The Fabrication Of Microelectrode Array System And Its Preliminary Application In Neural Network Research In-vitro

Cultured neural network is a simplified model of the nervous system, and it is avery important tool to the study of neural science such as learning, memory, plasticity,connectivity and information processing etc. Microelectrode array is integrateddozens or more micro-acquisition units. It has a lot of advantages such as harmless tocells, environmental controlled and operate easily, and available for long-term,real-time recording. So far microelectrode array system has been used to study onneural synaptic plasticity, regeneration of neural network function, the law ofdevelopment of neural network research, circadian rhythms, nerve pharmacology andtoxicology etc., and have became an important support tool on neurobiology research.The first microelectrode array appeared in1972, a few decades later, manyresearchers and companies have been involved in studying microelectrode arraysystem, and have finished a lot of work. Part of the conventional micro-electrodearray and the system has reached a higher level. So micro-electrode company, such asGermany MCS system, appeared. Commercial system has finer design, beautiful anduser-friendly software interface, powerful data processing ability, but their systemsare expensive, their chips are easily to wear out. In addition, commercial systemfunction is relatively fixed, and it is difficult to upgrade for the specific requirementsof researchers. Based on this, many laboratories have been trying to develop their ownsystem, for their specific experimental purpose.The first step of neural network research on microelectrode array is to form acultured neural network, it is often limited by several important issues. The neuronadhesion ability on the microelectrode array surface is one of the key issues. Peopleusually used silicon dioxide, silicon nitride, or several polymer materials for wireinsulation of microelectrode arrays. These materials typically cannot be guaranteedexcellent insulation and good biocompatibility at the same time. In addition, due tothe poor adhesion ability of the neurons, the surface of microelectrode array has to becoated with adhesion molecules to enhance cell adhesion and the ability to grow cells.The coating materials used for the microelectrode array include: matrigel, collagen,poly-L-lysine, laminin, fibronectin, and polyethyleneimine. Matrigel is a natural extracellular matrix component, rich in growth factors and extracellular matrixproteins, having wonderful biocompatibility and bioactivity. Therefore it is a idealsurface coating material. Although matrigel has been used to coat the microelectrodearray surface, and have achieved good results, research is not sufficient, many of thekey issues have not been stated, such as the best matrigel coating concentration.The appropriate seeding density of cell is another important factor for neuralnetwork formation on the surface of the microelectrode array. In order to enhancesurvival and the adhesion quantity of neurons, researchers usually seed cells withhigher density. However, studies indicate that the inappropriate density of neuronswould influence the neuron survival duration, development and quality of electricalactivity of neural network, etc. How do the seeding density of neurons affect neuralnetwork electrical signal quality? What is the best seeding density of neurons? Stillnone unified conclusion available.Based on the above analysis, the first subject of this study is to develop alow-cost and suitable system for our study. Based on the home-made system, we carryout some further neural networks stimulation and neurological drug evaluationresearch. This paper is divided into the following sections:Part I: The fabrication of microelectrode array and multi-channelelectrophysiological acquisition systems.In this study, we fabricate a multi-channel electrophysiological acquisitionsystems. The hardware is made based on printed circuit board technology, softwaresystem for data acquisition is developed based on LabVIEW, and a planarmicroelectrode array, the agarose well microelectrode microelectrode array, andstamp printing microelectrode array is fabricated based on micro-processingtechnology.Our multi-channel acquisition system’ parameter: input noise Vrms <2mV, thebandwidth is10to10000Hz, the magnification is1000times, the multi-channelsampling frequency up to20KHz,32-channel data acquisition, could filter and storagedata, the total cost is less than30,000yuan lower than the price of commercialsystems (500thousand yuan). Our planar microelectrode array has very goodelectrical properties, can be used for neural network research, in addition, in this studywe successfully fabricate the agarose well microelectrode array and the stamp printingmicroelectrode array. Part II: The preparation of the two-dimensional neural network and theoptimization options based on the planar microelectrode arrays.In this study we coat the microelectrode with different concentration of matrigelto find the best concentration of matrigel for cell adhesion and vitality. Then, based onthe best concentration of matrigel, we culture cortical cell at five plating densities(500cells/mm2,1000cells/mm2,2000cells/mm2,4000cells/mm2and8000cells/mm2) to obtain a neural network with high signal quality.We have successfully dissociate cortices into singe cells and could form neuralnetwork. Immunofluorescence results show that the cortical cultures containedMAP2-positive neurons and GFAP-positive astrocytes. The microelectrode arraycoated with different concentrations of matrigel leads to different cell adhesion type.Low concentration of matrigel leads to poor cell adhesion, while higher concentrationof matrigel is prone to cell aggregation on the microelectrode array. Therefore, themicroelectrode array coated with0.2-0.4mg/mL matrigel results in a large number ofneurons adhered and the best uniformity. MAP2staining results show that thenumber of MAP2-positive cell increases along with the increase of seeding density,but the MAP2-positive cell number of8000cells/mm2is less than2times that of2000cells/mm2. The nucleus staining results show that neural networks built withdifferent cell inoculation density have similar cell density after surviving14days. Theelectrophysiology recording results show that electrical activity of the culture appearsa week after cell seeding, and gradually increases over time. In comparison, the neuralnetwork constructed with the cell seeding density of2000/mm2gains the mostabundant electrical activity.Part III: Electrical and drug stimulus-response characteristics of neuralnetwork based on planar microelectrode array.Electrical stimulation characteristics of neural network based onmicro-electrode array:In this study, we use home-made micro-electrode array system to stimulate neuralnetwork in different electrical intensity with different mode (first negative thenpositive or first positive then negative) to determine the best current pulse stimulationparameter. To investigate characteristic of electric stimulus-response of culturedneural network during spontaneous neural network development, we stimulate16electrodes and record the response of others electrodes respectively in10,14,18,22days. The results show that first negative then positive10A current stimulation(duration of positive and negative phase is400s) could obtain almost100%of theinduced response.10times-repeated stimulation is applied to get a responseconclusion that the results of the response signals are not spontaneous activities. Toget the characteristic map of neural network stimulus–response, we stimulate16electrode repeatedly in10,14,18,22days cultured neural network. the connectionnumbers of the neural network is increase with the culture days, almost all electrodesshow stimulus–response when it is come to22day. This result is consistent withprevious report, indicating that the self-developing micro-electrode array system hasgood reliability and stability, and can be used in the study of neurobiology researchwith electric stimulation.Drugs stimulation characteristics of neural network based onmicro-electrode array:To investigate the effect of drugs on neural network electrical activity, we useddifferent concentration of L-glutamate and GABA interact with mature neuralnetwork culture on self-developing micro-electrode array system,then to verify theeffectiveness of home-made micro-electrode array system on drug evaluation.The results show that the increasing trend of average pulse rate and averagebursting rate are consistence with the increase of sodium L-glutamate. However,when the drug concentration reaches a certain plateau, increase in drug concentrationwould leads to depression in network activity due to sodium L-glutamateexcitotoxicity. Gamma-amino butyric acid (GABA) is an important inhibitory neuraltransmitter in brain. This study indicates that its impact on the neural network isassociated with its concentration. The average firing rate and average burstelectricity rate decrease with the rising concentration, and the signals in the networkare completely inhibited at a concentration of10M. In conclusion, the drugevaluation results correspond to the results of the effectiveness of drug action andliterature results, indicating that the home-made microelectrode array system isreliable and stable and can be used for drug evaluation based on neural network andother related research.