High-speed volumetric imaging technique is a useful tool to record and analyze biological activity. However, current volumetric imaging methods suffer from low imaging speed, poor contrast or resolution. Point-scanning microscopy and light-sheet fluorescence microscopy (LSFM) can effectively suppress the background noise, but their mechanical scanning strategy limits the speed of volumetric imaging. Light-field microscopy (LFM) captures 3D information without any scanning device, but constrained by the whole-volume illumination and the fixed microlens array, the contrast is low and the resolution is fixed and impossible to be optimized for different imaging scenarios. Here we report a selective volume microscopy (SVM) based on programmable light field microscopy (PLFM) with an engineered point spread function (PSF) strategy, which achieves flexible resolution tuning combined with selective illumination for optimized spatiotemporal resolution. By selectively illuminating and programmably detecting the volumes of interest (VOI) of the sample, SVM improved reconstruction contrast and spatial resolution by ∼2.6 times and ∼1.2 times in comparison with PLFM. The improvement of reconstruction quality of SVM is demonstrated by imaging the fluorescent beads and larval zebrafish across 665 μm × 665 μm × 200 μm at 100 Hz.

Neurons represent changes in external and internal environments by altering their activity patterns. While coherent brain-wide patterns of neural activity have been observed in neuronal populations, very little is known about their dimensionality, geometry, and how they are correlated with sensory inputs. Here, we recorded the activity of most head neurons in Caenorhabditis elegansexperiencing changes in bacterial or control buffer stimuli around their nose. We first classified active neurons into six functional clusters: two sensory neuron clusters (ON and OFF responding to addition and removal of stimuli, respectively) and four motor/command neuron clusters (AVA, RME, SMDD, and SMDV). Next, we estimated stimulus selectivity for each cluster and found that while sensory neurons exhibit their maximal responses within 15 s, changes in bacterial stimuli drive maximal responses in command and motor neuron clusters after tens of seconds. Furthermore, we show that bacterial stimuli induce neural dynamics that are best described by a hyperbolic, not Euclidean, space indicating hierarchical organization in C. elegans neural population dynamics. This space was 8-dimensional, with dimensions that aligned in one sensory direction (ON and OFF neurons), two motor directions (forward-backward and dorsal-ventral), and the rest of dimensions corresponding with unclassified neurons. Collectively, we show that C. elegans neural activity can be effectively represented in low-dimensional hyperbolic space and describe how sensory information is processed by the neural network.

To understand neural computations, one would like to relate circuit dynamics to neuronal intrinsic excitability and connectivity. We developed tools for large-scale all-optical electrophysiology and applied them to approximately 300 layer 1 interneurons per mouse in vivo. Across two mice, targeted stimulation and voltage imaging identified 1,469 inhibitory and 5,752 directed electrical connections among 14,312 tested pairs. Presynaptic identity was more strongly associated with inhibitory post-synaptic potential (IPSP) amplitude, whereas postsynaptic identity was more strongly associated with IPSP decay time. Gap junctional and synaptic interactions had distinct spatiotemporal kernels, with corresponding signatures in spontaneous activity. Excitability measured under optogenetic stimulus predicted spiking responses to subthreshold voltage, depolarization rate, and spike history during spontaneous firing. Genetic subtypes also showed different behavioral-state-dependent voltage-to-spike relationships despite similar population-average subthreshold voltages. These measurements link cellular properties and pairwise interactions to spontaneous circuit dynamics, establishing an approach for functional circuit mapping in vivo.

The endocytic pathway plays key roles in facilitating and regulating synaptic plasticity, but how its components are organized at spines undergoing plasticity is not fully understood. Here we use live-cell imaging and single-particle tracking to examine the localization of endosomes and lysosomes in response to plasticity-inducing stimuli. We find that endosomes and lysosomes are positioned and retained near the bases of stimulated spines after the induction of structural plasticity in a manner dependent on actin polymerization in the dendritic shaft. Spines with endocytic compartments near their bases exhibit greater glutamate receptor turnover and more sustained enlargement after stimulation than spines lacking them. These results suggest that actin polymerization in the dendritic shaft acts as a hub to organize critical cellular machinery at the bases of spines undergoing synaptic plasticity.

Synaptojanin 1 is a brain enriched phosphoinositide phosphatase implicated in endocytosis at the synapse. A mutation (R258Q) that selectively impairs its Sac1 phosphatase domain causes early onset familial Parkinsonism. Neurons of mice with this mutation display synaptic vesicle traffic defects across the brain, but selective dystrophic changes in a subset of dopaminergic axons in the dorsolateral striatum. Using correlative light microscopy-FIB-SEM of mutant mouse striata to visualize in 3D these abnormal structures we show that they represent clusters of focal axonal dilations harboring massive, onion-like DAT enriched plasma membrane infoldings, generally localized next to cell bodies of neighboring cells, often engulfing evaginations of such cells. This dysmorphia was associated with a deficit in dopamine release in the same striatal region. Given the involvement of Synj1 in endocytic mechanisms, these structures may reflect an imbalance between exocytosis and endocytosis. Their occurrence only in a subset of axons suggests a vulnerability threshold of these axons beyond which the expansion of the plasma membrane is not counteracted by compensatory mechanisms.

Effective navigation is critical for survival, but how animals contend with the scale, complexity, and self-similarity of natural landscapes is unknown. Here, we investigate spontaneous exploratory mouse behavior, both outdoors and in dark self-similar landscapes made from tens to hundreds of tiles of varying heights. Within 2 hours of exploration, mice detected small covert changes to the landscape and jumped across ledges to invisible landing spots, revealing detailed spatial knowledge. In this process, mice converged on a sparse set of memorized paths. These routes depended on prior experience and increased the compressibility of sensory information over time. Our results demonstrate that mice rapidly learn sparse internal representations of their surroundings and suggest that animals use structured behavioral sequences to reduce the complexity of natural navigation.

Cortical positioning of oocyte meiotic spindles is required for reducing chromosome number by segregating chromosomes into polar bodies. In C. elegans, kinesin-1 transports membrane-bound organelles inward into a central ball that is hypothesized to push the meiotic spindle outward. Using focused ion beam scanning electron microscopy, we found that the percent volume occupied by membrane-bound organelles was 28% in a mature oocyte that has completed endocytosis of the yolk precursor, vitellogenin, compared to 17% in a less mature oocyte that has not yet endocytosed vitellogenin. We therefore hypothesized that blocking endocytosis of vitellogenin would reduce the size of the central ball of organelles packed inward by kinesin-1 and limit outward movement of the spindle. Depletion of vitellogenin or proteins required for endocytosis resulted in a smaller central volume of kinesin-coated organelles and defective spindle positioning. A computer simulation of a simple exclusion model faithfully recapitulated wildtype and mutant spindle position phenotypes. These results support a volume exclusion model for positioning acentriolar meiotic spindles.

Proteomics is a powerful tool for profiling the protein landscape underlying cellular and physiological processes. Advances in mass spectrometry-based methods, such as tandem-mass-tags (TMT) isobaric labeling-based proteomics at both MS2 and MS3, label-free quantification (LFQ) with data-dependent-acquisition (DDA) or data-independent acquisition (DIA)-based proteomics, and stable isotope labeling with amino acid (SILAC) spike-in-assisted proteomics, enable quantitative proteome comparisons across biological conditions. These five quantitative proteomic methods offer distinct advantages in coverage, throughput, and quantitative reproducibility. Here, we treated them as orthogonal approaches, retaining protein candidates with significant, consistent-direction changes supported across multiple independently instrumented methods to identify high-confidence protein-level changes associated with longevity. Insulin/insulin-like growth factor signaling (IIS) is a highly conserved longevity regulatory pathway from Caenorhabditis elegans to humans. By quantitatively profiling IIS mutants in C. elegans and performing genetic screening, we obtained a systems-view of proteomic changes linked to longevity and identified new longevity regulators. This systematic cross-method comparison also serves as a valuable resource for guiding the selection of quantitative proteomics approaches.

Neuromodulators are generally understood to act through expansive projections that broadcast signals relatively indiscriminately, in stark contrast to the precise, synapse-bound organization of fast neurotransmitters like glutamate and GABA. This dichotomy has left the local architecture of neuromodulatory release and receptor engagement largely obscure. Here, using dopamine as a model system, we reveal that neuromodulatory signaling can be guided by precise molecular specificity. We show that dopamine axons exhibit a tropism for neurons expressing D1 or D2 dopamine receptors. Single-bouton-resolved optical imaging of release shows that dopamine is not broadcast stochastically but is highly localized to varicosities that form direct, synapse-like appositions with receptor-expressing somata and dendrites. To map endogenous receptor organization relative to release sites, we generated ALFA-tagged D1 and D2 receptor knock-in mice (ALFADoR mice). In cultures and intact tissue, D1 and D2 receptors formed discrete puncta rather than exhibiting diffuse membrane distributions. Across striatum, amygdala, and prefrontal cortex, receptor puncta were preferentially positioned near dopamine varicosities, and apposed receptor puncta were larger than nonapposed puncta. These findings reveal a synapse-like architecture for dopamine signaling in which release sites and receptor clusters are spatially coupled to support a precise and efficient neuromodulation.

We release an enhanced FIB-SEM volume and associated neuron, mitochondria, synapse and glia automated segmentations for the complete, gap- and artifact-free central nervous system of the first instar female larval Drosophila melanogaster. Segmentation data is largely unproofread besides select neurons in the premotor and proprioceptive system and select subvolumes where synapses have been manually annotated, serving as training and evaluation data. The dataset represents the first complete central nervous system of the larval fly, and now awaits substantial improvement of machine vision approaches that effectively leverage domain knowledge for the automated yet data-guided proofreading, or substantial and costly human proofreading. All the data and methods are publicly available for further improvement and analysis.