Neuronal network morphology and electrophysiologyof hippocampal neurons cultured on surface-treated multielectrode arrays (original) (raw)
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Low-density neuronal networks cultured using patterned poly-l-lysine on microelectrode arrays
Journal of Neuroscience Methods, 2007
Synaptic activity recorded from low-density networks of cultured rat hippocampal neurons was monitored using microelectrode arrays (MEAs). Neuronal networks were patterned with poly-Llysine (PLL) using microcontact printing (µCP). Polydimethysiloxane (PDMS) stamps were fabricated with relief structures resulting in patterns of 2 µm-wide lines for directing process growth and 20 µm-diameter circles for cell soma attachment. These circles were aligned to electrode sites. Different densities of neurons were plated in order to assess the minimal neuron density required for development of an active network. Spontaneous activity was observed at 10-14 days in networks using neuron densities as low as 200 cells/mm 2. Immunocytochemistry demonstrated the distribution of dendrites along the lines and the location of foci of the presynaptic protein, synaptophysin, on neuron somas and dendrites. Scanning electron microscopy demonstrated that single fluorescent tracks contained multiple processes. Evoked responses of selected portions of the networks were produced by stimulation of specific electrode sites. In addition, the neuronal excitability of the network was increased by the bath application of high K + (10-12 mM). Application of DNQX, an AMPA antagonist, blocked all spontaneous activity, suggesting that the activity is excitatory and mediated through glutamate receptors.
Neuronal cell patterning on a multi-electrode array for a network analysis platform
Biomaterials, 2013
We studied neuronal cell patterning on a commercial multi-electrode array (MEA). We investigated the surface chemical modification of MEA in order to immobilize Poly-D-lysine (PDL) and then to pattern PDL with a photolithographic method using vacuum ultraviolet light (VUV). We have clarified that the PDL layer was not fully decomposed but was partially fragmented by short-time irradiation with VUV, resulting in a change in the cell adhesiveness of the PDL. We succeeded in patterning primary rat cortex cells without manipulating the cells on MEA more than two months. This cell-adhesiveness change induced by VUV can be applied to any immobilized PDL on other kinds of MEA and culturing substrate. We conducted electrophysiological measurements and found that the patterned neuronal cells were sufficiently matured and developed neural networks, demonstrating that our patterning method is useful for a neuronal network analysis platform.
Brain Research Protocols, 1998
Spatiotemporally coordinated activity of neural networks is crucial for brain functioning. To understand the basis of physiological information processing and pathological states, simultaneous multisite long-term recording is a prerequisite. In a multidisciplinary Ž . approach we developed a novel system of organotypically cultured rat hippocampal slices on a planar 60-microelectrode array MEA . This biohybrid system allowed cultivation for 4 weeks. Methods known from semiconductor production were employed to fabricate and Ž . characterize the MEA. Simultaneous extracellular recording of local field potentials LFPs and spike activity at 60 sites under sterile conditions allowed the analysis of network activity with high spatiotemporal resolution. To our knowledge this is the first realization of hippocampus cultured organotypically on multi-microelectrode arrays for simultaneous recording and electrical stimulation. This biohybrid system promises to become a powerful tool for drug discovery and for the analysis of neural networks, of synaptic plasticity, and of pathophysiological conditions such as ischemia and epilepsy. q 1998 Elsevier Science B.V. All rights reserved.
Microelectrode Array Recordings of Patterned Hippocampal Neurons for Four Weeks
Biomedical Microdevices, 2000
Recent advances in cell biology and surface patterning make possible the construction of in vitro neural networks for long-term, multichannel recording studies. Towards this goal, we have demonstrated the recording of spontaneous electrical activity from rat embryonic hippocampal neurons confined to parallel lines which overlay the microelectrode array. The neurons adhered to adsorbed poly-D-lysine patterns and remained alive on the pattern for up to one month. Recordable, extracellular electrical activity began as early as 6 days in vitro and continued for the duration of the culture. Average amplitude of detected action potentials ranged between 70 μ V to 150 μ V measured from baseline to peak, consistent with results from unpatterned culture technologies.
Conference proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual Conference, 2005
Rectangular networks of rat hippocampal neurons have been produced on microelectrode arrays (MEAs). The crossing points of networks were located at the recording electrode sites by aligned microcontact printing (μCP) technique. Polydimethysiloxane (PDMS) stamp was fabricated to print fine poly-L-lysine (PLL) patterns of 2 -width lines for neurites and 20 -diameter circles for cell bodies. Different densities of neurons were applied on the PLL-stamped MEAs to find how a low density of neurons still has the functional connectivity. From the neural network applied with a density of 200 cells/mm2, we could observe signal propagation among spontaneous activities. Electrical responses were also evoked by 200 current pulse stimulation with 50 pulse width. Immunocytostaining was employed to identify dendrites, synapses, and nuclei in the patterned neurons.
Patterning to enhance activity of cultured neuronal networks
IEE Proceedings - Nanobiotechnology, 2004
Embryonic rat hippocampal neurons were cultured in order to gain insights into how small networks of neurons interact. The principal observations are the electrical activities recorded with the electrode arrays, primarily action potentials both spontaneous and evoked. Several lithographic techniques were developed for controlling with micrometer precision the patterns of surface molecules in order to control neuronal attachment and growth. Cytophilic polylysine against protein repellent and hence cytophobic polyethylene glycol were used. By combining the cellular lithography with the microelectrode arrays it was possible to guide neurons preferentially to electrodes and to begin to investigate the question as to whether the geometric pattern of a neuronal network influences the patterns of its neuroelectric activity. It is clear that the techniques are adequate to ensure contact of neurons to electrodes but not to ensure the recording of signals, even when neurons lie directly on top of electrodes. The maturation of neuroelectric activity depends on the growth of glia within the culture, such that spontaneous activity appears to become robust when the number of glia is roughly the same as the number of neurons.
Neuroscience Letters, 1997
We have studied the electrophysiological properties of hippocampal neurons grown on surfaces of organic thin films formed on glass or silicon substrates and on microelectronic device surfaces in culture. Hippocampal neurons were dissociated from embryonic rats and plated on substrates chemically modified with laminin peptide in a chemically defined medium. The electrophysiological properties of the neurons were studied using patch-clamp amplifier technique. We observed that the neurons grown on these substrates develop resting membrane potentials more negative than −33 mV after 3 days in culture and are able to produce action potentials. More interestingly we found that the neurons when grown on the microelectronic surfaces develop similar electrophysiological characteristics as those on the glass surfaces. Passive electrical properties (C m = 27 ± 5 pF, R m у 1 GQ) of the neurons studied by impedance spectroscopy did not change considerably during the first week in culture.
Multichannel recording and stimulation of neuronal cultures grown on microstamped poly-D-lysine
The 26th Annual International Conference of the IEEE Engineering in Medicine and Biology Society
We report progress toward designable, reproducible, patterned in vitro neuronal cultures. Cell adhesive proteins were directly microcontact printed on microelectrode arrays (MEAs) used as substrates for hippocampal neurons grown in a defined culture medium. The patterned neuronal network circuits were maintained up to one month. The recorded activity was comparable to that of cultures grown on unpatterned uniform surfaces. Time-locked evoked responses were recorded across the networks.
Patch method for culture of primary hippocampal neurons
Microelectronic Engineering, 2017
In vitro culture of primary neurons, especially hippocampal neurons, is important for understanding cellular mechanisms in neurobiology. Actually, this is achieved by using culture dish or glass slide with surface coated proteins. Here, we proposed a patch method to culture primary neurons on a monolayer of gelatin nanofibers, electrospun and crosslinked on a microfabricated honeycomb frame of poly (ethylene glycol) diacrylate (PEGDA). By using such a patch method, neural networks could be formed with a minimal cell-exogenous materials contact and a maximal exposure of the cells to the medium. Interestingly, hippocampal cells, especially astrocytes, showed in-vivo like morphology and most of neurons were found in the porous areas inside the honeycomb compartments although the nanofibers were deposited everywhere of the frame. Finally, calcium imaging showed that primary neurons have a higher degree of neural activity on the patch than on glass.
Journal of Nanoscience and Nanotechnology, 2012
We have prepared gold nanowire arrays inside nanoporous alumina templates with the goal towards neuronal interfacing and electrical recording from neurons. We have investigated biofunctionalization of such gold nanowire arrays (GNWs) and gold nanofilm (GNF) platforms to understand its impact on neuronal attachment and growth. Poly-D-Lysine (PDL) was coated on the nano-templates surfaces for adhesion of neurons which also enhanced the neuronal growth. Optical microscopy and scanning electron microscopy images revealed strong affinity and improved growth of neurons on PDL-coated surfaces. Such results will impact future investigation of stimulation and recording of electrical activity on nanoscale surfaces.