From the star-nosed mole’s eponymous mechanosensory organ to the platypus’ electroreceptive bill, the expansion of sensory neuron populations detecting important environmental cues is a widespread evolutionary phenomenon in animals1–6. How such neuron increases contribute to improved sensory detection and behaviour remain largely unexplained. Here we address this question through comparative analysis of olfactory pathways in Drosophila melanogaster and its close relative Drosophila sechellia, which feeds and breeds exclusively on Morinda citrifolia noni fruit7–9. We show that D. sechellia displays selective, large expansions of noni-detecting olfactory sensory neuron (OSN) populations, and that this trait has a multigenic basis. These expansions are accompanied by an increase in synaptic connections between OSNs and their projection neuron (PN) partners that transmit information to higher brain centres. Quantification of odour-evoked responses of partner OSNs and PNs reveals that OSN population expansions do not lead to heightened PN sensitivity, beyond that due to sensory receptor tuning differences. Rather, these pathways – but not those with conserved OSN numbers – exhibit non-adapting PN activity upon odour stimulation. In noni odour plume-tracking assays, D. sechellia exhibits enhanced performance compared to D. melanogaster. Through activation and inhibition of defined proportions of a noni-sensing OSN population, we establish that increased neuron numbers contribute to this behavioural persistence. Our work reveals an unexpected functional impact of sensory neuron expansions that can synergise with peripheral receptor tuning changes to explain ecologically-relevant, species-specific behaviour.

Motor systems flexibly implement diverse motor programs to pattern behavioral sequences, yet their neural underpinnings remain unclear. Here, we investigated the neural circuit mechanisms of flexible courtship behavior in Drosophila. Courting males alternately produce two types of courtship song. By recording calcium signals in the ventral nerve cord (VNC) in behaving flies, we found that different songs are produced by activating overlapping neural populations with distinct motor functions in a combinatorial manner. Recordings from the brain suggest that song is driven by two descending pathways – one defines when to sing and the other specifies what song to sing. Connectomic analysis reveals that these “when” and “what” descending pathways provide structured input to VNC neurons with different motor functions. These results suggest that dynamic changes in the activation patterns of descending pathways drive different combinations of motor modules, thereby flexibly switching between different motor actions.

Healthy mitochondria are critical for reproduction. During aging, both reproductive fitness and mitochondrial homeostasis decline. Mitochondrial metabolism and dynamics are key factors in supporting mitochondrial homeostasis. However, how they are coupled to control reproductive health remains unclear. We report that mitochondrial GTP (mtGTP) metabolism acts through mitochondrial dynamics factors to regulate reproductive aging. We discovered that germline-only inactivation of GTP- but not ATP-specific succinyl-CoA synthetase (SCS) promotes reproductive longevity in Caenorhabditis elegans. We further identified an age-associated increase in mitochondrial clustering surrounding oocyte nuclei, which is attenuated by GTP-specific SCS inactivation. Germline-only induction of mitochondrial fission factors sufficiently promotes mitochondrial dispersion and reproductive longevity. Moreover, we discovered that bacterial inputs affect mtGTP levels and dynamics factors to modulate reproductive aging. These results demonstrate the significance of mtGTP metabolism in regulating oocyte mitochondrial homeostasis and reproductive longevity and identify mitochondrial fission induction as an effective strategy to improve reproductive health.

Ionic driving forces provide the net electromotive force for ion movement across membranes and are therefore a fundamental property of all cells. In the nervous system, chloride driving force (DFCl) determines inhibitory signaling, as fast synaptic inhibition is mediated by chloride-permeable GABAA and glycine receptors. Here we present a new tool for all-Optical Reporting of CHloride Ion Driving force (ORCHID). We demonstrate ORCHID’s ability to provide accurate, high-throughput measurements of resting and dynamic DFCl from genetically targeted cell types over a range of timescales. ORCHID confirms theoretical predictions about the biophysical mechanisms that establish DFCl, reveals novel differences in DFCl between neurons and astrocytes under different network conditions, and affords the first in vivo measurements of intact DFCl in mouse cortical neurons. This work extends our understanding of chloride homeostasis and inhibitory synaptic transmission and establishes a precedent for utilizing all-optical methods to assess ionic driving force.

Optical microscopy methods such as calcium and voltage imaging enable fast activity readout of large neuronal populations using light. However, the lack of corresponding advances in online algorithms has slowed progress in retrieving information about neural activity during or shortly after an experiment. This gap not only prevents the execution of real-time closed-loop experiments, but also hampers fast experiment-analysis-theory turnover for high-throughput imaging modalities. Reliable extraction of neural activity from fluorescence imaging frames at speeds compatible with indicator dynamics and imaging modalities poses a challenge. We therefore developed FIOLA, a framework for fluorescence imaging online analysis that extracts neuronal activity from calcium and voltage imaging movies at speeds one order of magnitude faster than state-of-the-art methods. FIOLA exploits algorithms optimized for parallel processing on GPUs and CPUs. We demonstrate reliable and scalable performance of FIOLA on both simulated and real calcium and voltage imaging datasets. Finally, we present an online experimental scenario to provide guidance in setting FIOLA parameters and to highlight the trade-offs of our approach.

Methods of three-dimensional electron microscopy have been actively developed recently and open up great opportunities for morphological work. This approach is especially useful for studying microinsects, since it is possible to obtain complete series of high-resolution sections of a whole insect. Studies on the genus Megaphragma are especially important, since the unique phenomenon of lysis of most of the neuron nuclei was discovered in species of this genus. In this study we reveal the anatomical structure of the head of Megaphragma viggianii at all levels from organs to subcellular structures. Despite the miniature size of the body, most of the organ systems of M. viggianii retain the structural plan and complexity of organization at all levels. The set of muscles and the well-developed stomatogastric nervous system of this species correspond to those of larger insects, and there is also a well-developed tracheal system in the head of this species. Reconstructions of the head of M. viggianii at the cellular and subcellular levels were obtained, and of volumetric data were analyzed. A total of 689 nucleated cells of the head were reconstructed. The ultrastructure of M. viggianii is surprisingly complex, and the evolutionary benefits of such complexity are probably among the factors limiting the further miniaturization of parasitoid wasps.

A growing community is constructing a next-generation file format (NGFF) for bioimaging to overcome problems of scalability and heterogeneity. Organized by the Open Microscopy Environment (OME), individuals and institutes across diverse modalities facing these problems have designed a format specification process (OME-NGFF) to address these needs. This paper brings together a wide range of those community members to describe the format itself – OME-Zarr – along with tools and data resources available today to increase FAIR access and remove barriers in the scientific process. The current momentum offers an opportunity to unify a key component of the bioimaging domain — the file format that underlies so many personal, institutional, and global data management and analysis tasks.

Animal sounds are produced by patterned vibrations of specific organs, but the neural circuits that drive these vibrations are not well defined in any animal. Here we provide a functional and synaptic map of most of the neurons in the Drosophila male ventral nerve cord (the analog of the vertebrate spinal cord) that drive complex, patterned song during courtship. Male Drosophila vibrate their wings toward females during courtship to produce two distinct song modes – pulse and sine song – with characteristic features that signal species identity and male quality. We identified song-producing neural circuits by optogenetically activating and inhibiting identified cell types in the ventral nerve cord (VNC) and by tracing their patterns of synaptic connectivity in the male VNC connectome. The core song circuit consists of at least eight cell types organized into overlapping circuits, where all neurons are required for pulse song and a subset are required for sine song. The pulse and sine circuits each include a feed-forward pathway from brain descending neurons to wing motor neurons, with extensive reciprocal and feed-back connections. We also identify specific neurons that shape the individual features of each song mode. These results reveal commonalities amongst diverse animals in the neural mechanisms that generate diverse motor patterns from a single set of muscles.

PIEZOs are mechanosensitive ion channels that convert force into chemoelectric signals and have essential roles in diverse physiological settings. In vitro studies have proposed that PIEZO channels transduce mechanical force through the deformation of extensive blades of transmembrane domains emanating from a central ion-conducting pore. However, little is known about how these channels interact with their native environment and which molecular movements underlie activation. Here we directly observe the conformational dynamics of the blades of individual PIEZO1 molecules in a cell using nanoscopic fluorescence imaging. Compared with previous structural models of PIEZO1, we show that the blades are significantly expanded at rest by the bending stress exerted by the plasma membrane. The degree of expansion varies dramatically along the length of the blade, where decreased binding strength between subdomains can explain increased flexibility of the distal blade. Using chemical and mechanical modulators of PIEZO1, we show that blade expansion and channel activation are correlated. Our findings begin to uncover how PIEZO1 is activated in a native environment. More generally, as we reliably detect conformational shifts of single nanometres from populations of channels, we expect that this approach will serve as a framework for the structural analysis of membrane proteins through nanoscopic imaging.

Small unilamellar vesicles (SUVs) are indispensable model membranes, organelle mimics, and drug and vaccine carriers. However, the lack of robust techniques to functionalize or organize preformed SUVs limits their applications. Here we use DNA nanostructures to coat, cluster, and pattern sub-100-nm liposomes, generating distance-controlled vesicle networks, strings and dimers, among other configurations. The DNA coating also enables attachment of proteins to liposomes, and temporal control of membrane fusion driven by SNARE protein complexes. Such a convenient and versatile method of engineering premade vesicles both structurally and functionally is highly relevant to bottom-up biology and targeted delivery.