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ABSTRACT: A graphene-based flexible microprobe developed by microelectromechanical system technology shows high resolution for the detection of electrophysiological signals from various bio-objects. The hydrophilization post-treatment using steam plasma was performed on the graphene surface to decrease the interfacial impedance between graphene/electrolyte, and thus improve the signal-to-noise ratio during neural and cardiac recording. The signal-to-noise ratio of the action potentials from axons of a crayfish measured by hydrophilic-modified graphene microprobe (27.8 ± 4.0 dB) is higher than that of untreated device (20.3 ± 3.3 dB). And the form of the QRS complex and T wave in the electrocardiogram of the zebrafish heart can be clearly distinguished using the modified device. The total measured noise levels of the overall stability of the system were 4.2 μV(rms) (hydrophilic graphene) and 7.64 μV(rms) (hydrophobic graphene). The graphene-based implant can be further used for in vivo, long term recording and retina prosthesis.
Nanomedicine: nanotechnology, biology, and medicine 01/2013; · 5.44 Impact Factor
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ABSTRACT: Axons are long, slender processes of neurons that have various functions at different stages of development. Here, we report the use of a chip device to study the effects of various exogenous proteins on the growth and presynaptic differentiation of axons in a high-throughput manner. The device consists of a glass chip whose surface contains a protein-coated micropattern. When neurons are maintained on the chip, a specific region of the chip surface will be occupied exclusively by axons. The axons and clusters of release-competent synaptic vesicles, a presynapse-like specialization in the axon, can be quantified as the proportions of this specific region's area occupied respectively by these subcellular structures. By using chips with this specific region coated with different proteins, these proteins' effects on the growth and presynaptic differentiation of the axon were investigated by comparing the amounts of axons and clusters of release-competent synaptic vesicles in this region of the chip. We also demonstrate another application of this chip device by investigating the effective range of the signal produced by the interaction between neurons and neuroligin 1 in neurons. These results indicate the diverse applications of the chip device in exploring various issues pertaining to axonal functions.
Analytical Biochemistry 04/2012; 427(1):1-9. · 3.00 Impact Factor
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ABSTRACT: The interaction between the synaptic adhesion molecules neuroligins and neurexins is essential for connecting the pre- and post-synaptic neurons, modulating neuronal signal transmission, and facilitating neuronal axogenesis. Here, we describe the simultaneous expression of the extracellular domain of rat neuroligin-1 (NL1) proteins along with the enhanced green fluorescent protein (EGFP) using the bi-cistronic baculovirus expression vector system (bi-BEVS). Recombinant rat NL1 protein, fused with signal sequence derived from human Azurocidin gene (AzSP), was secreted into the culture medium and the optimum harvest time for NL1 protein before the lysis of infected cells was determined through the release of cytosolic EGFP. The NL1 protein (0.129±0.013 mg/8×10(7) High Five cells; ~96% purity by metal affinity chromatography) was obtained from the supernatant of the recombinant virus-infected insect cells. A novel chip was employed to address whether the recombinant NL1 is functional in axogenesis. The purified rat NL1 promoted and enhanced the growth rate (137.07±9.74 μm/day) of the axon on NL1/PLL (poly-L-lysine)-coated fine lines on the chip compared to those lines that were coated with PLL alone (105.53±4.53 μm/day). These results were confirmed by fluorescence immunocytochemistry and demonstrated that the recombinant protein can be purified by a one-step process using IMAC combined with monitoring of cell lysis by bi-BEVS. This technique along with our novel chip offers a simple, cost-effective and useful platform for understanding the roles of NL1 protein in neuronal regeneration and synaptic formation studies.
Protein Expression and Purification 09/2011; 81(1):18-24. · 1.59 Impact Factor
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ABSTRACT: Chikungunya virus infection has emerged in many countries over the past decade. There are no effective drugs for controlling the disease. To develop cell-based system for screening anti-virus drugs, a bi-cistronic baculovirus expression system was utilized to co-express viral structural proteins C (capsid), E2 and E1 and the enhanced green fluorescence protein (EGFP) in Spodoptera frugiperda insect cells (Sf21). The EGFP-positive Sf21 cells fused with each other and with uninfected cells to form a syncytium, allowing characterization of cholesterol and low pH requirements for syncytium formation. Western blot analysis showed three structural proteins were expressed in baculovirus infected cells. The structural proteins of Chikungunya virus that is required for cell fusion was determined with various recombinant baculoviruses bearing different lengths of the viral structural protein genes. Protein E1 was required for cell fusion and indicating that Chikungunya viral membrane fusion was a class II membrane fusion. It was also demonstrated that the heterologous expression of alphavirus monomeric E1 can induce insect cell fusions. Furthermore, this cell-based system provides a model for studying class II viral membrane fusion.
Journal of virological methods 08/2011; 175(2):206-15. · 2.13 Impact Factor
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ABSTRACT: A bi-cistronic baculovirus-insect/larval system containing a polyhedron promoter, an internal ribosome entry site (IRES), and an egfp gene was developed as a cost-effective platform for the production of recombinant human interferon gamma (rhIFN-γ). There was no significant difference between the amounts of rhIFN-γ produced in the baculovirus-infected Spodoptera frugiferda 21 cells grown in serum-free medium and the serum-supplemented medium, while the Trichoplusia ni (T. ni) and Spodoptera exigua (S. exigua) larvae afforded rhIFN-γ amounting to 1.08±0.04 and 9.74±0.35 µg/mg protein respectively. The presence of non-glycosylated and glycosylated rhIFN-γ was confirmed by immunoblot and lectin blot. The immunological activity of purified rhIFN-γ, with 96% purity by Nickel (II)-nitrilotriacetic acid (Ni-NTA) affinity chromatography, was similar to that commercially available. Moreover, the rhIFN-γ protein from T. ni had more potent antiviral activity. These findings suggest that this IRES-based expression system is a simple and inexpensive alternative for large-scale protein production in anti-viral research.
Bioscience Biotechnology and Biochemistry 07/2011; 75(7):1342-8. · 1.28 Impact Factor
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ABSTRACT: Postsynaptic density (PSD) is a protein supramolecule lying underneath the postsynaptic membrane of excitatory synapses and has been implicated to play important roles in synaptic structure and function in mammalian central nervous system. Here, PSDs were isolated from two distinct regions of porcine brain, cerebral cortex and cerebellum. SDS-PAGE and Western blotting analyses indicated that cerebral and cerebellar PSDs consisted of a similar set of proteins with noticeable differences in the abundance of various proteins between these samples. Subsequently, protein localization in these PSDs was analyzed by using the Nano-Depth-Tagging method. This method involved the use of three synthetic reagents, as agarose beads whose surface was covalently linked with a fluorescent, photoactivable, and cleavable chemical crosslinker by spacers of varied lengths. After its application was verified by using a synthetic complex consisting of four layers of different proteins, the Nano-Depth-Tagging method was used here to yield information concerning the depth distribution of various proteins in the PSD. The results indicated that in both cerebral and cerebellar PSDs, glutamate receptors, actin, and actin binding proteins resided in the peripheral regions within ∼ 10 nm deep from the surface and that scaffold proteins, tubulin subunits, microtubule-binding proteins, and membrane cytoskeleton proteins found in mammalian erythrocytes resided in the interiors deeper than 10 nm from the surface in the PSD. Finally, by using the immunoabsorption method, binding partner proteins of two proteins residing in the interiors, PSD-95 and α-tubulin, and those of two proteins residing in the peripheral regions, elongation factor-1α and calcium, calmodulin-dependent protein kinase II α subunit, of cerebral and cerebellar PSDs were identified. Overall, the results indicate a striking similarity in protein organization between the PSDs isolated from porcine cerebral cortex and cerebellum. A model of the molecular structure of the PSD has also been proposed here.
Molecular & Cellular Proteomics 06/2011; 10(10):M110.007138. · 7.40 Impact Factor
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ABSTRACT: This paper reports the success of amino-functionalization on multi-walled carbon nanotubes (MWCNTs) to promote neuronal cells growth on MWCNT electrode for extracellular recording, attributed to the formation of positive charge of NH(2) molecules on their surfaces. Besides, the surface of MWCNT electrode becomes hydrophilic after amino-functionalization (AF-MWCNTs) which can enhance electrical conductivity because of lower MWCNT/electrolyte interfacial impedance and higher interfacial capacitance. Durability tests show that electrical characteristics of the MWCNTs treated by 2 wt% 1,4-diaminobutane solution (2 wt%-AF-MWCNTs) can last for at least six months in air ambient. The neural recording of crayfish shows that 2 wt%-AF-MWCNTs can provide better capability on detecting action potentials of caudal photoreceptor (CPR) interneuron compared to suction glass pipette from the evidence of a higher S/N ratio (126 versus 23). The amino-functionalized MWCNT electrode is feasible for long-term recording application according to the results of biocompatibility tests. As the MWCNTs were directly synthesized on Si-based substrates by catalyst-assisted thermal chemical vapor deposition (CVD) at a low temperature (400 °C), these self-aligned MWCNT electrodes could be friendly implemented in integrated circuits fabrications.
Biosensors & bioelectronics 06/2011; 26(10):4124-32. · 5.43 Impact Factor
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ABSTRACT: Electrodes on planar type microelectromechanical system (MEMS) microprobes mainly record neurons on the top-side of probe shaft (called a top-side electrode). However, it is often necessary to record neurons other than those on the top-side of the probe shaft. This study uses the glass reflowing technique to embed silicon-vias in a glass probe to implement a microprobe capable of recording neurons around the shaft. The proposed technology makes it possible to fabricate, distribute, and integrate four types of electrodes on the shaft: top-side, back-side, double-side, and sidewall electrodes. These electrodes have different recording characteristics. The in vitro and in vivo (using crayfish and rat brain) experiments in this study shows that the top-side and back-side electrodes are respectively more sensitive to neurons on the top-side and back-side of the probe shaft. In contrast, signals recorded by double-side electrode and sidewall electrode are equally sensitive to neurons around the probe shaft. This study enables the implementation and integration of these four types of electrodes, meeting the requirements of various neural applications.
Biosensors & bioelectronics 06/2011; 26(12):4739-46. · 5.43 Impact Factor
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Yung-Chan Chen,
Hui-Lin Hsu,
Yu-Tao Lee,
Huan-Chieh Su,
Shiang-Jie Yen,
Chang-Hsiao Chen,
Wei-Lun Hsu,
Tri-Rung Yew,
Shih-Rung Yeh,
Da-Jeng Yao, Yen-Chung Chang,
Hsin Chen
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ABSTRACT: A variety of microelectrode arrays (MEAs) has been developed for monitoring intra-cortical neural activity at a high spatio-temporal resolution, opening a promising future for brain research and neural prostheses. However, most MEAs are based on metal electrodes on rigid substrates, and the intra-cortical implantation normally causes neural damage and immune responses that impede long-term recordings. This communication presents a flexible, carbon-nanotube MEA (CMEA) with integrated circuitry. The flexibility allows the electrodes to fit on the irregular surface of the brain to record electrocorticograms in a less invasive way. Carbon nanotubes (CNTs) further improve both the electrode impedance and the charge-transfer capacity by more than six times. Moreover, the CNTs are grown on the polyimide substrate directly to improve the adhesion to the substrate. With the integrated recording circuitry, the flexible CMEA is proved capable of recording the neural activity of crayfish in vitro, as well as the electrocorticogram of a rat cortex in vivo, with an improved signal-to-noise ratio. Therefore, the proposed CMEA can be employed as a less-invasive, biocompatible and reliable neuro-electronic interface for long-term usage.
Journal of Neural Engineering 06/2011; 8(3):034001. · 3.84 Impact Factor
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ABSTRACT: When rat fetuses grew from embryonic day (E) 18 to the day of birth (P0), the corticothalamic (CT) neurons, as identified by back labeling with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI), in the somatosensory cortex underwent gradual changes in the shape of their cell bodies, in their distribution in the cortical plate and in the complexity of dendritic branching. Fluorescence immunocytochemical studies indicated that in the marginal zone (MZ) the apical dendrites of the CT neurons formed contacts with horizontally oriented axons and contained putative glutamatergic, as clusters exhibiting both synaptophysin and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR1 subunit immunoreactivities, and γ-aminobutyric acid (GABA)-ergic synapses, as clusters exhibiting both synaptophysin and gephyrin immunoreactivities. Quantitative analyses further revealed that during this perinatal period, the proportion of CT neurons containing glutamatergic synapses increased significantly, whereas the proportion of CT neurons containing GABAergic synapses remained virtually unchanged. Our results indicate that glutamatergic and GABAergic synapses between the CT neurons and the axons in the MZ are already formed in rat cortices as early as E18 and further suggest that the activities of the neural networks in the somatosensory cortex could be conveyed to their targets in the thalamus in rat brains at least 3 days before birth.
Cerebral Cortex 04/2011; 21(4):884-95. · 6.54 Impact Factor
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ABSTRACT: We designed, fabricated and tested a novel three-dimensional flexible microprobe to record neural signals of a lateral giant nerve fiber of the escape circuit of an American crayfish. An electrostatic actuation folded planar probes into three-dimensional neural probes with arbitrary orientations for neuroscientific applications. A batch assembly based on electrostatic forces simplified the fabrication and was non-toxic. A novel fabrication for these three-dimensional flexible probes used SU-8 and Parylene technology. The mechanical strength of the neural probe was great enough to penetrate into a bio-gel. A flexible probe both decreased the micromotion and alleviated tissue encapsulation of the implant caused by chronic inflammation of tissue when an animal breathes or moves. The cortex consisted of six horizontal layers, and the neurons of the cortex were arranged in vertical structures; the three-dimensional microelectrode arrays were suitable to investigate the cooperative activity for neurons in horizontal separate layers and in vertical cortical columns. With this flexible probe we recorded neural signals of a lateral giant cell from an American crayfish. The response amplitude of action potentials was about 343 µV during 1 ms period; the average recorded data had a ratio of signal to noise as great as 30.22 ± 3.58 dB. The improved performance of this electrode made feasible the separation of neural signals according to their distinct shapes. The cytotoxicity indicated a satisfactory biocompatibility and non-toxicity of the flexible device fabricated in this work.
Lab on a Chip 03/2011; 11(9):1647-55. · 5.67 Impact Factor
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Chang-Hsiao Chen,
Huan-Chieh Su,
Shih-Chang Chuang,
Shiang-Jie Yen,
Yung-Chan Chen,
Yu-Tao Lee,
Hsin Chen,
Tri-Rung Yew, Yen-Chung Chang,
Shih-Rung Yeh,
Da-Jeng Yao
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ABSTRACT: To decrease the impedance of microelectrode arrays, for neuroscience applications we have fabricated and tested MEA based on multi-walled carbon nanotubes. With decreasing physical size of a microelectrode, its impedance increases and charge-transfer capability decreases. To decrease the impedance, the effective surface area of the electrode must generally be increased. We explored the effect of plasma treatment on the surface wettability of MWCNT. With a steam-plasma treatment the surface of MWCNT becomes converted from superhydrophobic to superhydrophilic; this hydrophilic property is attributed to -OH bonding on the surface of MWCNT. We reported the synthesis at 400 °C of MWCNT on nickel-titanium multilayered metal catalysts by thermal chemical vapor deposition. Applying plasma with a power less than 25 W for 10 s improved the electrochemical and biological properties, and circumvented the limitation of the surface reverting to a hydrophobic condition; a hydrophilic state is maintained for at least one month. The MEA was used to record neural signals of a lateral giant cell from an American crayfish. The response amplitude of the action potential was about 275 µV with 1 ms period; the recorded data had a ratio of signal to noise up to 40.12 dB. The improved performance of the electrode makes feasible the separation of neural signals and the recognition of their distinct shapes. With further development the rapid treatment will be useful for long-term recording applications.
Nanotechnology 11/2010; 21(48):485501. · 3.98 Impact Factor
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Huan-Chieh Su,
Chia-Min Lin,
Shiang-Jie Yen,
Yung-Chan Chen,
Chang-Hsiao Chen,
Shih-Rung Yeh,
Weileun Fang,
Hsin Chen,
Da-Jeng Yao, Yen-Chung Chang,
Tri-Rung Yew
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ABSTRACT: A novel cone-shaped 3D carbon nanotube (CNT) probe is proposed as an electrode for applications in neural recording. The electrode consists of CNTs synthesized on the cone-shaped Si (cs-Si) tip by catalytic thermal chemical vapor deposition (CVD). This probe exhibits a larger CNT surface area with the same footprint area and higher spatial resolution of neural recording compared to planar-type CNT electrodes. An approach to improve CNT characteristics by O(2) plasma treatment to modify the CNT surface will be also presented. Electrochemical characterization of O(2) plasma-treated 3D CNT (OT-CNT) probes revealed low impedance per unit area (∼64.5 Ω mm(-2)) at 1 kHz and high specific capacitance per unit area (∼2.5 mF cm(-2)). Furthermore, the OT-CNT probes were employed to record the neural signals of a crayfish nerve cord. Our findings suggest that OT-CNT probes have potential advantages as high spatial resolution and superb electrochemical properties which are suitable for neural recording applications.
Biosensors & bioelectronics 09/2010; 26(1):220-7. · 5.43 Impact Factor
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ABSTRACT: We have designed, fabricated, and tested a novel three-dimensional (3-D) flexible microprobe used for recording the neural signals of lateral giant (LG) on the escape system of American crayfish. We report an electrostatic actuation process to fold the planar probes to be the arbitrary orientations of 3-D probes for neuroscience application. The batch assembly method based on electrostatic force techniques gave more simple fabrication compared to others. A flexible probe could reduce both the chronic inflammation response and material fracture when animal breathes or moves. Furthermore, the cortex corresponds to hypothetical cortical modules with mostly vertically organized layers of neurons. Therefore, the 3-D flexible probe suits to understand how the cooperative activity for different layers of neurons. Advisedly, we present a novel fabrication for the 3-D flexible probe by using Parylene technology. The mechanical strength of the neural probe is strong enough to penetrate into a biogel. At the end, the flexible probe was used to record neural signals of the LG cell from American crayfish.
Journal of Micro/ Nanolithography, MEMS, and MOEMS 07/2010; 9(3):031007. · 1.00 Impact Factor
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ABSTRACT: We have designed, fabricated, and tested a novel three-dimensional (3-D) flexible microprobe used for recording the neural signals of lateral giant (LG) on the escape system of American crayfish. We report an electrostatic actuation process to fold the planar probes to be the arbitrary orientations of 3-D probes for neuroscience application. The batch assembly method based on electrostatic force techniques gave more simple fabrication compared to others. A flexible probe could reduce both the chronic inflammation response and material fracture when animal breathes or moves. Furthermore, the cortex corresponds to hypothetical cortical modules with mostly vertically organized layers of neurons. Therefore, the 3-D flexible probe suits to understand how the cooperative activity for different layers of neurons. Advisedly, we present a novel fabrication for the 3-D flexible probe by using Parylene technology. The mechanical strength of the neural probe is strong enough to penetrate into a biogel. At the end, the flexible probe was used to record neural signals of the LG cell from American crayfish.
Journal of Microlithography Microfabrication and Microsystems 07/2010; 9(3):031007. · 0.54 Impact Factor
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ABSTRACT: The effect of microwave (MW) treatment to improve the adhesion of carbon nanotubes (CNTs) to a Ni/Ti/Au/SiO2/Si substrate was examined. CNTs were synthesized at a low temperature (400 °C) by thermal chemical vapor deposition to avoid metal peeling. Results demonstrated that nearly 100% of MW-treated CNTs remained on the substrates even after sonication in a buffer solution. This was a significant improvement of adhesion compared to preparations not undergoing MW treatment, where almost no CNTs remained. Transmission electron microscopy of cross sections showed that before MW treatment, CNTs with Ni nanoparticles were located on the upper part of the Ti underlayer, whereas after MW treatment they were embedded in the Ti underlayer. Based on these results, a mechanism of adhesion improvement by MW treatment is proposed.
Carbon 03/2010; 48(3):805-812. · 5.38 Impact Factor
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ABSTRACT: Axons are long, slender processes extending from the cell bodies of neurons and play diverse and crucial roles in the development and function of nervous systems. Here, we describe the development of a chip device that can be used to produce large quantities of axons for proteomic and RNA analyses. On the chip surface, bundles of axons of rat hippocampal neurons in culture are guided to grow in areas distinct and distant from where their cell bodies reside. Fluorescence immunocytochemical studies have confirmed that the areas where these axons are guided to grow are occupied exclusively by axons and not by neuronal somatodendrites or astroglial cells. These axon-occupied parts are easily separated from the remainder of the chip and collected by breaking the chip along the well-positioned linear grooves made on the backside. One- and two-dimensional gel electrophoresis and Western blotting analyses reveal that the axons and whole cells differ in their protein compositions. RT-PCR analyses also indicate that the axons contain only a subset of neuronal RNAs. Furthermore, the chip device could be easily modified to address other issues concerning neuronal axons, such as the molecular composition of the axon substructure, the growth cone and shaft, the degeneration and regeneration processes associated with injured axons and the effects of extrinsic molecules, such as axon guidance cues and cell adhesion molecules, on the axon. With these diverse applications, the chip device described here will serve as a powerful platform for studying the functional proteome of neuronal axons.
Lab on a Chip 03/2010; 10(5):647-53. · 5.67 Impact Factor
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ABSTRACT: The effect of microwave (MW) treatment to improve the adhesion of carbon nanotubes (CNTs) to a Ni/Ti/Au/SiO2/Si substrate was examined. CNTs were synthesized at a low temperature (400 °C) by thermal chemical vapor deposition to avoid metal peeling. Results demonstrated that nearly 100% of MW-treated CNTs remained on the substrates even after sonication in a buffer solution. This was a significant improvement of adhesion compared to preparations not undergoing MW treatment, where almost no CNTs remained. Transmission electron microscopy of cross sections showed that before MW treatment, CNTs with Ni nanoparticles were located on the upper part of the Ti underlayer, whereas after MW treatment they were embedded in the Ti underlayer. Based on these results, a mechanism of adhesion improvement by MW treatment is proposed.
Carbon. 03/2010; 48(3):805-812.
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ABSTRACT: The distribution of cells expressing calcium-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (CP-AMPARs) in the somatosensory cortex of rats at different developmental stages was studied using a kainate-stimulated Co(2+)-labeling assay in a quantitative manner. The applicability of this assay for identifying CP-AMPAR-expressing cells was first verified using cultured rat cortical neurons by means of fluorescence Ca(2+) imaging and pharmacological tools. Cells positively identified by the Co(2+)-labelinig assay resided primarily in the marginal zone and subplate of young fetuses and became more widely distributed throughout the cortex as the fetus matured. The majority, >80%, of these Co(2+)-positive cells were neurons, exhibiting immunoreactivity with the neuronal marker NeuN. The proportion of neurons that were Co(2+)-positive increased from approximately 25% to approximately 60% as the rat fetus grew into adulthood. In contrast, less than 20% of nonneuronal cells were Co(2+)-positive. Of the Co(2+)-positive neurons, 15%-31% exhibited GABA immunoreactivity and nonpyramidal-shaped cell bodies; these were presumably GABAergic neurons. Most of the remaining non-GABAergic/Co(2+)-positive neurons had pyramidal-shaped cell bodies and were presumably excitatory principle neurons. Around 70% of GABAergic neurons in the cortex were Co(2+)-positive. Furthermore, in the cortex of neonatal rats the Co(2+)-positive neurons were found to be more susceptible to kainate toxicity than the Co(2+)-negative cells. The Co(2+)-positive neurons in the subplate of neonatal rats were more vulnerable to kainate toxicity than their counterparts in the remaining cortical areas. Together, the widespread distribution and distinct susceptibility to excitotoxicity of CP-AMPAR-expressing neurons suggest that they play various important roles in the development and physiology of the rat cerebral cortex.
The Journal of Comparative Neurology 01/2010; 518(1):75-91. · 3.81 Impact Factor
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ABSTRACT: This study presents a process for the assembling of a pseudo 3D glass microprobe array. A glass microprobe with embedded silicon (ES) is batch fabricated by a glass reflow process. The silicon fixture and carrier for the assembly are also batch fabricated by silicon micromachining processes. First, the chips with a glass microprobe array are bonded by parylene-C to form the pseudo 3D glass microprobe array. The pseudo 3D microprobe array is then mounted on the silicon carrier. ES is employed for alignment during the assembly, and also acts as the electrical routing for signal recording. In application, the impedance of this glass microprobe is measured, and at 1 kHz it is 1.1 MΩ. Action potentials from rat brain cortex are also successfully recorded.
Journal of Micromechanics and Microengineering 12/2009; 20(2):025014. · 2.11 Impact Factor
