The Study Of The Electro-excitability And The Related Molecular Mechanism Of The Effect Of External Factors On Neural Networks Through Voltage Threshold Measurement Method

In the work of this paper,a novel Voltage Threshold Measurement Method?VTMM?for quantitative neural excitability test was established and followed by verification.Firstly,three types of nerve-chips?the PC12 quasi-nerve-chip,hippocampal nerve-chip and hippocampal slice-chip?were composed based on micro-electrode array MEA biosensor.Based on the three types of nerve-chips,the VTMM was Introduced,verified and used to study the change rules of electrical excitability in the neural networks?including the PC12quasi-neuronal network,hippocampal neuronal network and hippocampal slice?under the effect of three kinds of factors?including acetylcholine?ACh?,ethanol?EtOH?,temperature?T??.Then,the High Content Screening?HCS?was further applied to study and analyze the changes of parameters under the mentioned three conditions.Herein,the recorded parameters were cell survival rate,length and area of??the protuberance,reactive oxygen species?ROS?and Ca²⁺content in PC12 quasi-neuronal network and hippocampal neuronal network..Finally,the conjoint analysis of the data from VTMM and HCS was investigated to analyze the relationships among cell survival rate,length and area of the protuberance,ROS and Ca²⁺content and the threshold voltage(V_Th)of the PC12 quasi-neuronal network and hippocampal neuronal network under the influence of the three factors.And the related molecular mechanisms that the three influencing factors affect the electrical excitability of neural network were explored.The main results of this thesis are shown as follows:1)The Normal V_Thh of the PC12 quasi-neuronal network,hippocampal neuronal network and hippocampal slice were 36,56 and 31 mV,respectively.2)The V_Thh of the three kinds of neural networks were inversely proportional to the concentration of ACh(C_ACh);when the C_AChCh in the culture medium was increased to 8 ?PC12?,6?Hippocampal Neuron?and 4?Hippocampus Slice??g/mL,the V_Thh of all three neuronal networks decreased to 0 mV.Under the action of ACh,the length and area of the protuberance,ROS and Ca²⁺content,and cell survival rates of PC12 quasi-neuronal network and hippocampal neuronal network showed no significant changes.The related molecular mechanism of the excitatory changes in the neural network under ACh action was discussed as follows:firstly,ACh acted on the cholinergic receptors on the linker or postsynaptic membrane,then,a variety of ion channels and the adenylate cyclase systems were activated,which not only change the content of various ions including Ca²⁺,affected the cell membrane potential,but also increased the activity of the"sodium band"and rised the excitability and droped the threshold voltage of the neural network eventually.3)The V_Thh of three neural networks were exponential with the EtOH concentration(C_EtOH).When the C_EtOHtOH in the culture medium was higher than 80?PC12?,110?Hippocampal Neuron?and 120?Hippocampus Slice?mmol/L,the three neural networks no longer had any electrical excitements.Under the action of EtOH,the cell survival rates of the PC12 quasi-neuronal network and hippocampal neuronal network had no significant changes,Meanwhile,the protuberance's length and area of two types of neural networks showed very significant decreases,and there were very significant increases in all of the ROS and Ca²⁺content in both two types of neural networks?a significant increase in the ROS content in the hippocampal neuronal network?.The related molecular mechanism of EtOH's effect on the electrical excitability of neural networks is roughly as follows:on the one hand,alcohol directly leaded to a decrease in the length and area of the protuberance,which inhibited the activity of the sodium band,on the other hand,it promoted rapid Ca²⁺overloading,resulting in a decrease in the length and area of the protuberance.At the same time,the rapid overloading of Ca²⁺also resulted in the rapid accumulation of ROS,further aggravating the reduction of the length and area of the protuberance,and directly inhibited the activity of the"sodium band."Finally,the excitability of the neural network was suppressed and the threshold of the stimulation voltage of the neural network increased.4)The relationship between the V_Thh and T of the culture medium?T_m?of three kinds of neural networks were presented as"U"curves.The V_Thh increased with the decreasing of T_m when T_m was below 37?,while when T_m was above 37?,the V_Thh decreased with the increasing of T_m.When the T_m was reduced to 33?PC12?,33?Hippocampal Neuron? and 32??Hippocampus Slice?,or increased to 42?PC12?,43?Hippocampal Neuron? and 44??Hippocampus Slice?,all of the neural networks losted electrical excitement.Under the action of different T_m,the survival rate of cells in both two types of neural networks increased first with the increasing of T_m,then decreased.While the overall change of the survival rate was not significant.When the T_m was raised from 37? to 42?,in both two types of neural networks,the length of the protuberance showed significant decreases,the area of the protuberance showed very significant decreases,and the ROS and Ca²⁺content showed very significant increases;When the T_m was decreased from 37? to 33?,in both two types of neural networks,the length and area of the protuberance had no significant changes,while the ROS and Ca²⁺content showed very significant increases?a significant increases in the ROS content in the PC12 quasi-neuronal network?.The related molecular mechanism of the effect of T_m on the electrical excitability of neural networks was roughly as follows:?1?Lowering the T_m on the one hand reduced the content of Ca²⁺and directly inhibited the nerve excitability;meanwhile,caused a decrease in ROS content.Finally,the decreasing of Ca²⁺and ROS content resulted in a decrease in the activity of the"sodium zone"and so on.Thus the threshold voltage increased and the excitability of the neural network was inhibited.?2?Increasing the T_m on the one hand increased Ca²⁺content.The increasing of Ca²⁺content not only directly interfered with nerve excitability,but also aggravated the accumulation of ROS;on the other hand,caused a rapid accumulation of ROS content,which interfered with the activity of the"sodium band"and increased the rise of Ca²⁺content.In addition,the increasing of T_m could also directly affect the length and area of the protuberance through physical effects.Finally,the physical effects of high T_m and the overload of Ca²⁺ and ROS caused a decrease in the length and area of the protuberance and an increasing of activities of the“sodium band”,etc.Therefore,the excitability of the neural network increased,and the threshold voltage decreased.The conclusion of this paper:1)Voltage Threshold Measuring Method?VTMM?was an accurate,simple and efficient new method to quantitatively evaluate the electrical excitability of neural networks,which could be used to study the different influences of factors on the excitability of the neural networks,such as environment?T?,food?EtOH?and drugs?ACh?,;2)The influence of the tested factors on the neural network could be detected accurately and quantitatively based on VTMM before the significant changes of cellular molecular indicators related to cell's electrical excitability had taken place,indicating that VTMM was very effective and sensitive;3)V_Thexisted in all the three types of neural networks.Meanwhile,the V_Thof three kinds of neural networks would change regularly under the influence of different external factors or different concentration?intensity?of the same external factor.But because different types of neural networks had different features,the V_Thand the concentration of external factors?strength?that reduced the V_Thto 0 or increased it to infinity were diverse.