Scanning electrochemical microscopy of model neurons: imaging and real-time detection of morphological changes

Living PC12 cells, a model cell type for studying neuronal function, were imaged using the negative feedback mode of a scanning electrochemical microscope (SECM). Six biocompatible redox mediators were successfully identified from a large pool of candidates and were then used for imaging PC12 cells before and after exposure to nerve growth factor (NGF). When exposed to NGF, cells differentiate into a neuron phenotype by growing narrow neurites (1-2 microm wide) that can extend > 100 microm from the cell proper. We demonstrate that carbon fiber electrodes with reduced tip diameters can be used for imaging both the cell proper and these neurites. Regions of decreased current, possibly resulting from raised features not identifiable by light microscopy, are clearly evident in the SECM images. Changes in the morphology of undifferentiated PC12 cells could be detected in real time with the SECM. After exposure to hypotonic and hypertonic solutions, reversible changes in cell height of <2 microm were measured.     

Novel thin-walled nerve conduit with microgrooved surface patterns for enhanced peripheral nerve repair

Randomly aligned nerve cells in vitro on conventional culture substrata do not represent the complex neuronal network in vivo and neurites growing in uncontrolled manner may form neuroma. It is of great importance to mimic the organised growth pattern of nerve cells in the study of peripheral nerve repair. The aim of this work was to modify and optimize the photolithographic technique in creating a reusable template in the form of a silicon wafer that could be used to produce contact guidance on biodegradable polymer surface for the orientated growth of nerve cells. Micro-grooves (approximately 3 μm in depth) were etched into the silicon template using KOH at increased temperature. The originality of this work lies in the low cost and high efficiency method in producing microgrooves on the surface of biodegradable ultra-thin polymer substrates (50–100 μm), which can be readily rolled up to form clinically implantable nerve conduits. The design of a pattern with small ridge width (i.e., 5 μm) and bigger groove width (i.e., 20 μm) favored the alignment of cells along the grooves rather than on the ridges of the patterns, which minimized the effect of cross growing of neurites between adjacent grooves. Effectively, enhanced nerve regeneration could be anticipated from these patterned conduits.     

Neuronal networks in vitro: formation and organization on biofunctionalized surfaces

Receptor-mediated recognition of substrate molecules is a prerequisite for nerve cells in order to find their target structures in vivo and leads to formation of neuronal connections and networks. In order to study these mechanisms under in vitro conditions, we cultured embryonic hippocampal neurons or neuronal cell lines, SH-SY5Y and PCC7-PCC7-Mz1, onto biofunctionalized surfaces. Micropatterning on polymer surfaces, glass- and silicone-oxide-based chip materials was performed in a micrometer range by microcontact printing using polydimethylsiloxane (PDMS) stamps. Hippocampal neurons were found to form networks on chip surfaces under serum-free conditions and remained functional for more than a week. Human neuroblastoma cells SH-SY5Y as well as PCC7-Mz1 stem cells were found to follow microcontact printed pattern on polystyrene surfaces. Both cell lines showed neuronal marker expression and were cultured for up to 7 days with serum containing culture medium. Widths of 3–5 µm of coating lines were found to enhance single cell spreading along the pattern. The techniques described in this study may be useful in promoting nerve cell regeneration and organization following transection due to trauma or surgery. The neuronal alignment and network formation in vitro may furthermore serve as a model system in the field of biosensors. © 1999 Kluwer Academic Publishers     

Surface modification of polydimethylsiloxane (PDMS) induced proliferation and neural-like cells differentiation of umbilical cord blood-derived mesenchymal stem cells

Stem cell-based therapy has recently emerged for use in novel therapeutics for incurable diseases. For successful recovery from neurologic diseases, the most pivotal factor is differentiation and directed neuronal cell growth. In this study, we fabricated three different widths of a micro-pattern on polydimethylsiloxane (PDMS; 1, 2, and 4 microm). Surface modification of the PDMS was investigated for its capacity to manage proliferation and differentiation of neural-like cells from umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs). Among the micro-patterned PDMS fabrications, the 1 microm-patterned PDMS significantly increased cell proliferation and most of the cells differentiated into neuronal cells. In addition, the 1 microm-patterned PDMS induced an increase in cytosolic calcium, while the differentiated cells on the flat and 4 microm-patterned PDMS had no response. PDMS with a 1 microm pattern was also aligned to direct orientation within 10 degrees angles. Taken together, micro-patterned PDMS supported UCB-MSC proliferation and induced neural like-cell differentiation. Our data suggest that micro-patterned PDMS might be a guiding method for stem cell therapy that would improve its therapeutic action in neurological diseases.     

Nanostructured Superhydrophobic Substrates Trigger the Development of 3D Neuronal Networks

The generation of 3D networks of primary neurons is a big challenge in neuroscience. Here, a novel method is presented for a 3D neuronal culture on superhydrophobic (SH) substrates. How nano‐patterned SH devices stimulate neurons to build 3D networks is investigated. Scanning electron microscopy and confocal imaging show that soon after plating neurites adhere to the nanopatterned pillar sidewalls and they are subsequently pulled between pillars in a suspended position. These neurons display an enhanced survival rate compared to standard cultures and develop mature networks with physiological excitability. These findings underline the importance of using nanostructured SH surfaces for directing 3D neuronal growth, as well as for the design of biomaterials for neuronal regeneration.     

monitoring activity-dependent peptide release from the CNS using single-bead solid-phase extraction and MALDI TOF MS detection

To investigate dynamic peptidergic cell-cell communication, single micrometer-sized solid-phase extraction (SPE) beads were used to collect peptides from specific locations of well-characterized neurosecretory structures and even individual neuronal processes for off-line MALDI MS analyses. Peptide binding parameters of single SPE beads, including limits of collection, detection, and saturation capacity, were tested with 14C-labeled cytochrome c as well as with mixtures of multiple neuropeptides (bradykinin, Aplysia acidic peptide 1-20, and insulin). MALDI MS detection of secreted peptides was demonstrated in two well-characterized neurosecretory structures, the rat pituitary gland and single cultured Aplysia bag cell neurons. With cultured cells, precise placement of SPE beads allowed peptide collection from distinct neurites with spatial localization on the order of 200 microm, and SPE beads could be replaced within time frames that allowed analyte collection before and after cell stimulation paradigms. Comparison between pre- and poststimulation peptide profiles in both model systems allowed a directed strategy to determine which compounds were released with neuronal activity. Single SPE bead MALDI MS offers a novel approach to investigate peptide signaling that allows the detection and discovery of unknown intercellular signals secreted from a large variety of biological tissues.     

Submicron-patterned fibronectin controls the biological behavior of human dermal fibroblasts

Cell adhesion is an important step in cell survival, and in the proliferation of anchorage-dependent cells, whose dimensions can be controlled by micro-patterning of the cell-adhesive extracellular matrix. To fabricate a micro-patterned fibronectin substrate with spacings ranging from 0.9 microm to 20 microm, we made a replica mold using e-beam lithography. The physiological behavior of human dermal fibroblasts (HDFs) on a substrate with a gradient of pattern spacings from 0.9 microm to 20 microm was evaluated after 4.5 hours and 2 days of culture. The number of proliferating cells on the fibronectin-patterned surface increased as the spacing between strip lines increased to 11 micron. However, the number of cells gradually decreased when the pattern spacing exceeded 11 microm. These findings demonstrate that the submicron-patterned topography of a substrate plays important roles in HDF survival and proliferation.     

嗅球中神经元间的相互作用及同步运动分析

嗅球对嗅觉信息的处理是嗅觉系统信号编码的一个重要环节,其中兴奋性的僧帽细胞（Mitral Cell, MC）与抑制性的颗粒细胞（Granular Cell, GC）的相互作用尤为关键。本文首先介绍了嗅觉系统网络中关于同步振荡的研究现状,然后建立嗅球中僧帽细胞及颗粒细胞的动力学模型,仿真得到了单个僧帽细胞、颗粒细胞以及僧帽细胞与颗粒细胞在耦合条件下神经元的发放模式。结果表明,僧帽细胞对颗粒细胞有兴奋性作用,而颗粒细胞对僧帽细胞有抑制性作用,细胞放电序列随着突触连接强度的改变而改变。此外,建立简单的嗅觉网络模型,分析了当颗粒细胞分别构成环形和网格状两种拓扑结构时,不同网络对两个僧帽细胞同步性的影响,用同步性指标ISI-distance刻画同步程度。数值分析表明颗粒细胞网格状的拓扑结构对僧帽细胞的同步性作用更为明显一些。     

Sheathless focusing of microbeads and blood cells based on hydrophoresis

This paper presents a microfluidic device for sheathless focusing of microbeads and blood cells based on a hydrophoretic platform comprising a V-shaped obstacle array (VOA). The VOA generates lateral pressure gradients that induce helical recirculations. Following the focusing flow particles passing through the VOA are focused in the center of the channel. In the device, the focusing pattern can be modulated by varying the gap height of the VOA. To achieve complete focusing within 4.4% coefficient of variation, the relative size differences between the gap and the particle were 3 and 4 microm for 10 and 15 microm beads, respectively. Red blood cells were used to study the hydrophoretic focusing pattern of biconcave, disk-shaped particles.     

Spatially resolved non-invasive chemical stimulation for modulation of signalling in reconstructed neuronal networks.

Functional coupling of reconstructed neuronal networks with microelectronic circuits has potential for the development of bioelectronic devices, pharmacological assays and medical engineering. Modulation of the signal processing properties of on-chip reconstructed neuronal networks is an important aspect in such applications. It may be achieved by controlling the biochemical environment, preferably with cellular resolution. In this work, we attempt to design cell-cell and cell-medium interactions in confined geometries with the aim to manipulate non-invasively the activity pattern of an individual neuron in neuronal networks for long-term modulation. Therefore, we have developed a biohybrid system in which neuronal networks are reconstructed on microstructured silicon chips and interfaced to a microfluidic system. A high degree of geometrical control over the network architecture and alignment of the network with the substrate features has been achieved by means of aligned microcontact printing. Localized non-invasive on-chip chemical stimulation of micropatterned rat cortical neurons within a network has been demonstrated with an excitatory neurotransmitter glutamate. Our system will be useful for the investigation of the influence of localized chemical gradients on network formation and long-term modulation.     

Temperature dependence of transport anisotropy of planar-type graphite nanostructures fabricated by focused ion beam

We report in this paper on the observation of temperature-dependent anisotropic transport behavior for planar-type nanostructures (in-plane and out-of-plane) fabricated on a thin graphite layer using a three-dimensional focused-ion-beam (FIB) etching technique. The transport characteristics were studied for several in-plane areas with sizes of 6 x 6 microm2, 6 x 4 microm2 and 6 x 2 microm2 planar-type structures/patterns and out-of-plane structures with the dimensions of 2 x 1 x 0.3 microm3. Both in-plane (rho(a)) and out-of-plane (rho(c)) resistivities are measured for these structures and the ratio of resistivity anisotropy is determined. The observed values of anisotropy ratio rho(c)/rho(a) were approximately 12.5 at 300 K and approximately 54 at 25 K. The room temperature value of rho(c)/rho(a) varies by a few orders from the values of previously reported anisotropy results of bulk pyrolytic graphite. However, the value of resistivity anisotropy increases with decreasing temperature, which is an identical behavior to bulk pyrolytic graphite. From current (I)-voltage (V) characteristics, we observed an ohmic behavior at 300 K for both low- and high-current biasing. This behavior turns into nonlinear characteristics when the temperature goes down. As these fabricated structures consist of multiple elementary junctions along the c-axis, nonlinear I-V characteristics result. The impurity assisted interlayer hopping conduction and thermal excitation of carriers play a key role in this effect.     

Inversion of multiwavelength Raman lidar data for retrieval of bimodal aerosol size distribution

We report on the feasibility of deriving microphysical parameters of bimodal particle size distributions from Mie-Raman lidar based on a triple Nd:YAG laser. Such an instrument provides backscatter coefficients at 355, 532, and 1064 nm and extinction coefficients at 355 and 532 nm. The inversion method employed is Tikhonov's inversion with regularization. Special attention has been paid to extend the particle size range for which this inversion scheme works to approximately 10 microm, which makes this algorithm applicable to large particles, e.g., investigations concerning the hygroscopic growth of aerosols. Simulations showed that surface area, volume concentration, and effective radius are derived to an accuracy of approximately 50% for a variety of bimodal particle size distributions. For particle size distributions with an effective radius of < 1 microm the real part of the complex refractive index was retrieved to an accuracy of +/- 0.05, the imaginary part was retrieved to 50% uncertainty. Simulations dealing with a mode-dependent complex refractive index showed that an average complex refractive index is derived that lies between the values for the two individual modes. Thus it becomes possible to investigate external mixtures of particle size distributions, which, for example, might be present along continental rims along which anthropogenic pollution mixes with marine aerosols. Measurement cases obtained from the Institute for Tropospheric Research six-wavelength aerosol lidar observations during the Indian Ocean Experiment were used to test the capabilities of the algorithm for experimental data sets. A benchmark test was attempted for the case representing anthropogenic aerosols between a broken cloud deck. A strong contribution of particle volume in the coarse mode of the particle size distribution was found.     

Photocurrent polarization anisotropy of randomly oriented nanowire networks

While the polarization sensitivity of single or aligned NW ensembles is well-known, this article reports on the existence of residual photocurrent polarization sensitivities in random NW networks. In these studies, CdSe and CdTe NWs were deposited onto glass substrates and contacted with Au electrodes separated by 30-110 microm gaps. SEM and AFM images of resulting devices show isotropically distributed NWs between the electrodes. Complementary high resolution TEM micrographs reveal component NWs to be highly crystalline with diameters between 10 and 20 nm and with lengths ranging from 1 to 10 microm. When illuminated with visible (linearly polarized) light, such random NW networks exhibit significant photocurrent anisotropies rho = 0.25 (sigma = 0.04) [rho = 0.22 (sigma = 0.04)] for CdSe (CdTe) NWs. Corresponding bandwidth measurements yield device polarization sensitivities up to 100 Hz. Additional studies have investigated the effects of varying the electrode potential, gap width, and spatial excitation profile. These experiments suggest electrode orientation as the determining factor behind the polarization sensitivity of NW devices. A simple geometric model has been developed to qualitatively explain the phenomenon. The main conclusion from these studies, however, is that polarization sensitive devices can be made from random NW networks without the need to align component wires.     

Electro-optical investigations of holographic-polymer-dispersed ferroelectric liquid crystals

Uniform alignment of ferroelectric liquid-crystal domains encapsulated by a polymer binder was established through a holographic exposure process. The refractive index modulation in these thin films is modeled as a phase grating that can be electrically addressed to erase the optical diffractive properties. A phenomenological model is developed to take into account a distribution of domain sizes and an effective field that stabilizes the ferroelectric liquid-crystal domains. A diffraction model successfully predicts changes in normalized intensities for first-order diffraction with applied field. These gratings demonstrate microsecond-scale response and relaxation times for various grating pitch sizes between approximately 3 and approximately 12 microm.     

Impact of neurite alignment on organelle motion

Intracellular transport is pivotal for cell growth and survival. Malfunctions in this process have been associated with devastating neurodegenerative diseases, highlighting the need for a deeper understanding of the mechanisms involved. Here, we use an experimental methodology that leads neurites of differentiated PC12 cells into either one of two configurations: a one-dimensional configuration, where the neurites align along lines, or a two-dimensional configuration, where the neurites adopt a random orientation and shape on a flat substrate. We subsequently monitored the motion of functional organelles, the lysosomes, inside the neurites. Implementing a time-resolved analysis of the mean-squared displacement, we quantitatively characterized distinct motion modes of the lysosomes. Our results indicate that neurite alignment gives rise to faster diffusive and super-diffusive lysosomal motion than the situation in which the neurites are randomly oriented. After inducing lysosome swelling through an osmotic challenge by sucrose, we confirmed the predicted slowdown in diffusive mobility. Surprisingly, we found that the swelling-induced mobility change affected each of the (sub-/super-)diffusive motion modes differently and depended on the alignment configuration of the neurites. Our findings imply that intracellular transport is significantly and robustly dependent on cell morphology, which might in part be controlled by the extracellular matrix.     

Spatial profiling with MALDI MS: distribution of neuropeptides within single neurons

MALDI MS imaging and single-cell profiling are important new capabilities for mass spectrometry. The distribution of neuropeptides within a cell plays an important role in the functioning of the cells in a neuronal network. Protocols for subcellular MALDI MS are described that allow comparative peptide profiling of cell bodies and the neuronal processes (neurites) using single isolated neurons from the neuronal model Aplysia californica. The seawater surrounding the neurons is problematic for mass spectrometry and so must be removed in a manner that does not cause morphological changes or a redistribution of the neuropeptides. Several protocols have been investigated for subcellular spatial profiling, including the use of air-drying, replacement of the seawater with deionized water, and substitution of the cell matrix with fluorinert, mineral oil and glycerol, as well as paraformaldehyde fixation. Glycerol stabilization offers the best combination of preservation of cell morphology and prevention of neuropeptide redistribution. The profiles of the peptides in specific neuronal processes and the cell bodies demonstrate a variety of differences that appear to be cell-specific. These methods are suitable for smaller cells and subcellular MS imaging.     

Formation of highly adherent nano-porous alumina on Ti-based substrates: a novel bone implant coating

Thin, nano-porous, highly adherent layers of anodised aluminium formed on the surface of titanium alloys are being developed as coatings for metallic surgical implants. The layers are formed by anodisation of a 1-5 microm thick layer of aluminium which has been deposited on substrate material by electron beam evaporation. The surface ceramic layer so produced is alumina with 6-8 wt % phosphate ions and contains approximately 5 x 10(8) cm(-2) pores with a approximately 160 nm average diameter, running perpendicular to the surface. Mechanical testing showed the coatings' shear and tensile strength to be at least 20 and 10 MPa, respectively. Initial cell/material studies show promising cellular response to the nano-porous alumina. A normal osteoblastic growth pattern with cell number increasing from day 1 to 21 was shown, with slightly higher proliferative activity on the nano-porous alumina compared to the Thermanox control. Scanning electron microscopy (SEM) examination of the cells on the porous alumina membrane showed normal osteoblast morphology. Flattened cells with filopodia attaching to the pores and good coverage were also observed. In addition, the pore structure produced in these ceramic coatings is expected to be suitable for loading with bioactive material to enhance further their biological properties.     

Bio-inducing growth of ZnO hollow spheres in lotus root

In this paper, ZnO hollow spheres are prepared via an antitoxic bio-inducing process in lotus roots. FESEM images indicate that the tannin cells coated with ZnO are spherical with diameters ranging from 1.5 microm to 3 microm. The spheres with approximately the same size aggregate on the surface of vessels in short-range. The new bio-inducing process for formation of oxides hollow spheres might exist for long time in nature. It is proved that plant antitoxic function is the key factor to induce these structures formation. And moreover, it could be predicted that this bio-inducing phenomenon is an ideal way for preparation of materials with special structures.