Limitations of fluorescent timer protein maturation kinetics to isolate transcriptionally synchronized human neural progenitor cells.

Differentiation of human pluripotent stem cells (hPSCs) into subtype-specific neurons holds substantial potential for disease modeling in vitro. For successful differentiation, a detailed understanding of the transcriptional networks regulating cell fate decisions is critical. The heterochronic nature of neurodevelopment, during which distinct cells in the brain and during in vitro differentiation acquire their fates in an unsynchronized manner, hinders pooled transcriptional comparisons. One approach is to "translate" chronologic time into linear developmental and maturational time. Simple binary promotor-driven fluorescent proteins (FPs) to pool similar cells are unable to achieve this goal, due to asynchronous promotor onset in individual cells. We tested five fluorescent timer (FT) molecules expressed from the endogenous paired box 6 (PAX6) promoter in 293T and human hPSCs. Each of these FT systems faithfully reported chronologic time in 293T cells, but none of the FT constructs followed the same fluorescence kinetics in human neural progenitor cells.

Limitations of fluorescent timer protein maturation kinetics to isolate transcriptionally synchronized cortically differentiating human pluripotent stem cells.

Differentiation of human pluripotent stem cells (hPSC) into distinct neuronal populations holds substantial potential for disease modeling in vitro, toward both elucidation of pathobiological mechanisms and screening of potential therapeutic agents. For successful differentiation of hPSCs into subtype-specific neurons using in vitro protocols, detailed understanding of the transcriptional networks and their dynamic programs regulating endogenous cell fate decisions is critical. One major roadblock is the heterochronic nature of neurodevelopment, during which distinct cells and cell types in the brain and during in vitro differentiation mature and acquire their fates in an unsynchronized manner, hindering pooled transcriptional comparisons. One potential approach is to "translate" chronologic time into linear developmental and maturational time. Attempts to partially achieve this using simple binary promotor-driven fluorescent proteins (FPs) to pool similar cells have not been able to achieve this goal, due to asynchrony of promotor onset in individual cells. Toward solving this, we generated and tested a range of knock-in hPSC lines that express five distinct dual FP timer systems or single time-resolved fluorescent timer (FT) molecules, either in 293T cells or in human hPSCs driving expression from the endogenous paired box 6 (PAX6) promoter of cerebral cortex progenitors. While each of these dual FP or FT systems faithfully reported chronologic time when expressed from a strong inducible promoter in 293T cells, none of the tested FP/FT constructs followed the same fluorescence kinetics in developing human neural progenitor cells, and were unsuccessful in identification and isolation of distinct, developmentally synchronized cortical progenitor populations based on ratiometric fluorescence. This work highlights unique and often surprising expression kinetics and regulation in specific cell types differentiating from hPSCs.

An unbiased AAV-STARR-seq screen revealing the enhancer activity map of genomic regions in the mouse brain in vivo.

Enhancers are important cis-regulatory elements controlling cell-type specific expression patterns of genes. Furthermore, combinations of enhancers and minimal promoters are utilized to construct small, artificial promoters for gene delivery vectors. Large-scale functional screening methodology to construct genomic maps of enhancer activities has been successfully established in cultured cell lines, however, not yet applied to terminally differentiated cells and tissues in a living animal. Here, we transposed the Self-Transcribing Active Regulatory Region Sequencing (STARR-seq) technique to the mouse brain using adeno-associated-viruses (AAV) for the delivery of a highly complex screening library tiling entire genomic regions and covering in total 3 Mb of the mouse genome. We identified 483 sequences with enhancer activity, including sequences that were not predicted by DNA accessibility or histone marks. Characterizing the expression patterns of fluorescent reporters controlled by nine candidate sequences, we observed differential expression patterns also in sparse cell types. Together, our study provides an entry point for the unbiased study of enhancer activities in organisms during health and disease.

BigNeuron: a resource to benchmark and predict performance of algorithms for automated tracing of neurons in light microscopy datasets.

BigNeuron is an open community bench-testing platform with the goal of setting open standards for accurate and fast automatic neuron tracing. We gathered a diverse set of image volumes across several species that is representative of the data obtained in many neuroscience laboratories interested in neuron tracing. Here, we report generated gold standard manual annotations for a subset of the available imaging datasets and quantified tracing quality for 35 automatic tracing algorithms. The goal of generating such a hand-curated diverse dataset is to advance the development of tracing algorithms and enable generalizable benchmarking. Together with image quality features, we pooled the data in an interactive web application that enables users and developers to perform principal component analysis, t-distributed stochastic neighbor embedding, correlation and clustering, visualization of imaging and tracing data, and benchmarking of automatic tracing algorithms in user-defined data subsets. The image quality metrics explain most of the variance in the data, followed by neuromorphological features related to neuron size. We observed that diverse algorithms can provide complementary information to obtain accurate results and developed a method to iteratively combine methods and generate consensus reconstructions. The consensus trees obtained provide estimates of the neuron structure ground truth that typically outperform single algorithms in noisy datasets. However, specific algorithms may outperform the consensus tree strategy in specific imaging conditions. Finally, to aid users in predicting the most accurate automatic tracing results without manual annotations for comparison, we used support vector machine regression to predict reconstruction quality given an image volume and a set of automatic tracings.

Rapid nucleus-scale reorganization of chromatin in neurons enables transcriptional adaptation for memory consolidation.

The interphase nucleus is functionally organized in active and repressed territories defining the transcriptional status of the cell. However, it remains poorly understood how the nuclear architecture of neurons adapts in response to behaviorally relevant stimuli that trigger fast alterations in gene expression patterns. Imaging of fluorescently tagged nucleosomes revealed that pharmacological manipulation of neuronal activity in vitro and auditory cued fear conditioning in vivo induce nucleus-scale restructuring of chromatin within minutes. Furthermore, the acquisition of auditory fear memory is impaired after infusion of a drug into auditory cortex which blocks chromatin reorganization in vitro. We propose that active chromatin movements at the nucleus scale act together with local gene-specific modifications to enable transcriptional adaptations at fast time scales. Introducing a transgenic mouse line for photolabeling of histones, we extend the realm of systems available for imaging of chromatin dynamics to living animals.

In vivo modeling of human neuron dynamics and Down syndrome.

Harnessing the potential of human stem cells for modeling the physiology and diseases of cortical circuitry requires monitoring cellular dynamics in vivo. We show that human induced pluripotent stem cell (iPSC)-derived cortical neurons transplanted into the adult mouse cortex consistently organized into large (up to ~100 mm3) vascularized neuron-glia territories with complex cytoarchitecture. Longitudinal imaging of >4000 grafted developing human neurons revealed that neuronal arbors refined via branch-specific retraction; human synaptic networks substantially restructured over 4 months, with balanced rates of synapse formation and elimination; and oscillatory population activity mirrored the patterns of fetal neural networks. Lastly, we found increased synaptic stability and reduced oscillations in transplants from two individuals with Down syndrome, demonstrating the potential of in vivo imaging in human tissue grafts for patient-specific modeling of cortical development, physiology, and pathogenesis.

Filopodyan: An open-source pipeline for the analysis of filopodia.

Filopodia have important sensory and mechanical roles in motile cells. The recruitment of actin regulators, such as ENA/VASP proteins, to sites of protrusion underlies diverse molecular mechanisms of filopodia formation and extension. We developed Filopodyan (filopodia dynamics analysis) in Fiji and R to measure fluorescence in filopodia and at their tips and bases concurrently with their morphological and dynamic properties. Filopodyan supports high-throughput phenotype characterization as well as detailed interactive editing of filopodia reconstructions through an intuitive graphical user interface. Our highly customizable pipeline is widely applicable, capable of detecting filopodia in four different cell types in vitro and in vivo. We use Filopodyan to quantify the recruitment of ENA and VASP preceding filopodia formation in neuronal growth cones, and uncover a molecular heterogeneity whereby different filopodia display markedly different responses to changes in the accumulation of ENA and VASP fluorescence in their tips over time.

Directional Emission from Dielectric Leaky-Wave Nanoantennas.

An important source of innovation in nanophotonics is the idea to scale down known radio wave technologies to the optical regime. One thoroughly investigated example of this approach are metallic nanoantennas which employ plasmonic resonances to couple localized emitters to selected far-field modes. While metals can be treated as perfect conductors in the microwave regime, their response becomes Drude-like at optical frequencies. Thus, plasmonic nanoantennas are inherently lossy. Moreover, their resonant nature requires precise control of the antenna geometry. A promising way to circumvent these problems is the use of broadband nanoantennas made from low-loss dielectric materials. Here, we report on highly directional emission from hybrid dielectric leaky-wave nanoantennas made of Hafnium dioxide nanostructures deposited on a glass substrate. Colloidal semiconductor quantum dots deposited in the nanoantenna feed gap serve as a local light source. The emission patterns of hybrid nanoantennas with different sizes are measured by Fourier imaging. We find for all antenna sizes a highly directional emission, underlining the broadband operation of our design.

Reflective Metasurfaces for Incoherent Light To Bring Computer Graphics Tricks to Optical Systems.

The normal mapping technique is widely used in computer graphics to visualize three-dimensional (3D) objects displayed on a flat screen. Taking advantage of optical properties of metasurfaces, which provide a highly efficient approach for manipulation of incident light wavefront, we have designed a metasurface to implement diffuse reflection and used the concept of normal mapping to control its scattering properties. As a proof of principle, we have fabricated and characterized a flat diffuse metasurface imitating lighting and shading effects of a 3D cube. The 3D image is displayed directly on the illuminated metasurface and it is brighter than a standard white paper by up to 2.4 times. The designed structure performs equally well under coherent and incoherent illumination. The normal mapping approach based on metasurfaces can complement traditional optical engineering methods of surface profiling and gradient refractive index engineering in the design of 3D security features, high-performance planar optical diffusers, novel optical elements, and displays.

Development and function of human cerebral cortex neural networks from pluripotent stem cells in vitro.

A key aspect of nervous system development, including that of the cerebral cortex, is the formation of higher-order neural networks. Developing neural networks undergo several phases with distinct activity patterns in vivo, which are thought to prune and fine-tune network connectivity. We report here that human pluripotent stem cell (hPSC)-derived cerebral cortex neurons form large-scale networks that reflect those found in the developing cerebral cortex in vivo. Synchronised oscillatory networks develop in a highly stereotyped pattern over several weeks in culture. An initial phase of increasing frequency of oscillations is followed by a phase of decreasing frequency, before giving rise to non-synchronous, ordered activity patterns. hPSC-derived cortical neural networks are excitatory, driven by activation of AMPA- and NMDA-type glutamate receptors, and can undergo NMDA-receptor-mediated plasticity. Investigating single neuron connectivity within PSC-derived cultures, using rabies-based trans-synaptic tracing, we found two broad classes of neuronal connectivity: most neurons have small numbers (<10) of presynaptic inputs, whereas a small set of hub-like neurons have large numbers of synaptic connections (>40). These data demonstrate that the formation of hPSC-derived cortical networks mimics in vivo cortical network development and function, demonstrating the utility of in vitro systems for mechanistic studies of human forebrain neural network biology.

Dynamics of dendritic spines in the mouse auditory cortex during memory formation and memory recall.

Long-lasting changes in synaptic connections induced by relevant experiences are believed to represent the physical correlate of memories. Here, we combined chronic in vivo two-photon imaging of dendritic spines with auditory-cued classical conditioning to test if the formation of a fear memory is associated with structural changes of synapses in the mouse auditory cortex. We find that paired conditioning and unpaired conditioning induce a transient increase in spine formation or spine elimination, respectively. A fraction of spines formed during paired conditioning persists and leaves a long-lasting trace in the network. Memory recall triggered by the reexposure of mice to the sound cue did not lead to changes in spine dynamics. Our findings provide a synaptic mechanism for plasticity in sound responses of auditory cortex neurons induced by auditory-cued fear conditioning; they also show that retrieval of an auditory fear memory does not lead to a recapitulation of structural plasticity in the auditory cortex as observed during initial memory consolidation.

Transgenic mouse models enabling photolabeling of individual neurons in vivo.

One of the biggest tasks in neuroscience is to explain activity patterns of individual neurons during behavior by their cellular characteristics and their connectivity within the neuronal network. To greatly facilitate linking in vivo experiments with a more detailed molecular or physiological analysis in vitro, we have generated and characterized genetically modified mice expressing photoactivatable GFP (PA-GFP) that allow conditional photolabeling of individual neurons. Repeated photolabeling at the soma reveals basic morphological features due to diffusion of activated PA-GFP into the dendrites. Neurons photolabeled in vivo can be re-identified in acute brain slices and targeted for electrophysiological recordings. We demonstrate the advantages of PA-GFP expressing mice by the correlation of in vivo firing rates of individual neurons with their expression levels of the immediate early gene c-fos. Generally, the mouse models described in this study enable the combination of various analytical approaches to characterize living cells, also beyond the neurosciences.