Optical nanoscopy of intact biological specimens has been transformed by recent advancements in hydrogel-based tissue clearing and expansion, enabling the imaging of cellular and subcellular structures with molecular contrast. However, existing high-resolution fluorescence microscopes are physically limited by objective-to-specimen distance, which prevents the study of whole-mount specimens without physical sectioning. To address this challenge, we developed a photochemical strategy for spatially precise sectioning of specimens. By combining serial photochemical sectioning with lattice light-sheet imaging and petabyte-scale computation, we imaged and reconstructed axons and myelin sheaths across entire mouse olfactory bulbs at nanoscale resolution. An olfactory bulb–wide analysis of myelinated and unmyelinated axons revealed distinctive patterns of axon degeneration and de-/dysmyelination in the neurodegenerative brain, highlighting the potential for peta- to exabyte-scale super-resolution studies using this approach. High-resolution microscopes have a short working distance, making it difficult to see deep within large biological samples such as an intact brain. Slicing the tissue with a blade can reach deeper, but this often distorts or destroys the fine structures that scientists want to study. By embedding a sample in a light-sensitive hydrogel, Wang et al. demonstrated a gentler approach using a precise ray or sheet of light to dissolve or cut away tissue layer by layer. After each layer is removed, the newly exposed surface is imaged, allowing for a complete, high-resolution, three-dimensional reconstruction without damaging physical contact. 

bioRxiv preprint: https://www.biorxiv.org/content/10.1101/2024.08.01.605857v1

Researchers have long noted the differences in synapse count between different EM reconstructions of similar circuitry. In this paper we attempt to determine the portion of these differences that may be due to different sample preparation and imaging techniques, in particular serial-section transmission imaging (SS-TEM) compared to focused ion beam with scanning electron microscopy (FIB-SEM). To do this, we compare synapse detection in the major Drosophila EM reconstructions - FANC, MANC, FAFB (with original and new synapses), male CNS, BANC, and HemiBrain, plus several smaller reconstructions. We look at raw synapse counts to avoid any dependence on proofreading, and compensate insofar as possible for the confounds of sample sizes differences and different software detection efficiency. The result are estimates, per compartment and for the sample as a whole, of the number of synapses that would be visible to a skilled human observer. These are then compared across all samples, using regions which are reconstructed in common for each sample pair. We find that in almost all known cases where a volume has been reconstructed by both techniques, isotropic FIB-SEM reconstructions show more human-visible synapses than microtome sliced reconstructions, typically by more than 40%. This strongly suggests, but does not conclusively prove, that synapses are easier to see in isotropic FIB-SEM data.

Primary cilia are microtubule-based sensory organelles that have been conserved throughout eukaryotic evolution. As discussed in this Review, a cilium is an elongated and highly specialized structure, and, together with its ability to selectively traffic and concentrate proteins, lipids and second messengers, it creates a signaling environment distinct from the cell body. Ciliary signaling pathways adopt a bow-tie network architecture, in which diverse inputs converge on shared effectors and second messengers before diverging to multiple outputs. Unlike other cellular bow-tie systems, cells exploit ciliary geometry, compartmentalization and infrastructure to enhance sensitivity at multiple scales, from individual molecular reactions to entire signaling pathways. In cilia, integration of the bow-tie network architecture with their specialized structure and unique environment confers robustness and evolvability, which enables cilia to acquire diverse signaling roles. However, this versatility comes with vulnerability - rare mutations that disrupt the features most essential for cilia robustness cause multisystem ciliopathies.

Two simple models—vaulting over stiff legs and rebounding over compliant legs—are employed to describe the mechanics of legged locomotion. It is agreed that compliant legs are necessary for describing running, and that leg compliance is also present during walking. Stiff legs continue to be employed to model walking under the assumption that the compliance of the leg during walking is low enough to be considered stiff. Here we study gait choice and walk-to-run transition in a biped with compliance and show that the principles underlying gait choice and transition are completely different from stiff legs. Two findings underpin our conclusions: First, at the same speed, step length and stance duration, multiple gaits that differ in the number of times the leg expands and contracts during a single stance are possible. Among them, humans and other animals choose the (normal) gait with M-shaped vertical ground reaction forces (vGRF) not just because of energy considerations but also constraints from forces. Second, the transition from walking to running occurs because of three factors: vGRF minimum at mid-stance characteristic of normal walking, synchronization of horizontal and vertical motions during single support, and velocity redirection during the double support. The insight above required an analytical approximation of the double spring-loaded pendulum (DSLIP) model describing the intricate oscillatory dynamics that relate single and double support phases. Additionally, we also examined DSLIP as a quantitative model for locomotion and conclude that DSLIP speed range is limited. However, insights gleaned from the analytical treatment of DSLIP are general and will inform the construction of more accurate models of walking.

bioRxiv preprint: https://doi.org/10.1101/2024.09.23.612940

fMRI signals were traditionally seen as slow and sampled in the order of seconds, but recent technological advances have enabled much faster sampling rates. We hypothesized that high-frequency fMRI signals can capture spontaneous neural activity that index brain states. Using fast fMRI (TR=378ms) and simultaneous EEG in 27 humans drifting between sleep and wakefulness, we found that fMRI spectral power increased during NREM sleep (compared to wakefulness) across several frequency ranges as fast as 1Hz. This fast fMRI power was correlated with canonical arousal-linked EEG rhythms (alpha and delta), with spatiotemporal correlation patterns for each rhythm reflecting a combination of shared arousal dynamics and rhythm-specific neural signatures. Using machine learning, we found that alpha and delta EEG rhythms can be decoded from fast fMRI signals, in subjects held-out from the training set, showing that fMRI as fast as 0.9Hz (alpha) and 0.7Hz (delta) contains reliable neurally-coupled information that generalizes across individuals. Finally, we demonstrate that this fast fMRI acquisition allows for EEG rhythms to be decoded from 3.8s windows of fMRI data. These results reveal that high-frequency fMRI signals are coupled to dynamically varying brain states, and that fast fMRI sampling allows for more temporally precise quantification of spontaneous neural activity than previously thought possible.

We have identified a Drosophila species in which males exhibit spontaneous, elaborate, and robust intermale sexual behavior. Males of D. santomea, a West African island endemic, distinguish conspecific sexes but court males and females promiscuously and seldom attack. Elevated intermale courtship derives from at least three changes in two separate pheromone systems. In males, the sexually monomorphic cuticular pheromone 7-tricosene promotes rather than inhibits courtship and the courtship-inhibiting olfactory pheromone cVA is reduced 84-92% compared to close relatives, including the sibling species D. yakuba. The third change is surprisingly in D. santomea females, where cVA suppresses rather than promotes sexual receptivity. The female cVA switch and male cVA reduction may have co-evolved to maintain efficient intraspecific mating in D. santomea but prevent sympatric hybridization with D. yakuba, or to reduce intraspecific aggression. We find that high intermale courtship and low cVA also co-occur and appear selectively derived in a distant monomorphic species D. persimilis, implying pheromonal and behavioral convergence in the two recently speciated taxa. The data suggest that sequential changes in the behavioral valence and levels of pheromones explain the recent evolutionary emergence of intermale sexual behavior in Drosophila.

Matriglycan is a linear glycan (xylose-β1,3-glucuronate), which binds proteins in the extracellular matrix that contain laminin-globular domains and Lassa Fever Virus. It is indispensable for neuromuscular function. Matriglycan of insufficient length can cause muscular dystrophy with abnormal brain and eye development. LARGE1 (Like-acetylglucosaminyltransferase-1) uniquely synthesizes matriglycan on dystroglycan. The mechanism of matriglycan synthesis is not obvious from cryo-EM reconstructions of LARGE1. However, by reconstituting activity in vitro on recombinant prodystroglycan we show that the presence of the dystroglycan N-terminal domain (DGN), phosphorylated core M3, and a xylose-glucuronate primer are necessary for matriglycan polymerization by LARGE1. By introducing active site mutations, we demonstrate that LARGE1 processively polymerizes matriglycan on prodystroglycan, with its length regulated by the dystroglycan prodomain, DGN. Our enzymatic analysis of LARGE1 uncovers the mechanism of matriglycan synthesis on dystroglycan, which can form the basis for therapeutic strategies to treat matriglycan-deficient neuromuscular disorders and arenaviral infections.

Sex differences in behaviour exist across the animal kingdom, typically under strong genetic regulation. In Drosophila, previous work has shown that fruitless and doublesex transcription factors identify neurons driving sexually dimorphic behaviour. However, the organisation of dimorphic neurons into functional circuits remains unclear.We now present the connectome of the entire Drosophila male central nervous system. This contains 166,691 neurons spanning the brain and ventral nerve cord, fully proofread and comprehensively annotated including fruitless and doublesex expression and 11,691 cell types. By comparison with a previous female brain connectome, we provide the first comprehensive description of the differences between male and female brains to synaptic resolution. Of 7,319 cross-matched cell types in the central brain, 114 are dimorphic with an additional 262 male- and 69 female-specific (totalling 4.8% of neurons in males and 2.4% in females).This resource enables analysis of full sensory-to-motor circuits underlying complex behaviours as well as the impact of dimorphic elements. Sex-specific and dimorphic neurons are concentrated in higher brain centres while the sensory and motor periphery are largely isomorphic. Within higher centres, male-specific connections are organised into hotspots defined by male-specific neurons or the presence of male-specific arbours on neurons that are otherwise similar between sexes. Numerous circuit switches reroute sensory information to form conserved, antagonistic circuits controlling opposing behaviours.

The study of foraging is central to a renewed interest in naturalistic behavior in neuroscience. Applying a foraging framework grounded in behavioral ecology has enabled probing of the mechanisms underlying cognitive processes such as decision-making within a more ecological context. Yet, foraging also involves myriad other aspects, including navigation of complex environments, sensory processing, and social interactions. Here, we first provide a brief overview of the neuroscience of foraging decisions, and then combine insights from behavioral ecology and neuroscience to review the role of these additional dimensions of foraging. We conclude by highlighting four opportunities for the continued development of foraging as an ethological framework for neuroscience: integrating normative and implementation-level models, developing new tools, enabling cross-species comparisons, and fostering interdisciplinary collaboration.

BACKGROUND: Kidney epithelial cells perform complex vectorial fluid and solute transport at high volumes and rapid rates. Their structural organization both reflects and enables these sophisticated physiological functions. However, our understanding of the nanoscale spatial organization and intracellular ultrastructure that underlies these crucial cellular functions remains limited.

METHODS: To address this knowledge gap, we generated and reconstructed an extensive electron microscopic dataset of renal proximal tubule (PT) epithelial cells at isotropic resolutions down to 4nm. We employed artificial intelligence-based segmentation tools to identify, trace, and measure all major subcellular components. We complemented this analysis with immunofluorescence microscopy to connect subcellular architecture to biochemical function.

RESULTS: Our ultrastructural analysis revealed complex organization of membrane-bound compartments in proximal tubule cells. The apical endocytic system featured deep invaginations connected to an anastomosing meshwork of dense apical tubules, rather than discrete structures. The endoplasmic reticulum displayed distinct structural domains: fenestrated sheets in the basolateral region and smaller, disconnected clusters in the subapical region. We identified, quantified, and visualized membrane contact sites between endoplasmic reticulum, plasma membrane, mitochondria, and apical endocytic compartments. Immunofluorescence microscopy demonstrated distinct localization patterns for endoplasmic reticulum resident proteins at mitochondrial and plasma membrane interfaces.

CONCLUSIONS: This study provides novel insights into proximal tubule cell organization, revealing specialized compartmentalization and unexpected connections between membrane-bound organelles. We identified previously uncharacterized structures, including mitochondria-plasma membrane bridges and an interconnected endocytic meshwork, suggesting mechanisms for efficient energy distribution, cargo processing and structural support. Morphological differences between 4nm and 8nm datasets indicate subsegment-specific specializations within the proximal tubule. This comprehensive open-source dataset provides a foundation for understanding how subcellular architecture supports specialized epithelial function in health and disease.