Expansion microscopy (ExM) is a method to expand biological specimens ~fourfold in each dimension by embedding in a hyper-swellable gel material. The expansion is uniform across observable length scales, enabling imaging of structures previously too small to resolve. ExM is compatible with any microscope and does not require expensive materials or specialized software, offering effectively sub-diffraction-limited imaging capabilities to labs that are not equipped to use traditional super-resolution imaging methods. Expanded specimens are ~99% water, resulting in strongly reduced optical scattering and enabling imaging of sub-diffraction-limited structures throughout specimens up to several hundred microns in (pre-expansion) thickness.

The connectome provides large scale connectivity and morphology information for the majority of the central brain of . Using this data set, we provide a complete description of the olfactory system, covering all first, second and lateral horn-associated third-order neurons. We develop a generally applicable strategy to extract information flow and layered organisation from connectome graphs, mapping olfactory input to descending interneurons. This identifies a range of motifs including highly lateralised circuits in the antennal lobe and patterns of convergence downstream of the mushroom body and lateral horn. Leveraging a second data set we provide a first quantitative assessment of inter- versus intra-individual stereotypy. Comparing neurons across two brains (three hemispheres) reveals striking similarity in neuronal morphology across brains. Connectivity correlates with morphology and neurons of the same morphological type show similar connection variability within the same brain as across two brains.

Fluorescence microscopy relies on dyes that absorb and then emit photons. In addition to fluorescence, fluorophores can undergo photochemical processes that decrease quantum yield or result in spectral shifts and irreversible photobleaching. Chemical strategies that suppress these undesirable pathways—thereby increasing the brightness and photostability of fluorophores—are crucial for advancing the frontier of bioimaging. Here, we describe a general method to improve small-molecule fluorophores by incorporating deuterium into the alkylamino auxochromes of rhodamines and other dyes. This strategy increases fluorescence quantum yield, inhibits photochemically induced spectral shifts, and slows irreparable photobleaching, yielding next-generation labels with improved performance in cellular imaging experiments.

Dopamine neuromodulation of neural synapses is a process implicated in a number of critical brain functions and diseases. Development of protocols to visualize this dynamic neurochemical process is essential to understanding how dopamine modulates brain function. We have developed a non-genetically encoded, near-IR (nIR) catecholamine nanosensor (nIRCat) capable of identifying ~2-µm dopamine release hotspots in dorsal striatal brain slices. nIRCat is readily synthesized through sonication of single walled carbon nanotubes with DNA oligos, can be readily introduced into both genetically tractable and intractable organisms and is compatible with a number of dopamine receptor agonists and antagonists. Here we describe the synthesis, characterization and implementation of nIRCat in acute mouse brain slices. We demonstrate how nIRCat can be used to image electrically or optogenetically stimulated dopamine release, and how these procedures can be leveraged to study the effects of dopamine receptor pharmacology. In addition, we provide suggestions for building or adapting wide-field microscopy to be compatible with nIRCat nIR fluorescence imaging. We discuss strategies for analyzing nIR video data to identify dopamine release hotspots and quantify their kinetics. This protocol can be adapted and implemented for imaging other neuromodulators by using probes of this class and can be used in a broad range of species without genetic manipulation. The synthesis and characterization protocols for nIRCat take ~5 h, and the preparation and fluorescence imaging of live brain slices by using nIRCats require ~6 h.

Typical patterned movements in animals are achieved through combinations of contraction and delayed relaxation of groups of muscles. However, how intersegmentally coordinated patterns of muscular relaxation are regulated by the neural circuits remains poorly understood. Here, we identify Canon, a class of higher-order premotor interneurons, that regulates muscular relaxation during backward locomotion of Drosophila larvae. Canon neurons are cholinergic interneurons present in each abdominal neuromere and show wave-like activity during fictive backward locomotion. Optogenetic activation of Canon neurons induces relaxation of body wall muscles, whereas inhibition of these neurons disrupts timely muscle relaxation. Canon neurons provide excitatory outputs to inhibitory premotor interneurons. Canon neurons also connect with each other to form an intersegmental circuit and regulate their own wave-like activities. Thus, our results demonstrate how coordinated muscle relaxation can be realized by an intersegmental circuit that regulates its own patterned activity and sequentially terminates motor activities along the anterior-posterior axis.

To thrive, organisms must maintain physiological and environmental variables in suitable ranges. Given that these variables undergo constant fluctuations over varying time scales, how do biological control systems maintain control over these values? We explored this question in the context of phototactic behavior in larval zebrafish. We demonstrate that larval zebrafish use phototaxis to maintain environmental luminance at a set point, that the value of this set point fluctuates on a time scale of seconds when environmental luminance changes, and that it is determined by calculating the mean input across both sides of the visual field. These results expand on previous studies of flexible phototaxis in larval zebrafish; they suggest that larval zebrafish exert homeostatic control over the luminance of their surroundings, and that feedback from the surroundings drives allostatic changes to the luminance set point. As such, we describe a novel behavioral algorithm with which larval zebrafish exert control over a sensory variable.

Through the corpus callosum, interhemispheric communication is mediated by callosal projection (CP) neurons. Using retrograde labeling, we identified a population of layer 6 (L6) excitatory neurons as the main conveyer of transcallosal information in the monocular zone of the mouse primary visual cortex (V1). Distinct from L6 corticothalamic (CT) population, V1 L6 CP neurons contribute to an extensive reciprocal network across multiple sensory cortices over two hemispheres. Receiving both local and long-range cortical inputs, they encode orientation, direction, and receptive field information, while are also highly spontaneous active. The spontaneous activity of L6 CP neurons exhibits complex relationships with brain states and stimulus presentation, distinct from the spontaneous activity patterns of the CT population. The anatomical and functional properties of these L6 CP neurons enable them to broadcast visual and nonvisual information across two hemispheres, and thus may play a role in regulating and coordinating brain-wide activity events.

A significant challenge for developmental systems biology is balancing throughput with controlled conditions that minimize experimental artifacts. Large-scale developmental screens such as unbiased mutagenesis surveys have been limited in their applicability to embryonic systems, as the technologies for quantifying precise expression patterns in whole animals has not kept pace with other sequencing-based technologies. Here, we outline an open-source semi-automated pipeline to chemically fixate, stain, and 3D-image Drosophila embryos. Central to this pipeline is a liquid handling robot, Flyspresso, which automates the steps of classical embryo fixation and staining. We provide the schematics and an overview of the technology for an engineer or someone equivalently trained to reproduce and further improve upon Flyspresso, and highlight the Drosophila embryo fixation and colorimetric or antibody staining protocols. Additionally, we provide a detailed overview and stepwise protocol for our adaptive-feedback pipeline for automated embryo imaging on confocal microscopes. We demonstrate the efficiency of this pipeline compared to classical techniques, and how it can be repurposed or scaled to other protocols and biological systems. We hope our pipeline will serve as a platform for future research, allowing a broader community of users to build, execute, and share similar experiments.