Axon-First Neuritogenesis on Vertical Nanowires.

In this work, we report that high-density, vertically grown silicon nanowires (vg-SiNWs) direct a new in vitro developmental pathway of primary hippocampal neurons. Neurons on vg-SiNWs formed a single, extremely elongated major neurite earlier than minor neurites, which led to accelerated polarization. Additionally, the development of lamellipodia, which generally occurs on 2D culture coverslips, was absent on vg-SiNWs. The results indicate that surface topography is an important factor that influences neuronal development and also provide implications for the role of topography in neuronal development in vivo.

Function of ezrin-radixin-moesin proteins in migration of subventricular zone-derived neuroblasts following traumatic brain injury.

Throughout life, newly generated neuroblasts from the subventricular zone migrate toward the olfactory bulb through the rostral migratory stream. Upon brain injury, these migrating neuroblasts change their route and begin to migrate toward injured regions, which is one of the regenerative responses after brain damage. This injury-induced migration is triggered by stromal cell-derived factor 1 (SDF1) released from microglia near the damaged site; however, it is still unclear how these cells transduce SDF1 signals and change their direction. In this study, we found that SDF1 promotes the phosphorylation of ezrin-radixin-moesin (ERM) proteins, which are key molecules in organizing cell membrane and linking signals from the extracellular environment to the intracellular actin cytoskeleton. Blockade of ERM activation by overexpressing dominant-negative ERM (DN-ERM) efficiently perturbed the migration of neuroblasts. Considering that DN-ERM-expressing neuroblasts failed to maintain proper migratory cell morphology, it appears that ERM-dependent regulation of cell shape is required for the efficient migration of neuroblasts. These results suggest that ERM activation is an important step in the directional migration of neuroblasts in response to SDF1-CXCR4 signaling following brain injury.

Directional neurite growth using carbon nanotube patterned substrates as a biomimetic cue.

Researchers have made extensive efforts to mimic or reverse-engineer in vivo neural circuits using micropatterning technology. Various surface chemical cues or topographical structures have been proposed to design neuronal networks in vitro. In this paper, we propose a carbon nanotube (CNT)-based network engineering method which naturally mimics the structure of extracellular matrix (ECM). On CNT patterned substrates, poly-L-lysine (PLL) was coated, and E18 rat hippocampal neurons were cultured. In the early developmental stage, soma adhesion and neurite extension occurred in disregard of the surface CNT patterns. However, later the majority of neurites selectively grew along CNT patterns and extended further than other neurites that originally did not follow the patterns. Long-term cultured neuronal networks had a strong resemblance to the in vivo neural circuit structures. The selective guidance is possibly attributed to higher PLL adsorption on CNT patterns and the nanomesh structure of the CNT patterns. The results showed that CNT patterned substrates can be used as novel neuronal patterning substrates for in vitro neural engineering.

Low- and high-thermogenic brown adipocyte subpopulations coexist in murine adipose tissue.

Brown adipose tissue (BAT), as the main site of adaptive thermogenesis, exerts beneficial metabolic effects on obesity and insulin resistance. BAT has been previously assumed to contain a homogeneous population of brown adipocytes. Utilizing multiple mouse models capable of genetically labeling different cellular populations, as well as single-cell RNA sequencing and 3D tissue profiling, we discovered a brown adipocyte subpopulation with low thermogenic activity coexisting with the classical high-thermogenic brown adipocytes within the BAT. Compared with the high-thermogenic brown adipocytes, these low-thermogenic brown adipocytes had substantially lower Ucp1 and Adipoq expression, larger lipid droplets, and lower mitochondrial content. Functional analyses showed that, unlike the high-thermogenic brown adipocytes, the low-thermogenic brown adipocytes have markedly lower basal mitochondrial respiration, and they are specialized in fatty acid uptake. Upon changes in environmental temperature, the 2 brown adipocyte subpopulations underwent dynamic interconversions. Cold exposure converted low-thermogenic brown adipocytes into high-thermogenic cells. A thermoneutral environment had the opposite effect. The recruitment of high-thermogenic brown adipocytes by cold stimulation is not affected by high-fat diet feeding, but it does substantially decline with age. Our results revealed a high degree of functional heterogeneity of brown adipocytes.

Optimizing tissue-clearing conditions based on analysis of the critical factors affecting tissue-clearing procedures.

Tissue-clearing techniques have received great attention for volume imaging and for the potential to be applied in optical diagnosis. In principle, tissue clearing is achieved by reducing light scattering through a combination of lipid removal, size change, and matching of the refractive index (RI) between the imaging solution and the tissue. However, the contributions of these major factors in tissue clearing have not been systematically evaluated yet. In this study, we experimentally measured and mathematically calculated the contribution of these factors to the clearing of four organs (brain, liver, kidney, and lung). We found that these factors differentially influence the maximal clearing efficacy of tissues and the diffusivity of materials inside the tissue. We propose that these physical properties of organs can be utilized for the quality control (Q/C) process during tissue clearing, as well as for the monitoring of the pathological changes of tissues.

BrainFilm, a novel technique for physical compression of 3D brain slices for efficient image acquisition and post-processing.

Tissue clearing enables us to observe thick tissue at a single cell resolution by reducing light scattering and refractive index matching. However, imaging of a large volume of tissue for 3D reconstruction requires a great deal of time, cost, and efforts. Few methods have been developed to transcend these limitations by mechanical compression or isotropic tissue shrinkage. Tissue shrinkage significantly lessens the imaging burden; however, there is an inevitable trade-off with image resolution. Here, we have developed the "BrainFilm" technique to compress cleared tissue at Z-axis by dehydration, without alteration of the XY-axis. The Z-axis compression was approximately 90%, and resulted in substantial reduction in image acquisition time and data size. The BrainFilm technique was successfully used to trace and characterize the morphology of thick biocytin-labelled neurons following electrophysiological recording and trace the GFP-labelled long nerve projections in irregular tissues such as the limb of mouse embryo. Thus, BrainFilm is a versatile tool that can be applied in diverse studies of 3D tissues in which spatial information of the Z-axis is dispensable.

Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors.

Neuromodulatory systems exert profound influences on brain function. Understanding how these systems modify the operating mode of target circuits requires spatiotemporally precise measurement of neuromodulator release. We developed dLight1, an intensity-based genetically encoded dopamine indicator, to enable optical recording of dopamine dynamics with high spatiotemporal resolution in behaving mice. We demonstrated the utility of dLight1 by imaging dopamine dynamics simultaneously with pharmacological manipulation, electrophysiological or optogenetic stimulation, and calcium imaging of local neuronal activity. dLight1 enabled chronic tracking of learning-induced changes in millisecond dopamine transients in mouse striatum. Further, we used dLight1 to image spatially distinct, functionally heterogeneous dopamine transients relevant to learning and motor control in mouse cortex. We also validated our sensor design platform for developing norepinephrine, serotonin, melatonin, and opioid neuropeptide indicators.

Cell-Type Dependent Effect of Surface-Patterned Microdot Arrays on Neuronal Growth.

Surface micropatterns have been widely used as chemical cues to control the microenvironment of cultured neurons, particularly for neurobiological assays and neurochip designs. However, the cell-type dependency on the interactions between neurons and underlying micropatterns has been rarely investigated despite the inherent differences in the morphology of neuronal types. In this study, we used surface-printed microdot arrays to investigate the effect of the same micropatterns on the growth of mouse spinal interneuron, mouse hippocampal neurons, and rat hippocampal neurons. While mouse hippocampal neurons showed no significantly different growth on control and patterned substrates, we found the microdot arrays had different effects on early neuronal growth depending on the cell type; spinal interneurons tended to grow faster in length, whereas hippocampal neurons tended to form more axon collateral branches in response to the microdot arrays. Although there was a similar trend in the neurite length and branch number of both neurons changed across the microdot arrays with the expanded range of size and spacing, the dominant responses of each neuron, neurite elongation of mouse spinal interneurons and branching augmentation of rat hippocampal neurons were still preserved. Therefore, our results demonstrate that the same design of micropatterns could cause different neuronal growth results, raising an intriguing issue of considering cell types in neural interface designs.

Synaptic compartmentalization by micropatterned masking of a surface adhesive cue in cultured neurons.

Functions of neuronal circuit are fundamentally modulated by its quality and quantity of connections. Assessment of synapse, the basic unit for a neuronal connection, is labor-intensive and time-consuming in conventional culture systems, due to the small size and the spatially random distribution. In the present study, we propose a novel 'synapse compartmentalization' culture system, in which synapses are concentrated at controlled locations. We fabricated a negative dot array pattern by coating the entire surface with poly-l-lysine (PLL) and subsequent microcontact printing of 1) substrates which mask positive charge of PLL (Fc, BSA and laminin), or 2) a chemorepulsive protein (Semaphorin 3F-Fc). By combination of physical and biological features of these repulsive substrates, functional synapses were robustly concentrated in the PLL-coated dots. This synapse compartmentalization chip can be combined with the various high-throughput assay formats based on the synaptic morphology and function. Therefore, this quantifiable and controllable dot array pattern by microcontact printing will be potential useful for bio-chip platforms for the high-density assays used in synapse-related neurobiological studies.

NeuroCa: integrated framework for systematic analysis of spatiotemporal neuronal activity patterns from large-scale optical recording data.

Optical recording facilitates monitoring the activity of a large neural network at the cellular scale, but the analysis and interpretation of the collected data remain challenging. Here, we present a MATLAB-based toolbox, named NeuroCa, for the automated processing and quantitative analysis of large-scale calcium imaging data. Our tool includes several computational algorithms to extract the calcium spike trains of individual neurons from the calcium imaging data in an automatic fashion. Two algorithms were developed to decompose the imaging data into the activity of individual cells and subsequently detect calcium spikes from each neuronal signal. Applying our method to dense networks in dissociated cultures, we were able to obtain the calcium spike trains of [Formula: see text] neurons in a few minutes. Further analyses using these data permitted the quantification of neuronal responses to chemical stimuli as well as functional mapping of spatiotemporal patterns in neuronal firing within the spontaneous, synchronous activity of a large network. These results demonstrate that our method not only automates time-consuming, labor-intensive tasks in the analysis of neural data obtained using optical recording techniques but also provides a systematic way to visualize and quantify the collective dynamics of a network in terms of its cellular elements.

Agarose-assisted micro-contact printing for high-quality biomolecular micro-patterns.

Micro-contact printing has been developed to print biomolecules, such as cell adhesive molecules, proteins, or DNAs, on a substrate, which can serve as experimental platforms for investigating biological issues and engineering biosensors. Despite the popularity of this method, it has been technically challenging to use a conventional stamp made of a hydrophobic polydimethoxysilane (PDMS) elastomer that often requires surface treatments to facilitate the inking and stamping of biomolecules. In this work, we proposed a new surface modification method for a PDMS stamp using agarose hydrogel and demonstrated the applications to the design of micro-patterned substrates with biomolecules. By using a simple bench-top dip-coating method with a commercial syringe pump to steadily pull out the stamp from boiled agarose solution, we coated an agarose layer on the stamp. It consequentially enhanced the transferability of ink molecules to the target substrate and the uniformity of printed patterns compared to the traditional methods for treating stamp surface such as surfactant coating and temporary oxidation with air plasma. In addition, this microstamping method was also used to produce patterns of proteins with the preservation of bioactivity, which could guide neuronal growth. Thus, we demonstrated the applicability to the interface designs of biochips and biosensors.

Surface-printed microdot array chips for the quantification of axonal collateral branching of a single neuron in vitro.

Precise and quantitative control of extracellular signalling cues using surface-engineered chips has facilitated various neurobiological assays in vitro. Although the formation of axon collateral branches is important for the establishment and refinement of the neuronal connections during the development and regeneration, surface designs for controlling branch phenotypes have been rarely proposed. In this work, we fabricated a surface-printed microdot array for controlling axon branch formation. Following the culture of hippocampal neurons on a 5 µm dot array patterned by micro-contact printing of poly-d-lysine, we found that most axon collateral branches were initiated from axonal regions on a microdot and terminated on neighboring dots. In addition, the length of branches increased as the spacing between dots increased. Surprisingly, other morphological features were not significantly different from the neurons cultured on a conventional unpatterned surface. Further investigation of this phenomenon indicated that the branch-forming machineries, such as actin patches, were focused on the dot. According to these investigations, we concluded that discontinuous adhesion spots given by dot arrays arranged the branching formation on the expectable location and direction. Therefore, microdot arrays will be applicable as the surface design parameter of bio-chip platforms to reduce branching complexity and quantize branching formation for the simple and easy assay in neurobiological studies.

Geometric effect of cell adhesive polygonal micropatterns on neuritogenesis and axon guidance.

Recent advances in nano- and micro-technology have made it possible to deliver surface-bound extracellular signaling cues to cultured neurons. In this study, we investigated the formation of neurites and axonal outgrowth using various types of polygonal micropatterns ('micropolygon arrays') on cell culture substrates and suggested a novel design principle of in vitro axon guidance. Ten different types of micropolygons (circle, triangle, square, pentagon, hexagon, stars and isosceles triangles) were printed on a culture substrate using micro-contact printing with a mixture of poly-l-lysine and laminin A chain synthetic peptide. E18 rat hippocampal neurons were cultured on the patterned substrates, and the relation between micropatterns and neurite outgrowth was analyzed. Micropolygon arrays had effects on the soma shape and neurite initiation. In the case of regular triangle patterns, neurons showed vertex preference in terms of neurite initiation: neurites were more frequently generated from the vertex region. In the case of isosceles triangles, a major neurite was formed from the sharpest vertex and axons were developed from the sharpest vertex. Thus, the direction of axon growth could be controlled by the orientation of the sharpest vertex in the isosceles triangles. This work suggests that the geometry of cell adhesive regions influences the development of a cultured neuron, and the structure of neural circuits can be designed by controlling axonal outgrowth with individual micropolygons.