Sleep is regulated by a homeostatic process and associated with an increased arousal threshold, but the genetic and neuronal mechanisms that implement these essential features of sleep remain poorly understood.To address these fundamental questions, we performed a zebrafish genetic screen informed by human genome-wide association studies.We found that mutation of serine/threonine kinase 32a (stk32a) results in increased sleep and impaired sleep homeostasis in both zebrafish and mice, and that stk32a acts downstream of neurotensin signaling and the serotonergic raphe in zebrafish. stk32a mutation reduces phosphorylation of neurofilament proteins, which are co-expressed with stk32a in neurons that regulate motor activity and in lateral line hair cells that detect environmental stimuli, and ablating these cells phenocopies stk32a mutation. Neurotensin signaling inhibits specific sensory and motor populations, and blocks stimulus-evoked responses of neurons that relay sensory information from hair cells to the brain.Our work thus shows that stk32a is an evolutionarily conserved sleep regulator that links neuropeptidergic and neuromodulatory systems to homeostatic sleep drive and changes in arousal threshold, which are implemented through suppression of specific sensory and motor systems.

A central challenge in single-cell biology is understanding how molecular programs drive changes in cellular architecture that enable specialized function. A striking example of cellular remodeling is the differentiation of airway stem cells into the respiratory multiciliated epithelium, a protective tissue barrier that clears inhaled pathogens and particulate matter. Here, we present its first three-dimensional nanometer-scale reconstruction, revealing coordinated changes in cellular organization, organelle topology, and inter-organelle contacts during multiciliogenesis. We uncover a structural and functional association between motile cilia and mitochondria mediated by rootlets, striated cytoskeletal fibers that remain poorly characterized in human airway multiciliated cells. Rootlets connect to basal bodies through a multiprotein linker containing the uncharacterized rootletin/CROCC homolog CROCC2, oscillate at frequencies comparable to ciliary beating, promote basal body alignment, and when lost, reduce maximal mitochondrial respiratory capacity. Altogether, this work integrates structural, dynamic, and functional analyses to elucidate mechanisms underlying airway mucociliary defense.

Direct identification of macromolecular complexes in their native context remains a major barrier to unbiased biological discovery. This challenge is particularly acute in mammalian sperm nuclei, in which condensed chromatin is interspersed with poorly understood phase-separated compartments termed nuclear vacuoles. Vacuoles are associated with reduced fertilization efficiency, yet their composition remains unclear. Here we combine high-resolution in situ cryo-electron tomography (cryo-ET) with AlphaFold docking to identify vacuole components as proteasomes, the proteasome activator PA200, and ferritin. In situ structures at resolutions up to 3.8 Å reveal distinct proteasome-PA200 associations and gating states, consistent with a stepwise activation mechanism. Ferritin assemblies exhibit heterogeneous mineralization states and directly contact chromatin. Together, these findings establish the molecular organization of sperm nuclear vacuoles and implicate protein turnover and metal homeostasis in shaping the nuclear landscape, while demonstrating the power of in situ cryo-ET to resolve protein identity and conformational dynamics in native cellular environments.

In the wild, a mouse must flexibly perform multiple computations at once, rapidly navigating, sampling its environment, forming and using memories, and balancing internal needs. Capturing the self-paced, low-repetition, complex nature of decision making in the wild while preserving the experimental control needed to interpret its process remains a challenge. Here we show that lab mice, without food restriction, learn to forage for hidden resources cued by ambiguous sounds in a dynamic environment by spatiotemporally reorganizing their existing behavioral repertoire. Using a novel closed-loop assay, we required mice to sit still to receive informative but hard-to-localize sound cues emanating from one correct location they must find, out of 157 possible locations in a large arena. With experience, mice become efficient hunters. Efficient hunting cannot be explained by an increase in a particular behavioral module, but instead, mice reorganize existing behaviors into clustered bouts of high-quality sampling and site-checking. Looking forward, this work establishes an approach for studying the neural, molecular, and evolutionary basis of naturalistic decision-making in mice.

Fluorescence microscopy applications often require specialized instruments that are optimized for different experimental goals. Here, we present a reconfigurable microscopy module that integrates highly inclined swept tile (HIST) illumination for high-sensitivity single-molecule imaging and line-scanning confocal microscopy for rapid and optically sectioned volumetric acquisition. The system shares major hardware components, including lasers, scanning optics, and detection hardware, while employing unique beam shaping pathways to enable rapid switching between modalities without realignment. We characterize the module performance by measuring the excitation beam profiles, the point spread functions (PSF), and the optical transfer functions (OTF) across 40x, 60x, and 100x magnifications and demonstrate imaging applications including diffraction-limited fixed and live-cell volumetric imaging, fluorescence recovery after photobleaching, and super-resolution DNA-PAINT and single particle tracking (SPT). We also demonstrate the capability to execute multimodal imaging workflows by performing confocal imaging for chromatin density classification correlated with SPT data of nuclear proteins with diverse functions. Together, these results demonstrate a versatile imaging platform capable of supporting complementary fluorescence imaging modalities within a single instrument.

Dendrites transform local electrical activity into intracellular Ca2+ signals that drive plasticity1,2, yet the voltage→Ca2+ mapping during natural behavior remains poorly defined. Here, we measure this transfer function via simultaneous voltage and Ca2+ imaging throughout the dendritic arbors of hippocampal CA2 pyramidal neurons in behaving mice. Dendritic Ca2+ exhibited a hierarchical activation pattern dominated by back-propagating action potentials: simple spikes primarily drove somatic and proximal Ca2+, whereas complex spikes produced larger somatic Ca2+ signals and propagated farther into distal dendrites, sometimes in a branch-selective manner. Dendrite-restricted co-activation of voltage and Ca2+ without concurrent somatic events was rare. A biophysics-inspired model accurately predicted local Ca2+ transients from local voltage waveforms. Our data and model provide a quantitative understanding of when – and why – dendritic Ca2+ signals in CA2 pyramidal cells arise during behavior.

Sickness behaviors are common in cancer-associated cachexia and affect up to half of lung cancer patients. We demonstrate that among the most common cancer mutations, loss of liver kinase B1 () promotes the development of cachexia in preclinical models of lung cancer. In an effort to improve caloric intake with an obesogenic high-fat diet, we paradoxically observed worsened cachexia-associated sickness. We found that local production of prostaglandin E2 (PGE), rather than circulating factors, promotes sickness and that genetic, dietary, and pharmacological inhibition of tumor-derived PGE suppresses sickness and cachexia. Notably, we demonstrate that lung sensory neuron abrogation prevents PGE-dependent cachexia. Our study establishes localized tumor-derived signals to sensory neurons, rather than circulating factors, as drivers of cachexia and highlights a previously unknown role of the peripheral nervous system in cancer cachexia.

Calcium imaging with miniature endoscopes has become an essential tool in neuroscience, but conventional miniscopes typically record signals from only a single calcium indicator. Here, we present a dual-color miniature endoscope (miniscope) that enables simultaneous calcium imaging from two neuronal populations using spectrally distinct genetically encoded indicators. In freely moving mice, we used this system to record activity from striatal neurons of the direct (dSPN) and indirect (iSPN) pathways. We showed that dSPNs were activated earlier than iSPNs during contraversive movements, with dSPNs preferentially active during acceleration and iSPNs during deceleration. During ipsiversive turns, however, this temporal relationship was reversed. These findings indicate that dSPNs and iSPNs are not concurrently active, but instead exhibit complementary, direction-dependent dynamics that govern movement velocity. Our dual-color miniscope provides a compact, cost-effective platform for simultaneous two-population imaging, offering new opportunities to dissect coordinated activity across neural circuits in freely behaving animals.

In natural environments, animals must allocate choices across multiple concurrently available resources when foraging, a complex decision-making process not fully captured by existing models. To understand how rodents learn to navigate this challenge, we developed a novel paradigm in which naive, water-restricted mice freely sampled six options of varying quality arranged around a large (∼2 m) arena. Mice exhibited rapid learning, matching their choices to integrated reward probabilities across six options within tens of minutes. A reinforcement learning model with distinct states for staying vs. leaving an option, as well as a dynamic global learning rate, accurately reproduced behavior. Fiber photometry recordings revealed that dopamine in the nucleus accumbens core (NAcC), but not the dorsomedial striatum (DMS), reflected this learning rate. Moreover, optogenetic manipulation of NAcC dopamine bidirectionally altered learning in quantitative agreement with model predictions. Together, we identified a neural substrate of a learning algorithm enabling efficient multi-option foraging in large spatial environments.

Most animals move intermittently. Of these, animals that fly and swim move in two stages: during active propulsion, when they beat their wings or swing their tails, and right after, when they glide or coast through the air or water. The kinematics of active movements are typically well studied, but the coasting period afterwards is less understood. We used larval zebrafish as a model of intermittent locomotion. Larval zebrafish swim in discrete bouts lasting \~200 ms, coast for short durations and distances, and then are largely stationary for \~800 ms. We find that larval zebrafish do not coast like a passive object. While they coast for distances that are correlated with the prior swim’s strength, as expected from physics, they seem to be able to modulate the mechanics of their coasting: the time constant is not a constant, but varies from coast-to-coast. Furthermore, the time constant is correlated with the duration of the interval between swim bouts. This suggests that larval zebrafish can either shorten or extend their coasting duration, and that coasting inhibits the initiation of the next swim. Finally, the time constant changes quickly, while other swimming kinematics (e.g., distance swum) are autocorrelated over the course of at least several seconds. Altogether, this suggests that fish, and perhaps other animals that move intermittently, actively modulate their post-propulsion movement, possibly to gather sensory feedback in the absence of blurry visual feedback associated with fast locomotion.