Zhuo Huang

Surface Coating as a Key Parameter in Engineering Neuronal Network Structures In Vitro

By quantitatively comparing a variety of macromolecular surface coating agents, we discovered that surface coating strongly modulates the adhesion and morphogenesis of primary hippocampal neurons and serves as a switch of somata clustering and neurite fasciculation in vitro. The kinetics of neuronal adhesion on poly-lysine-coated surfaces is much faster than that on laminin and Matrigel-coated surfaces, and the distribution of adhesion is more homogenous on poly-lysine. Matrigel and laminin, on the other hand, facilitate neuritogenesis more than poly-lysine does. Eventually, on Matrigel-coated surfaces of self-assembled monolayers, neurons tend to undergo somata clustering and neurite fasciculation. By replacing coating proteins with cerebral astrocytes, and patterning neurons on astrocytes through self-assembled monolayers, microfluidics and micro-contact printing, we found that astrocyte promotes soma adhesion and astrocyte processes guide neurites. There, astrocytes could be a versatile substrate in engineering neuronal networks in vitro. Besides, quantitative measurements of cellular responses on various coatings would be valuable information for the neurobiology community in the choice of the most appropriate coating strategy.

Evaluation of the effect of the structure of bacterial cellulose on full thickness skin wound repair on a microfluidic chip.

Bacterial cellulose (BC) is a kind of nanobiomaterial for tissue engineering. How the nanoscale structure of BC affects skin wound repair is unexplored. Here, the hierarchical structure of BC films and their different effects on skin wound healing were studied both in vitro and in vivo. The bottom side of the BC film had a larger pore size, and a looser and rougher structure than that of the top side. By using a microfluidics-based in vitro wound healing model, we revealed that the bottom side of the BC film can better promote the migration of cells to facilitate wound healing. Furthermore, the full-thickness skin wounds on Wistar rats demonstrated that, compared with gauze and the top side of the BC film, the wound covered by the bottom side of the BC film showed faster recovery rate and less inflammatory response. The results indicate that the platform based on the microfluidic chip provide a rapid, reliable, and repeatable method for wound dressing screening. As an excellent biomaterial for wound healing, the BC film displays different properties on different sides, which not only provides a method to optimize the biocompatibility of wound dressings but also paves a new way to building heterogeneous BC-based biomaterials for complex tissue engineering.

c-Jun NH2-terminal kinase (JNK)-interacting protein-3 (JIP3) regulates neuronal axon elongation in a kinesin- and JNK-dependent manner.

Background: The role of JIP3 in axon specification and elongation in addition to axon branching remains unknown. Results: JIP3 locally activates the JNK-cofilin pathway at axon tips and thus enhances axon elongation. Conclusion: JIP3 is essential for axon elongation. Significance: These results advance our understanding of the role of JIP3 in axon development. The development of neuronal polarity is essential for the establishment of the accurate patterning of neuronal circuits in the brain. However, little is known about the underlying molecular mechanisms that control rapid axon elongation during neuronal development. Here, we report that c-Jun NH2-terminal kinase (JNK)-interacting protein-3 (JIP3) is highly expressed at axon tips during the critical period for axon development. Using gain- and loss-of-function approaches, immunofluorescence analysis, and in utero electroporation, we find that JIP3 can enhance axon elongation in primary hippocampal neurons and cortical neurons in vivo. We further demonstrate that JIP3 promotes axon elongation in a kinesin- and JNK-dependent manner using several deletion mutants of JIP3. Next, we demonstrate that the successful transportation of JIP3 to axon tips by kinesin is a prerequisite for enhancing JNK phosphorylation in this area and therefore promotes axon elongation, constituting a novel mechanism for coupling JIP3 anterograde transport with JNK signaling at the distal axons and axon elongation. Finally, our immunofluorescence data suggest that the activation of JNK at axon tips facilitates axon elongation by modulating cofilin activity and actin filament dynamics. These findings may have important implications for our understanding of neuronal axon elongation during development.

Development of neurons on micropatterns reveals that growth cone responds to a sharp change of concentration of laminin

In this report we fabricated laminin (LN) stripes on a background of poly‐L‐lysine as substrates for the growth of rat hippocampal neurons, and found that a sharp change of the concentration of LN guides the growth of neurites by leading the growth cones in a time‐ and space‐dependent manner. The percentage of neurites that grow along the edge of LN stripes (where there is a sharp change of concentration) decreases as a function of the concentration of LN under a threshold value. The actin cytoskeleton plays an important role in the process of growth cones response to the sharp change of concentration of LN on micropatterns. We believe that the findings here are useful for not only fundamental studies in neuroscience, but also helpful for the design of devices or chips for nervous prosthesis.

Self-Organizing Circuit Assembly through Spatiotemporally Coordinated Neuronal Migration within Geometric Constraints

Background Neurons are dynamically coupled with each other through neurite-mediated adhesion during development. Understanding the collective behavior of neurons in circuits is important for understanding neural development. While a number of genetic and activity-dependent factors regulating neuronal migration have been discovered on single cell level, systematic study of collective neuronal migration has been lacking. Various biological systems are shown to be self-organized, and it is not known if neural circuit assembly is self-organized. Besides, many of the molecular factors take effect through spatial patterns, and coupled biological systems exhibit emergent property in response to geometric constraints. How geometric constraints of the patterns regulate neuronal migration and circuit assembly of neurons within the patterns remains unexplored. Methodology/Principal Findings We established a two-dimensional model for studying collective neuronal migration of a circuit, with hippocampal neurons from embryonic rats on Matrigel-coated self-assembled monolayers (SAMs). When the neural circuit is subject to geometric constraints of a critical scale, we found that the collective behavior of neuronal migration is spatiotemporally coordinated. Neuronal somata that are evenly distributed upon adhesion tend to aggregate at the geometric center of the circuit, forming mono-clusters. Clustering formation is geometry-dependent, within a critical scale from 200 µm to approximately 500 µm. Finally, somata clustering is neuron-type specific, and glutamatergic and GABAergic neurons tend to aggregate homo-philically. Conclusions/Significance We demonstrate self-organization of neural circuits in response to geometric constraints through spatiotemporally coordinated neuronal migration, possibly via mechanical coupling. We found that such collective neuronal migration leads to somata clustering, and mono-cluster appears when the geometric constraints fall within a critical scale. The discovery of geometry-dependent collective neuronal migration and the formation of somata clustering in vitro shed light on neural development in vivo.

Micro/nano-scale materials and structures for constructing neuronal networks and addressing neurons

In recent years, a number of new materials and techniques at the micro/nano-scale for neuroscience have been reported, in particular in studies of neuronal development and electronic addressing. They offer new capabilities to fabricate tools in controlling the surface properties (such as topography and chemistry) as well as executing electrical stimulations and measurements. Here we review the basic principles of these micro/nano-scale materials and highlight the important advances in this field, and finally provide some perspectives for the future.

Assembly of Functional Three‐Dimensional Neuronal Networks on a Microchip

Herein we describe an on-chip method by which, for the fi rst time, we construct a kind of long-term 3D neuronal networks with ordered somata patterns and guided neurite connections, and we monitored the neuronal activities with functional imaging simultaneously. We used gravity-driven, microchamber-assisted assembly of borosilicate glass microspheres to fabricate scaffolds onto which dissociated neurons develop 3D neuronal networks. These neuron-carrying microspheric building blocks bear the following advantages: i) The relatively large surfaceto-volume ratio of each microsphere facilitates neuronal adhesion, polarity as well as synaptogenesis; ii) these microspheres can be stabilized in a highly ordered manner with the geometrical confi nement of microchambers, allowing easy manipulation of structural enlargement or re-assembly without damaging neuronal adhesion or neurite extension; iii) the spherical shape supports suffi cient nutritional exchange through the spaces between the building blocks; iv) the stiffness of glass provides good long-term stability of the 3D structure, which is the prerequisite for functional studies in neuronal networks. In comparison, the polymeric/hydrogel systems [ 12 ] tend to deform and collapse. We also utilized an array of aligned microgrooves for neurite guidance. These microgrooves support ordered neurite connections between the assembled 3D neuronal networks in different microchambers, connecting several 3D sub-networks into an interconnected one. These microgroove-guided neurites were set up in a way such that the 3D structures resemble the topology of long-range projecting nerve fi bers that connect nuclei in the brain. Finally, we applied calcium imaging to test the neuronal activities in the 3D networks, confi rming that the on-chip neuronal networks are functionally alive. We assembled borosilicate glass microspheres around 60 μm in diameter as scaffolds, where microspheres from 40 μm to 120 μm are all applicable in providing considerable neuronal density, [ 16 ] to support the survival and development of neuronal networks. The microspheres were pre-coated with poly-D-lysine (PDL) to enhance the adhesion as well as to promote the maturation of neurons. [ 13 ] We placed PDLcoated microspheres onto a large substrate to allow them to spontaneously assemble under gravity. After a compact monolayer was formed, we plated primary cortical neurons onto them and re-collected the microspheres after neuronal adhesion. These neuron-carrying microspheres were transferred onto a smaller coverslip to assemble layered structures ( Figure 1 A). The number of layers is determined by DOI: 10.1002/smll.201400513 Neuronal Networks