The actin cytoskeleton is a fundamental and highly conserved structure that functions in diverse cellular processes, yet its direct contribution to organismal aging remains unclear. Here, we systematically interrogated how genetic and pharmacologic perturbations of actin structure and function influence lifespan and various hallmarks of aging in Caenorhabditis elegans. Whole-animal and tissue-specific knockdown of actin and key actin-binding proteins (ABPs) - arx-2 (Arp2/3), unc-60 (cofilin), and lev-11 (tropomyosin) - led to premature disruption of filament organization, reduced lifespan, and tissue-specific physiological defects. Bulk and single-nucleus RNA-sequencing revealed that ABP knockdowns elicited a strongly “aged” transcriptome. Actin dysfunction broadly exacerbated many age-associated phenotypes, including mitochondrial dysfunction, lipid dysregulation, loss of proteostasis, impaired autophagy, and intestinal barrier failure. Pharmacological destabilization with Latrunculin A mirrored genetic knockdowns, while mild stabilization with Jasplakinolide modestly extended lifespan, emphasizing that optimal and finely-tuned actin function is critical for healthy aging. Finally, analysis of human genome-wide association data revealed that common ACTB polymorphisms correlate with differences in age-related decline in gait speed, suggesting evolutionary conservation of actin’s role in healthy aging. Taken together, our results provide a comprehensive and publicly accessible resource that maps, for the first time, how actin integrity intersects with diverse aging pathways across tissues and scales. This descriptive framework is intended to enable future mechanistic discovery by offering a deep, unbiased dataset that can be integrated with emerging studies to define how actin dynamics contribute to aging.

The ability to use generalized prior experience to guide behavior in novel situations is a fundamental cognitive function. While recent evidence suggests that the hippocampus supports generalization how this is accomplished is poorly understood. Here we combined longitudinal optical imaging in head-fixed mice with computational modeling to examine generalization in hippocampal area CA1. We found that prior training accelerated behavioral adaptation to a novel environment and that this was accompanied by highly stable hippocampal representations. We identified putative memory traces from prior experience that enabled this generalization at multiple levels. At the population level, novel-context network dynamics rapidly aligned with low-dimensional neural subspaces established during prior experience. At the cellular level, spatially-informative weak "residual" activity reflecting generalizable information about the task structure appeared to bias which neurons form place fields (PFs) and where via behavioral timescale synaptic plasticity (BTSP). Finally, this was an active process as many PFs changed their reference frame in the novel environment to reflect the consistent task structure. In sum, the influence of memory traces on new PF formation may allow past experience to guide new learning such that representations are based on generalizable features, thus enabling rapid adaptive behavior in new contexts.

Most behaviors involve neural dynamics in high-dimensional activity spaces. A common approach is to extract dimensions that capture task-related variability, such as those separating stimuli or choices, yielding low-dimensional, task-aligned neural activity subspaces (“coding dimensions”). However, whether these dimensions actively drive decisions or merely reflect underlying computations remains unclear. Moreover, neural activity outside these coding subspaces (“residual dimensions”) is often ignored, though it could also causally shape neural dynamics driving behavior. We developed a recurrent neural network model that fits population activity and uncovers the dynamic interactions between coding and residual subspaces on single trials. Applied to electrophysiological recordings from the anterior lateral motor cortex (ALM) and motor thalamus in mice performing a delayed response task, our model demonstrates that perturbations of residual dimensions reliably alter behavioral choices, whereas perturbations of the choice dimension, which strongly encodes the animal’s upcoming decision, are largely ineffective. These perturbation effects arise because residual dimensions drive transient amplification across an intermediate number of coding and residual dimensions (\~10), before the dynamics collapse into discrete attractor states corresponding to the animal’s choice. By dissecting the low-dimensional variability underlying error trials, we find that it primarily shifts trajectories along residual dimensions, biasing single decisions. Residual activity in thalamus shapes cortical decision dynamics, implicating weakly selective thalamic populations in the emergence of cortical selectivity. Our findings challenge the conventional focus on low-dimensional coding subspaces as sufficient framework for understanding neural computations, demonstrating that dimensions previously considered task-irrelevant and accounting for little variance can have a critical role in driving behavior.

The first step to probing any potential interaction between two biomolecules is to determine their spatial association. In other words, if two biomolecules localize similarly within a cell, then it is plausible they could interact. Traditionally, this is quantified through various colocalization metrics. These measures infer this association by estimating the degree to which fluorescent signals from each biomolecule overlap or correlate. However, these metrics are, at best, proxies, and they depend strongly on various experimental choices. Alternatively, here we define a new strategy which leverages multispectral imaging and phasor analysis, termed the Phasor Mixing Coefficient (PMC). PMC measures the precise mixing of fluorescent signals in each pixel. We demonstrate how PMC captures complex biological subtlety by offering two distinct values, a global measure of overall color mixing and the homogeneity thereof. We additionally show that PMC exhibits less sensitivity to signal-to-noise ratio, intensity threshold, and background signal compared to canonical methods. Moreover, this method provides a means to visualize color mixing at each pixel. We show that PMC offers users a nuanced and robust metric to quantify biological association.

Geroscience aims to target the aging process to extend healthspan. However, even isogenic individuals show heterogeneity in natural aging rate and responsiveness to pro-longevity interventions, limiting translational potential. Using RNAseq analysis of young, isogenic, subpopulations of Caenorhabditis elegans selected solely on the basis of the splicing pattern of an in vivo minigene reporter that is predictive of future life expectancy, we find a strong correlation in young animals between predicted life span and alternative splicing of mRNAs related to lipid metabolism. The activity of two RNA splicing factors, Reversed Polarity-1 (REPO-1) and Splicing Factor 1 (SFA-1), early in life is necessary for C. elegans response to specific longevity interventions and leads to context-specific changes to fat content that is mirrored by knockdown of their direct target POD-2/ACC1. Moreover, POD-2/ACC1 is required for the same longevity interventions as REPO-1/SFA-1. In addition, early inhibition of REPO-1 renders animals refractory to late onset suppression of the TORC1 pathway. Together, we propose that splicing factor activity establishes a cellular landscape early in life that enables responsiveness to specific longevity interventions and may explain variance in efficacy between individuals.

Movement-related activity has been detected across much of the brain, including sensory and motor regions. However, much remains unknown regarding the distribution of movement-related activity across brain regions, and how this activity relates to neural computation. Here we analyzed movement-related activity in brain-wide recordings of more than 50,000 neurons in mice performing a decision-making task. We used multiple machine learning methods to predict neural activity from videography and found that movement-related signals differed across areas, with stronger movement signals close to the motor periphery and in motor-associated subregions. Delineating activity that predicts or follows movement revealed fine-scale structure of sensory and motor encoding across and within brain areas. Through single-trial video-based predictions of behavior, we identified activity modulation by uninstructed movements and their impact on choice-related activity analysis. Our work provides a map of movement encoding across the brain and approaches for linking neural activity, uninstructed movements and decision-making.

Cortical neurogenesis proceeds through a precise temporal program in which radial glia sequentially generate distinct neuronal subtypes and later glia, yet how post-transcriptional regulators coordinate these transitions remain poorly understood. We previously identified that a decreasing temporal gradient of the RNA-binding protein Imp encodes neural stem cell age in Drosophila. In this work, we extend our investigation to Imp1, a mammalian homologue of Imp, and its role in murine neocortical development. Using TEMPO to track birth-order dynamics, we demonstrate that sustained Imp1 overexpression during early neurogenesis arrests temporal fate progression, shifting neuronal populations toward deeper cortical layers V-VI. Immunostaining with layer-specific transcription factors Cux1 and Ctip2 confirmed that laminar repositioning results from genuine changes in neuronal identity rather than migratory defects, with neurons adopting molecular identities matching their final positions. Temporal window-specific manipulations reveal distinct stage-specific effects where early-stage Imp1 induction produces cascading effects on fate specification and moderately delays the neuronal-to-gliogenic transition, while mid-stage induction induces neuronal accumulation in the subplate region. Live imaging of organotypic cultures reveals continuous neuronal recruitment within intermediate and ventricular zones, with mid-stage-born neurons accumulating at significantly faster rates than earlier cohorts. Strikingly, mid-stage Imp1 overexpression also induces ectopic glial-like foci distributed throughout the cortical plate, featuring dramatic cellular expansion and morphological heterogeneity. These findings establish Imp1 as a dosage- and stage-dependent temporal rheostat orchestrating developmental transitions in radial glial progenitors, controlling neuronal fate decisions and spatial organization. This work advances our understanding of molecular timing mechanisms governing neuronal diversity in the mammalian cortex.

The brain microvascular functions are strongly influenced by the local microenvironment and cellular organization. Intracellular organelles, including primary cilia and centrioles, play critical roles in sensing and transmitting environmental cues and maintaining vascular integrity. However, their distribution across the brain vasculature remains poorly understood. In this study, we utilized publicly available large-volume electron microscopy datasets encompassing the cerebral vasculature from pial arterioles through parenchymal capillaries to pial venules. We systematically analyzed the cellular organization and characterized the distribution of primary cilia and centrioles in the mouse and human brain microvasculature. We found primary cilia exclusively on human cortical endothelial cells (ECs), indicating inter-species differences between mouse and human. Primary cilia were frequently present on mural cells (MCs, smooth muscle cells or pericytes) surrounding venules and capillaries but rarely observed on arterioles in both mouse and human brains. These MC primary cilia exhibited heterogeneity in ciliogenesis, including cells with ciliary pockets, surface cilia, and a hybrid configuration we refer as a partial pocket. In the mouse brain, many MC primary cilia were closely ensheathed by astrocytic endfeet and occasionally extended between them to establish proximity to synapses, whereas all primary cilia in the human brain were confined within the basal lamina. Our analysis of cellular density revealed similar EC densities between arterioles and venules in mice, but not in human. EC centrioles were consistently positioned against the direction of blood flow relative to the nuclei, suggesting that they may serve as a structural marker for flow direction. Collectively, these findings provide a comprehensive characterization of primary cilia and centrioles, highlighting distinct interspecies differences between mouse and human brain microvasculature. The proximity to neural cells and gradient distribution of these subcellular structures suggest that they may act as antennae for sensing mechanical and chemical signals within the brain microvascular environment.

Quantitative analysis of neuron morphology is essential in order to develop our understanding of circuit organisation and development. The recent acquisition of whole-brain electron microscopy-based (EM) reconstructions of the Drosophila melanogaster nervous system now provide the resolution needed to examine morphology at scale. Utilising these data, together with new computational tools, we extract and analyse the dendrites of all T4 and T5 neurons within one hemisphere (n \~ 6000).T4 and T5 neurons are the first uniquely direction-selective neurons in the visual pathway, and are classified into four subtypes (a,b,c, and d). Each subtype encodes one of four cardinal motion directions (up, down, forwards, backwards). The dendrites of these neurons form in two distinct neuropils, the Medulla (T4) and the Lobula (T5), and are asymmetrically oriented in a direction inverse to the direction of motion which they encode. However, their densely overlapping and compact arbours has made rigorous morphological quantification challenging. The presence of differences beyond their characteristic orientation, both between T4 and T5, as well as within subtypes, has remained poorly understood.Our analysis reveals a high degree of structural similarity across both types and subtypes. Particularly, measures of geometry and graph topology show only minor variation, with no consistent separation between T4 and T5, or their subtypes.These results indicate that, despite forming in different neuropils, and serving distinct motion directions, T4 and T5 dendrites follow closely aligned morphological patterns. This suggests that their arborization may be governed by shared developmental constraints and mechanisms.

The timing of spikes dictates a neuron’s impact on downstream circuits and behavior, and spike timing is determined by the membrane potential (Vm). However, due to technical challenges, it has been impossible to analyze the relative timing of Vm dynamics between neurons during behavior. Using large scale membrane voltage imaging, we simultaneously recorded Vm from many individual hippocampal neurons in animals engaged in a virtual spatial task. We found that relative phase of Vm theta oscillations across neurons exhibit gradual or discrete shifts depending on spatial position. This finding extends beyond previous studies showing Vm dynamics in single neurons or spiking activity in multiple neurons, revealing previously unknown evidence for consistent coding of space by spike-independent relative phase of Vm theta dynamics between neurons.