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MINISTRY OF EDUCATION AND TRAINING MINISTRY OF NATIONAL DEFENCE ACADEMY OF MILITARY SCIENCE AND TECHNOLOGY TA QUOC GIAP RESEARCH ON ESTABLISHING THE NEURAL STIMULATION SYSTEM AND APPLY FOR EVALUATING THE SPATIAL RESPONSE OF HIPPOCAMPAL PLACE CELLS Specialization: Electronic engineering Code: 52 02 03 The abstract of dissertation Hanoi – 2019 This dissertation was completed at: ACADEMY OF MILITARY SCIENCE AND TECHNOLOGY Scientific supervisors: Dr Nguyen Le Chien Dr Le Ky Bien Reviewer 1: Tran Duc Tan, Ph.D., Assoc.Prof Reviewer 2: Nguyen Minh Phuong, Ph.D., Assoc.Prof Reviewer 3: Le Manh Hai, Ph.D The doctoral thesis was examined by the Doctoral Evaluating Council of Academy level held at Military Science and Technology at … on …, … This thesis can be found at: The library of Academy of Military Science and Technology Vietnam National Library INTRODUCTION The necessity of the project Biomedical engineering is an applied science field, which connects different sciences from physics, chemistry, and biology to electrical, control, information, micro and nano technologies in order to provide biomedical solutions for improving human health Neural engineering is an important subfield of biomedical engineering, which uses engineering techniques to treat, replace, or restore the functions of the neural system It requires a device possessed controllable and stable properties for studying the mechanism of memory storing in the brain This plays an important role in a comprehensive understanding of physiological neural system Therefore, the development of systems that allow studying the physiology of the nervous system has highly practical applications In this dissertation, a neural stimulation and recording sytem is developed for evaluating behavioral and spatial responses of mice from electrical stimulations with proper algorithms This system allows deeper understanding of the working principles of neurons and the brain In addtion, this is fundamental to study the structure and function of hippocampus, which may be associated with some neurodegenerative diseases such as Alzheimer’s, Amnesia, and Schizophrenia The practical exercises with their respective algorithms are first built on animals in order to develop the electrical stimulating and recording system for neurons The built stimulation system allows evaluating the electrical activity of neurons in their surrounding environment and the whole living organism correlations The electrical recording of neurons in hippocampus is fundamental to assess cells’ behavior in this place Importantly, specific working principles of the central nervous system will be elucidated to better understand feeling, memory, and autonomic nervous mechanisms Therefore, the project “research on essurrtablishing the neural stimulation system and apply for evaluating the spatial response on hippocampal place cells” has a practical role in comprehensive studies of neuronal physiology Research objectives - Developing a system for stimulating and recording the electrical activity of neurons based on electronics engineering - Building mathematical algorithms of neuronal stimulation for practical exercises on mice Research subjects and scope - Research subjects: Neurons in a defined place of the Hippocampus is electrically stimulated and recorded by using the built system Supporting devices and integrated programs for stimulating and recording are synchronous to form a complete system The stimulating and recording processes are automatically performed and analyzed - Research scope: Developing a system that allows stimulating and recording neurons The stimulating programs associated with mathematical algorithms are integrated into the system Simulation and analyzing the results are based on electronic engineering Research Methodology Data collecting programs, simulations, and practical exercises are used on mice for evaluating the system and exercises Research contents and thesis structure The main research contents: - The overview of the electrical activity of neurons - Modeling neurons with equivalent circuits and building algorithms of electrical simulations for neurons - Evaluating the algorithms and system by simulation programs and practical exercises on mice Scientific and practical significance - Proposing proper stimulating parameters for studying subjects - Developing a system for stimulating and recording the electrical activity of neurons with algorithms and practical exercises on animals - Performing simulations and practical exercises on mice to evaluate the proposed system and programs - Providing fundamentals of medical issues for studying the central nervous system CHAPTER 1: THE OVERVIEW OF THE ELECTRICAL ACTIVITY OF NEURONS 1.1 Membrane potential of neurons Neurons are analogous to other cells, which have structural components of cell membranes, nuclei and organelles The electrical activity of normal cells as well as neurons is highly related to the structure and characteristics of the cell membrane 1.2 Electrical nerve stimulation and medical significance The development of nerve stimulating and recording system with proper algorithms is based on studying electrical properties of the cell membrane, the influence of electrical stimulating parameters, the response of cell membranes, and electrical stimulations in medical research Figure 1.1 The change in membrane potential by the influence of stimulating pulses 1.3 The response of cell membrane to the electrical stimulation The plasma membrane potential changes when neurons are stimulated The membrane potential will return to its initial resting value after responding to the stimulus If the electrical stimulation is insufficient to create a transmembrane potential larger than a threshold, the membrane will not be activated The amplitude and frequency of the electrical stimulation mainly influence the intracranial electrical stimulation, which are used to determine the stimulating threshold and maximum response of cells In this work, electrical stimulating pulses are positive pulses with their variable amplitudes and frequencies 1.4 The recording methods of the neuronal action potential The neuronal potential recording technique was developed in the 1940s During this period, extracellular microelectrodes were used to determine the potential characteristics of a neuron Recent studies of neurons associated with the neural stimulation and response have proven the relation of neurons in different places within brain Current technologies and equipment have also been developed for more accurate and convenient analyses of neuronal activities 1.5 Hippocampus and hippocampal place cells Neuron studies have proven that hippocampal place cells play a vital role in information store, short-term to long-term memory conversion and spatial orientation 1.6 The basic electronic circuit of neurons To more comprehensively study and understand the action potential of cell membrane on electric stimulation, an equivalent conducting model of neurons has been modeled as an electronic circuit 1.7 Relevant research - National: currently, there is not any neuronal stimulating system, which allows both stimulating and recording the electrical activity of neurons - Global: systems of manual neuronal stimulation and recording have been often found, however most of them are not synchronized and complete systems, which leads to the lack of accuracy in data analysis Some advanced systems can only describe the system function and stimulation results, but not evaluate the system 1.8 Chapter conclusion CHAPTER 2: THE EQUIVALENT ELECTRICAL CIRCUITS OF NEURONS AND ALGORITHMS FOR ELECTRICAL NEURAL STIMULATION 2.1 The electronic circuit of neuron membrane and the investigation of electrical stimulating parameters The action potential of cell membrane can be analogously modelled as an electronic circuit 2.1.2 The simulation of electrical stimulating parameters with the Maeda-Makino model XSC1 XFG2 SC1 Ext Trig + COM _ IO1 IO3 IO2 B A + R2 200Ω PWM I1 _ + _ R8 100kΩ C1 0.5µF R1 100kΩ Q3 2N3904 Q1 2N3904 0.07mA R3 100kΩ R5 10kΩ R6 300Ω Q2 2N3906 Q4 2N3904 R4 1kΩ V1 5V Q5 2N3904 XMM1 C4 0.2µF C3 1µF V2 0.4V Figure 2.3 The electronic circuit model of neurons by Maeda and Makino 2.1.3 Simulation results and discussions The amplitude and frequency parameters of stimulation pulses are applied to the electric circuit for simulating the action membrane potential based on the Maeda – Makino model by the NI Multisim 14.0 progam 2.1.3.1 The relationship between current and the membrane potential of stimulation pulses at a fixed 80Hz frequency The simulation results show that the membrane potential is directly proportional to the stimulating intensity (Figure 2.6) However, the potential only dramatically rises over the current of less than about 10μA (the "bursting" range of potential response) before gradually increasing in the current range from 10 to 110 μA In addition, while the stimulating current increases 110 μA, the potential increases suddenly and oscillates Theoretically, this explains the risk of breakdown voltage of electronic components and the demolition of cell membrane Figure 2.6 The change of potential depends on the stimulating intensity at the frequency of 80Hz 2.1.3.2 The membrane potential depends on the frequency of stimulating current at a fixed current Figure 2.7 shows the dependence of the membrane potential over the frequencies of current from to 180 Hz when the current intensity is fixed It can be seen that the membrane potential increases and reaches a maximum value at the frequency of 100 Hz before slightly reducing at higher frequencies Figure 2.7 The change of potential depends on the stimulating frequency at a fixed intensity of 70μA 2.2 The stimulation and recording system for electrical activity of neurons Figure 2.8 The illustration of the stimulation and recording system for electrical activity of neurons - The behavioral observation system: Consisting of a CCD camera for monitoring movements, behavior and positions of mice - The stimulation system: a pulse generator establishes the form and parameters of pulses (Stimulator), which are sent to the isolator and DAC via USB 6501 before delivering to the nerve cells of mice by stimulation electrodes -The recording system: The neuronal membrane potential is also recorded by stimulating electrodes The recorded signal from neurons is doubly amplified and processed by a signal processing unit (Plexon) The action potential is recorded and synchronously counted together with the stimulation pulse as well as the co-ordinates of the mice by control signals (TTL form) from the processing program installed in the computer to the Plexon system via USB device 6501 The developed system with integrated programs forms a complete device, which allows both electrically stimulating neurons and recording the neuronal membrane potential with designed task algorithms 2.3 Algorithms of electrical stimulations for neurons 2.3.1.1 The model of electrical stimulations for neurons with the NPT task Processing Circuit Isolator Stimulator Monitor USB 6501 Central processing system Stimulating electrode Sensor Mice Figure 2.10 The model of electrical stimulations and nose – poking responses The input is the nose-poking behavior of the mice, which is transmitted via an optical sensor hiddenly located in a circular hole with the size diameter of 1.5 cm inside the chamber The sensor operating mode is set at a high logic status When the nose-poking happens, the sensor will be switched to a lower logic level The response signal from the sensor is sent to a processing circuit for counting the number of nosepoking if conditions of the task are completed as described in model 2.10 2.3.1.2 The electrical stimulations for neurons with NPT task a) The significance of the NPT exercise The NPT task algorithm is based on the strict requirements of reward conditions The intensity and frequency parameters of electrical stimulating pulses are evaluated from practical tasks in order to compare with the simulated parameters The NPT stimulation algorithm is shown in Figure 2.11 with variable intensity and frequency parameters The program completely monitors the reward conditions and automatically rewards when the conditions of the task are reached The number of rewards or nose-poking behaviour is updated and visually displayed on a bar graph These values are stored in a file and objectively analyzed to evaluate the most appropriate parameters for spatial response tasks The expectation of the NPT task is to find the optimal parameters of the stimulating electrical pulse, which makes mice interest and poking the most in a period time of the task 11 2.3.2.3 The model of electrical stimulations for neurons with the RRPST and PLT tasks Monitor CCD camera Isolator Stimulator USB 6501 Central processing system Plexon AMP recording electrode Stimulating electrode Mice Figure 2.14 system for stimulating and recording the electrical activity of neurons on the mice The algorithm of electrical stimulations for the RRPST task a) The significance of the RRPST exercise The algorithm of the RRPST task (Random Reward Place Search Task) is based on the strict requirements of reward conditions The movement and reward motivation of mice are evaluated by the algorithm of electrical stimulations for building the program and content of the RRPST task This experimental exercise will train the mice to move for searching rewards, which appear randomly The number of rewards or moving distances will be simulated to display the tracking path of mice and to update the reward number The obtained results are stored in a file and objectively analyzed for assessing the movements of mice in a particular space b) The RRPST task c) The algorithm flowchart of stimulations for the RRPST task 12 Start Pt = 0; t= 0; xt = x0; yt = y0; xzt = xz0; yzt = yz0; wz deltaTime = 0; delayTime; maxwidth; maxPt; maxT; taovungpt = false t++; deltaTime++; x t; y t Reading data yes deltaTime>= delayTime & taovungpt = true xzt = rand(0,maxwidth) yzt = rand(0,maxwidth) no delta = sqrt[(xt – xzt)2 + (yt – yzt)2 ] delta