compared to the representative neutralizing antibodies analyzed in this study. This highlights its potential as an alternative to existing COVID-19 antibodies. Our work provides a robust approach for the efficient design of a relatively large number of high-affinity aptamers with complicated topologies. This approach paves the way for the development of aptamer-based RNA diagnostics and therapeutics.

Endosomes generate tubulo-vesicular carriers to redistribute proteins between plasma membrane, Golgi, and lysosomes. These transport routes employ distinct combinations of sorting nexins with complexes such as Retromer or Retriever. We now show that, while Retromer associates with the PROPPIN WIPI1 to form the previously described CROP complex, Retriever associates with WIPI2, forming CROP2. WIPI2 integrates into Retriever-dependent coat complexes since it interacts both with the Commander subunit CCDC93 and its cognate sorting nexin SNX17. CROP and CROP2 are exclusive in their physical associations and pathway selective. Whereas CROP2 is required for endosomal exit of Integrin β1, it does not affect CROP-dependent cargos such as EGFR or GLUT1. Vice versa, CROP is not required for Integrin β1 trafficking. WIPI1 and WIPI2 rely on similar molecular features. Their activity depends on the same FSSS motif to integrate into Retromer and Retriever complexes, respectively, and on an amphipathic membrane-inserting α-helix, which conveys membrane fission activity to PROPPINs. This suggests that Retromer and Retriever coats integrate distinct PROPPIN isoforms to promote fission of the respective endosomal carriers formed by them.

When a population of bacteria is exposed to a bactericidal antibiotic, most cells die rapidly. However, a subpopulation of antibiotic-tolerant cells known as 'persister cells' can survive for prolonged periods. In addition, antibiotic tolerance can be broadly induced throughout the population by stresses such as nutrient deprivation. However, the pathways required to maintain viability in this setting and how stress induces antibiotic tolerance are both poorly understood. To identify genetic determinants of antibiotic tolerance in mycobacteria, we carried out transposon insertion sequencing (Tn-Seq) screens in Mycobacterium abscessus (Mabs) exposed to bactericidal translation-inhibiting antibiotics. This analysis identified genes essential for the survival of both spontaneous persister cells, as well as for stress-induced tolerance, allowing the first genetic comparison of these states in mycobacteria. Pathway analysis identified multiple genes involved in the detoxification of reactive oxygen species (ROS), including the catalase-peroxidase katG, which contributed to survival in both unstressed and nutrient-starved cells. In addition, we found that endogenous ROS were generated by translation-inhibiting antibiotics, and that hypoxia impaired bacterial killing. KatG specifically contributed to survival following exposure to transcription or translation inhibitors, but not other antibiotic classes tested. Thus, the lethality of some antibiotics is amplified by toxic ROS accumulation, and antibiotic-tolerant cells require detoxification systems in order to remain viable. These findings further demonstrate that antibiotic-induced ROS plays a broad role in mediating antibiotic lethality across diverse organisms.

Despite many years of research, the quest to identify neural correlates of perceptual consciousness (NCC) remains unresolved. One major obstacle lies in methodological limitations: most studies rely on non-invasive neural measures with limited spatial or temporal resolution, making it difficult to disentangle proper NCCs from concurrent cognitive processes. Additionally, the relatively low sensitivity of non-invasive neural measures limits the interpretation of null findings in studies targeting proper NCCs. In this review, we discuss how human intracranial recordings can advance the search for NCCs by offering high spatiotemporal resolution, improved signal sensitivity, and broad cortical and subcortical coverage. We review studies that have examined NCCs at the level of single neurons and populations of neurons, and evaluate their implications on the debates between cognitive and sensory theories of consciousness. Finally, we highlight the limits of current intracranial human recordings and propose future directions based on emerging technologies and novel experimental paradigms.

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Morphogenesis of the anterior segment (AS) is crucial for healthy ocular physiology and vision, but is only partially understood. The Schlemm's canal (SC) and trabecular meshwork (TM) are essential drainage tissues within the AS, and their proper development and function are critical for maintaining normal intraocular pressure; abnormalities in either tissue can result in elevated pressure and glaucoma. Here, we use single-cell transcriptomic profiling to provide high-resolution molecular detail of mouse AS development with a particular focus on SC and TM. We report transcriptomes for ~130,000 single cells at key developmental stages from postnatal day 2 (P2) to P60. We provide the first annotation of cell types across these developmental stages and crucial information about dynamic changes in pathways/gene expression. Further, we trace developmental trajectories for TM cell and SC endothelial cell (SEC) subtypes and determine genes and signaling networks driving their specific cell fates. We demonstrate dynamic changes in signaling interactions between SC and the TM cells during their synchronized development. Collectively, our data lay a deep molecular foundation for AS development that will direct understanding of normal ocular physiology, glaucoma, and other AS conditions.

A novel computational pipeline reveals patterns of tissue movement and growth in early heart formation and advances virtual modeling of development.