Current techniques for monitoring GABA (γ-aminobutyric acid), the primary inhibitory neurotransmitter in vertebrates, cannot follow transients in intact neural circuits. To develop a GABA sensor, we applied the design principles used to create the fluorescent glutamate receptor iGluSnFR. We used a protein derived from a previously unsequenced Pseudomonas fluorescens strain and performed structure-guided mutagenesis and library screening to obtain intensity-based GABA sensing fluorescence reporter (iGABASnFR) variants. iGABASnFR is genetically encoded, detects GABA release evoked by electric stimulation of afferent fibers in acute brain slices and produces readily detectable fluorescence increases in vivo in mice and zebrafish. We applied iGABASnFR to track mitochondrial GABA content and its modulation by an anticonvulsant, swimming-evoked, GABA-mediated transmission in zebrafish cerebellum, GABA release events during interictal spikes and seizures in awake mice, and found that GABA-mediated tone decreases during isoflurane anesthesia.

Dividing cells reorganize their microtubule cytoskeleton into a bipolar spindle, which moves one set of sister chromatids to each nascent daughter cell. Early spindle assembly models postulated that spindle pole-derived microtubules search the cytoplasmic space until they randomly encounter a kinetochore to form a stable attachment. More recent work uncovered several additional, centrosome-independent microtubule generation pathways, but the contributions of each pathway to spindle assembly have remained unclear. Here, we combined live microscopy and mathematical modeling to show that most microtubules nucleate at noncentrosomal regions in dividing human cells. Using a live-cell probe that selectively labels aged microtubule lattices, we demonstrate that the distribution of growing microtubule plus ends can be almost entirely explained by Augmin-dependent amplification of long-lived microtubule lattices. By ultrafast 3D lattice light-sheet microscopy, we observed that this mechanism results in a strong directional bias of microtubule growth toward individual kinetochores. Our systematic quantification of spindle dynamics reveals highly coordinated microtubule growth during kinetochore fiber assembly.

Hox genes pattern the anterior-posterior axis of animals and are posited to drive animal body plan evolution, yet their precise role in evolution has been difficult to determine. Here, we identified evolutionary modifications in the Hox gene Abd-Bthat dramatically altered its expression along the body plan of Drosophila santomea. Abd-B is required for pigmentation in Drosophila yakuba, the sister species of D. santomea, and changes to Abd-B expression would be predicted to make large contributions to the loss of body pigmentation in D. santomea. However, manipulating Abd-B expression in current-day D. santomea does not affect pigmentation. We attribute this epistatic interaction to four other genes within the D. santomea pigmentation network, three of which have evolved expression patterns that do not respond to Abd-B. Our results demonstrate how body plans may evolve through small evolutionary steps distributed throughout Hox-regulated networks. Polygenicity and epistasis may hinder efforts to identify genes and mechanisms underlying macroevolutionary traits.

We advance two-photon microscopy for near-diffraction-limited imaging up to 850 µm below the pia in awake mice. Our approach combines direct wavefront sensing of light from a guidestar (formed by descanned fluorescence from Cy5.5-conjugated dextran in brain microvessels) with adaptive optics to compensate for tissue-induced aberrations in the wavefront. We achieve high signal-to-noise ratios in recordings of glutamate release from thalamocortical axons and calcium transients in spines of layer 5b basal dendrites during active tactile sensing.

With the development of neural recording technology, it has become possible to collect activities from hundreds or even thousands of neurons simultaneously. Visualization of neural population dynamics can help neuroscientists analyze large-scale neural activities efficiently. In this letter, Laplacian eigenmaps is applied to this task for the first time, and the experimental results show that the proposed method significantly outperforms the commonly used methods. This finding was confirmed by the systematic evaluation using nonhuman primate data, which contained the complex dynamics well suited for testing. According to our results, Laplacian eigenmaps is better than the other methods in various ways and can clearly visualize interesting biological phenomena related to neural dynamics.

Neuromodulation plays a critical role in brain function in both health and disease, and new tools that capture neuromodulation with high spatial and temporal resolution are needed. Here, we introduce a synthetic catecholamine nanosensor with fluorescent emission in the near infrared range (1000–1300 nm), near infrared catecholamine nanosensor (nIRCat). We demonstrate that nIRCats can be used to measure electrically and optogenetically evoked dopamine release in brain tissue, revealing hotspots with a median size of 2 µm. We also demonstrated that nIRCats are compatible with dopamine pharmacology and show D2 autoreceptor modulation of evoked dopamine release, which varied as a function of initial release magnitude at different hotspots. Together, our data demonstrate that nIRCats and other nanosensors of this class can serve as versatile synthetic optical tools to monitor neuromodulatory neurotransmitter release with high spatial resolution.

Point-scanning two-photon microscopy enables high-resolution imaging within scattering specimens such as the mammalian brain, but sequential acquisition of voxels fundamentally limits imaging speed. We developed a two-photon imaging technique that scans lines of excitation across a focal plane at multiple angles and uses prior information to recover high-resolution images at over 1.4 billion voxels per second. Using a structural image as a prior for recording neural activity, we imaged visually-evoked and spontaneous glutamate release across hundreds of dendritic spines in mice at depths over 250 microns and frame-rates over 1 kHz. Dendritic glutamate transients in anaesthetized mice are synchronized within spatially-contiguous domains spanning tens of microns at frequencies ranging from 1-100 Hz. We demonstrate high-speed recording of acetylcholine and calcium sensors, 3D single-particle tracking, and imaging in densely-labeled cortex. Our method surpasses limits on the speed of raster-scanned imaging imposed by fluorescence lifetime.

We present a method combining affinity prediction with region agglomeration, which improves significantly upon the state of the art of neuron segmentation from electron microscopy (EM) in accuracy and scalability. Our method consists of a 3D U-net, trained to predict affinities between voxels, followed by iterative region agglomeration. We train using a structured loss based on MALIS, encouraging topologically correct segmentations obtained from affinity thresholding. Our extension consists of two parts: First, we present a quasi-linear method to compute the loss gradient, improving over the original quadratic algorithm. Second, we compute the gradient in two separate passes to avoid spurious gradient contributions in early training stages. Our predictions are accurate enough that simple learning-free percentile-based agglomeration outperforms more involved methods used earlier on inferior predictions. We present results on three diverse EM datasets, achieving relative improvements over previous results of 27%, 15%, and 250%. Our findings suggest that a single method can be applied to both nearly isotropic block-face EM data and anisotropic serial sectioned EM data. The runtime of our method scales linearly with the size of the volume and achieves a throughput of ~2.6 seconds per megavoxel, qualifying our method for the processing of very large datasets.

Serotonin plays different roles across networks within the same sensory modality. Previously, we used whole-cell electrophysiology in Drosophila to show that serotonergic neurons innervating the first olfactory relay are inhibited by odorants (Zhang and Gaudry, 2016). Here we show that network-spanning serotonergic neurons segregate information about stimulus features, odor intensity and identity, by using opposing coding schemes in different olfactory neuropil. A pair of serotonergic neurons (the CSDns) innervate the antennal lobe and lateral horn, which are first and second order neuropils. CSDn processes in the antennal lobe are inhibited by odors in an identity independent manner. In the lateral horn, CSDn processes are excited in an odor identity dependent manner. Using functional imaging, modeling, and EM reconstruction, we demonstrate that antennal lobe derived inhibition arises from local GABAergic inputs and acts as a means of gain control on branch specific inputs that the CSDns receive within the lateral horn.