The functional features of neural circuits are determined by a combination of properties that range in scale from projections systems across the whole brain to molecular interactions at the synapse. The burgeoning field of neurocartography seeks to map these relevant features of brain structure—spanning a volume ∼20 orders of magnitude—to determine how neural circuits perform computations supporting cognitive function and complex behavior. Recent technological breakthroughs in tissue sample preparation, high-throughput electron microscopy imaging, and automated image analyses have produced the first visualizations of all synaptic connections between neurons of invertebrate model systems. However, the sheer size of the central nervous system in mammals implies that reconstruction of the first full brain maps at synaptic scale may not be feasible for decades. In this review, we outline existing and emerging technologies for neurocartography that complement electron microscopy-based strategies and are beginning to derive some basic organizing principles of circuit hodology at the mesoscale, microscale, and nanoscale. Specifically, we discuss how a host of light microscopy techniques including array tomography have been utilized to determine both long-range and subcellular organizing principles of synaptic connectivity. In addition, we discuss how new techniques, such as two-photon serial tomography of the entire mouse brain, have become attractive approaches to dissect the potential connectivity of defined cell types. Ultimately, principles derived from these techniques promise to facilitate a conceptual understanding of how connectomes, and neurocartography in general, can be effectively utilized toward reaching a mechanistic understanding of circuit function.

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.

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.

mRNA translation is a key step in decoding genetic information. Genetic decoding is surprisingly heterogeneous, as multiple distinct polypeptides can be synthesized from a single mRNA sequence. To study translational heterogeneity, we developed the MoonTag, a new fluorescence labeling system to visualize translation of single mRNAs. When combined with the orthogonal SunTag system, the MoonTag enables dual readouts of translation, greatly expanding the possibilities to interrogate complex translational heterogeneity. By placing MoonTag and SunTag sequences in different translation reading frames, each driven by distinct translation start sites, start site selection of individual ribosomes can be visualized in real-time. We find that start site selection is largely stochastic, but that the probability of using a particular start site differs among mRNA molecules, and can be dynamically regulated over time. Together, this study provides key insights into translation start site selection heterogeneity, and provides a powerful toolbox to visualize complex translation dynamics.

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.

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.

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.

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.