Nghiên cứu xây dựng hệ thống kích thích tế bào thần kinh và ứng dụng trong đánh giá đáp ứng không gian của tế bào vị trí hồi hải mã

Model and algorithm of electrical stimulation of neurons with spatial response tests...47 2.4.. hippocampus of mice, and the cell potential field was recorded as animalsmoved through C++

TA QUOC GIAP

RESEARCH ON ESTABLISHING THE NEURAL

STIMULATION SYSTEM AND APPLY FOR EVALUATING THE SPATIAL RESPONSE OF

HIPPOCAMPAL PLACE CELLS

DOCTOR OF ENGINEERING DISSERTATION

HANOI - 2020

TA QUOC GIAP

RESEARCH ON ESTABLISHING THE NEURAL

STIMULATION SYSTEM AND APPLY FOR EVALUATING THE SPATIAL RESPONSE OF

HIPPOCAMPAL PLACE CELLS

Specialization: Electronic engineering

I hereby declare that this dissertation is my original work The data andresults presented in the dissertation are honest and have not been published inany other work References are fully cited

10 th January, 2020

giả luận án

TA Quoc Giap

First and foremost, I would like to express my deep appreciation to mydirect supervisors, Dr NGUYEN Le Chien, Dr LE Ky Bien and AssociationProfessor TRAN Hai Anh, who enthusiastically guided me during my whole PhDtime Thank you very much for many meaningful advices and discussion for mywork I learnt from the mentors not only techniques for fulfilling my PhD work,but also methods for solving problems in a lab as well as in the life Thank youvery much for revising my thesis, giving me helpful comments and advices

My sincere appreciations must go to other teachers in the Departmentsfor their encouragement, knowledge sharing, supports and helps in our courseand conduct the thesis

I would like to express my sincere thanks to the Institute of Electronics –Academy of Military Science and Technology; Department of Physiology,Department of Material Equipment – VietNam Military Medical University,where I study, live and work for creating favorable conditions for me toparticipate in studying and researching during my time as a PhD student

I want to express my special thank to the leader of Academy of Militaryscience and technology and other collaborator centers for their support andhelp for this work

Finally, I would like to thank my family members for their love,encouragement And especially, I would thank my wife who have sacrificed alot of things for supporting me to fulfill my PhD work

TABLE OF CONTENTS

Page

LIST OF SYMBOL AND ABBREVIATION……… v

LIST OF FIGURES AND TABLES………ix

INTRODUCTION 1

CHAPTER 1 OVERVIEW ABOUT ELECTRICAL ACTIVITY OF NEURONS 6

1.1 Membrane potential of neurons 6

1.1.1 Structure of nerve cells membrane 6

1.1.2 Resting and action potential 9

1.2 Electrical nerve stimulation and medical significance 12

1.3 The response of cell membranes to electrical stimulation 16

1.4 The recording methods of the neuronal action potential 18

1.5 Hippocampus and hippocampal place cells 21

1.5.1 Structural characteristics 21

1.5.2 Function of the Hippocampus 21

1.6 Fundamentals of electronic circuit model of neuron 23

1.7 Related research to this dissertation 26

1.8 Chapter conclusion 29

CHAPTER 2 EQUIVALENT ELECTRICAL CIRCUIT MODEL

AND NEURONAL ELECTRICAL STIMULATION ALGORITHMS 31

2.1 Electronic model of neuron membrane and assessment of electric stimulation parameters 32

2.1.1 Electronic circuit model of neurons 32

2.1.2 Simulation of stimulating parameters on Maeda and Makino models .34

2.1.3 Simulation results and discussion 36

2.2 The system for stimulation and recording the electrical activity of neurons 39

2.3 Building electrical stimulation algorithm model for neurons 41

2.3.1 Model and algorithm of electrical stimulation of neurons with NPT

test 41

2.3.2 Model and algorithm of electrical stimulation of neurons with spatial response tests 47

2.4 Chapter conclusion 63

CHAPTER 3 EVALUATING THE STIMULATION ALGORITHMS AND

THE SYSTEM BY BEHAVIOURAL RESPONSES AND

PRACTICAL EXERCISES ON MICE 64

3.1 Materials and methods 64

3.2 Simulation results 67

3.2.1 Simulation of the NPT task 68

3.2.2 Response simulation in spatial exercises 69

3.3 Analyze and evaluate experimental results on mice 74

3.3.1 Experimental results performed on NPT test 74

3.3.2 Experimental results performed on the spatial response tests 79

3.4 The results of stimulating and recording experiments of the neuronal electronic activity in the hippocampus on mice………80

3.4.1 Unit isolation and recording……… 80

3.4.2 Common characteristics of hippocampal place cells……… 82

3.5 The evaluation of the algorithms, stimulation and recording systems for the electrical activity of neurons………83

3.5.1 The evaluation of algorithms……… 83

3.5.2 The evaluation of stimulating and recording system for the electrical activity of neurons 86

3.6 Chapter conclusion 94

REFERENCES 100

APPENDICES ………

LIST OF SYMBOLS AND ABBREVIATIONS

Ions concentrationCapacitance of the membrane per unit plane

countInterVal Number of stops to adjust the parameter

delayTime The minimum time from when the mouse receives the

reward until the new reward area appears

deltaTime The time it takes to count from the time the mouse receives

the prize until the new reward area appears

delta Limits the distance the mouse moves to get the reward

The distance the mouse moves over a certain period of time

in the DMT testThe distance the mouse moves over a certain period of time

in the RRPST testThe distance the mouse moves over a certain period of time

in the PLT testDiameter on the horizontal axis of the virtual environmentDiameter on the vertical axis of the virtual environmentAction potential of cell

Resting potential of cell

Faraday constantConductivity of Na+ ion channelsConductivity of K+ ion channelsConductivity of secondary ion channels

Interval Interval to stop for parameter adjustment

Intra-axonal current

70 percent of the optimal

80 percent of the optimalValence of ions

Extracellular Na+ concentrationIntracellular Na+ concentrationConstant

Membrane resistance per unit areaAbsolute temperature

Time to stimulateRewarding eligible timeReward receiving timeTotal amount of exercise time for the mouse

Training time (also the total time of sessions)

Rest time to adjust the value of the stimulating parameterMembrane potential

Number of rewards

Transmembrane potential of Na+ channel

The mean of movement speed of the mouse in the open environment

Maximum diameter in the horizontal axis of the virtual environment

Minimum diameter in the horizontal axis of the virtual environment

Reward coordinates of mouse before t

The coordinates of the mice at the time t is assigned with

x0, y0 which is the original position of the mice Reward coordinates of mouse at

The x and y coordinates of the center of the reward area 1 The x and y coordinates of the center of the reward area 2

x, y coordinates of the center of the current reward area Maximum diameter in the vertical axis of the virtual environment

Minimum diameter in the vertical axis of the virtualenvironment

Reward region 1Reward region 2Radius of the reward areaSystem latency

System latency in DMT testSystem latency in NPT testSystem latency in RRPST test

Direct current

Distance movement taskElectrical brain stimulationExtracellular field

Frames per secondHippocampal network modelIntracranial self – stimulationMild cognitive impairmentMedial forebrain bundleMesial temporal lobe epilepsyNose – poking task

Open – fieldPlace learning taskRandom task, random reward place search taskSpike potential field

Signal to noise ratio

LIST OF FIGURES

page

Figure 1.1 Basic structure of nerve cell……… 7

Figure 1.2 Concentration and potential of ions at rest……… 9

Figure 1.3 Direction of potential field lines around a neuron……… 11

Figure 1.4 Changes in membrane potential under the effect of stimulation pulses……… 13

Figure 1.5 Dopamine transmission pathways of mesolimbic……… 14

and mesocortical systems……… 14

Figure 1.6 Cell membrane’s response to stimulus signals……… 16

Figure 1.7 Demonstration of extracellular potential recording technique and the data form 19

Figure 1.8 Diagram of rodent brain and the location of the hippocampus… 21 Figure 1.9 Experimental equipment for the formation of the axon cable equation……… 23

Figure 1.10 Electronic circuit model and voltage chart of neurons…………24

Figure 2.1 Electric model of neron and the theory of action potential………32

Figure 2.2 Electrical neuron model according to Maeda and Makino………34

Figure 2.3 Electric model of a neuron under the stimulation of direct current……… 35

Figure 2.4 One-dimensional stimulation pulse form with specified parameter……… 36

Figure 2.5 The voltage response pattern of the model……… 37

Figure 2.6 Voltage change by stimulating intensity at 80Hz……… 38

Figure 2.7 Change in voltage by stimulation frequency, at the intensity of 70μA……… 39

Figure 2.8 Model of stimulating and recording the potential of neurons… 40

Figure 2.9 The integrated control pulse pattern of the system and the neuron

stimulation pulse……… 41

Figure 2.10 Model of system for stimulating and responding to nose-poke behavior……… 42

Figure 2.11 Flow chart of the NPT test……… 45

Figure 2.13 Stimulating algorithm flowchart for DMT test……… 51

Figure 2.14 The system for stimulation and recording the action potential of neurons on mice……… 53

Figure 2.15 Algorithm flowchart for the RRPST test……… 57

Figure 2.16 Flowchart of electric stimulation algorithm for PLT test…… 61

Figure 3.2 The recording chamber for the ICSS response and………

nose-poking behaviors of mice………66

Figure 3.3 The illutration of the model and the arrangement of the spatial tasks……… 66

Figure 3.5 Program interface in DMT test……… 70

Figure 3.6 Program interface in RRPST test……… 71

Figure 3.7 Program interface in PLT test……… 72

Figure 3.8 Relationship between nasal poking behavioral response and intensity of stimulation……… 77

Figure 3.9 The dependence of nose-poking response on the stimulating frequency……… ……… 78

Figure 3.10 Experimental results are analyzed for the spatial response tests………80

Figure 3.11 The neuron activity are recorded and isolated using an offline-sorter program (Plexon)……… 81

Figure 3.12 Electrical activity of neurons recorded at hippocampus……… 82

Figure 3.13 Model of evaluating the stability and latency of the system for NPTtask by labchart Pro v8.1.8……… 86Figure 3.14 The illustration for pulses of the reward condition, rewarddelivery, and the delay time of the system……… 87Figure 3.15 The evaluation of the stability and delay of the system for theDMT, RRPST and PLT tasks……… 87Figure 3.16 Program to evaluate the stability and latency of DMT test…… 88Figure 3.17 Graph of system latency time in DMT test……… 89Figure 3.18 Program to evaluate systemic stability and latency in RRPSTtest……… 90Figure 3.19 Graph of system latency time in RRPST test……… 90Figure 3.20 Program to evaluate systemic stability and latency in PLT test 91Figure 3.21 Graph of system latency time in PLT test……… 92

1 The necessity of the dissertation

Biomedical engineering is an applied science field, which connectsdifferent sciences from physics, chemistry, and biology to electrical, control,information, micro and nano technologies in order to provide biomedicalsolutions for improving human health Neural engineering is an importantsubfield of biomedical engineering, which uses engineering techniques totreat, replace, or restore the functions of the neural system One of the centralfield of neurophysiology is the study of the mechanisms of memory andinformation storage in the brain [8], [48], [73], [87 - 89] It requires a devicepossessed controllable and stable properties for studying the mechanism ofmemory storing in the brain This plays an important role in a comprehensiveunderstanding of physiological neural system Therefore, the development ofsystems that allow studying the physiology of the nervous system has highlypractical applications

Based on the available but functionally limited equipments andprograms, many supportive equipment and programs is needed for the system

to be functionally competent

In this dissertation, a neural stimulation and recording sytem isdeveloped for evaluating behavioral and spatial responses of mice fromelectrical stimulations with proper algorithms This system allows deeperunderstanding of the working principles of neurons and the brain In addition,this is fundamental to study the structure and function of hippocampus, whichmay be associated with some neurodegenerative diseases such as Alzheimer’s,mild cognitive impairment, mesial temporal lobe epilepsy, and Schizophrenia

[5 - 6], [23], [41], [68], [78]

The practical exercises with their respective algorithms are first built onanimals in order to develop the electrical stimulating and recording system for

neurons The built stimulation system allows the electrical activities ofneurons to be evaluated in environment and whole living organismcorrelations The electrical recording of neurons in hippocampus isfundamental to assess cells’ behavior in this place Importantly, specificworking principles of the central nervous system will be elucidated to betterunderstand feeling, memory, and autonomic nervous mechanisms.

Therefore, the project “research on establishing 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.

2 Objectives

-Developing a system for stimulating and recording the electrical activity

of neurons based on electronics engineering

- Building mathematical algorithms of neuronal stimulation for 4

practical exercises on mice

3 Subjects and scope of research

In order to build an electrical stimulation system which targets the

"reward" mechanism of the central nervous system, the study anddevelopment of a stimulating control program system with appropriateequipments including:

- Single - channel Stimulator SEN - 3401 (Nihon Kohden, Japan)

- Digital - Analog converter (DAC) and Isolator SS - 203J (Nihon Kohden, Japan)

- Nose - poking chamber

- Control program is built on C++ language, version 2010 (Microsoft Inc.,USA); data structure and data collection program is built on C# language,version 2010 (Microsoft Inc., USA)

Recording the response potential of the hippocampal place cell when theanimal moved in given environment Microelectrodes were placed in the

hippocampus of mice, and the cell potential field was recorded as animalsmoved through C++ and C# - based drivers developed for research purposes,the experimental tests are built based on the corresponding algorithm.Equipment used in recording neuron electrical activity and programs forrecording, analyzing data and evaluating system activities and developedalgorithms, including:

- Plexon HLK2 system (Plexon Inc., USA) could record the actionpotential of the hippocampal place cells and the spatial location ofanimals in the open environment

- Measure the resistance of the recording electrode: Electronic Balance (Shimadzu Corporation, Japan)

- Programs have been developed and applied in the characteristic analysis

of hippocampal place cells activities

4 Methodology

The thesis uses circuit theory to simulate electric stimulation parameters

by NI Multisim program version 14.0 (National Instruments Inc., Australia);mathematical statistical theory in experimental tests on mice; biomedicaltechniques in implementing research systems, especially in setting upstimulating electrodes and electrodes for recording the electrical activity ofneurons; theory of digital signal processing in signal visualization andmathematical model formulation of the problem Simulation program,algorithmic models building, experimental methods description on mice anddata results with C# programming language (Microsoft, USA) Systemcontrolling and synchronization with C++ programming language (Microsoft,USA) Using intensive developed software to analyze the collected data as abasis for evaluating built algorithms and system Moreover, these results showcharacteristics of hippocampal place cells in relation to a given environment

5 Content and structure

Apart from Introduction, Conclusion and References, this dissertation contains 3 chapters as follow:

 Chapter 1 OVERVIEW ABOUT ELECTRICAL ACTIVITY OF NEURONSChapter 1 presents an overview of the membrane potential of neuron, suchas: the structure and function of the cell, the membrane of the neuron; theory

of resting and action potentials; the function of hippocampal place cells

In assessing the electrical activity of neuron, it is necessary to build asystem capable of evaluating the neuronal electrical activity characteristicsunder the influence of stimulating factors Chapter 1 introduces the modeling

of the response of the nervous system in relation to the "reward" mechanismfor electrical stimulation, which is the basis for simulating the electricalstimulation and response of the cell membrane carried out in Chapter 2

The electrical activity of the cell membrane induces changes in theextracellular potential field Therefore, chapter 1 also provides the technicalknowledge as well as the electrical activity recording system of neurons

 Chapter 2 EQUIVALENT ELECTRICAL CIRCUIT MODEL AND

NEURONAL ELECTRICAL STIMULATION ALGORITHMS

In chapter 2, using electronic models of neurons to examine electricalstimulation parameters and select appropriate parameters as the basis forbuilding experimental stimulating parameters on animals

Besides, chapter 2 also proposes 4 models and 4 algorithms to apply inthe tests related to brain stimulation reward (BSR) from suitable parameters(frequency, amplitude) simulated and verified through experiments in buildingmodel, algorithm for intracranial self-stimulation (ICSS) with response tonose-poking through NPT test Algorithms and drivers are applied to developreward-seeking exercise test in an open field, thereby assessing the potentialactivity related to spatial memory of hippocampal place cells

Chapter 3 EVALUATING THE STIMULATION ALGORITHMS AND THESYSTEM BY BEHAVIOURAL RESPONSES AND PRACTICALEXERCISES ON MICE

This chapter presents the simulation results before the experiments andthe experimental results on the system through exercises performed on mice,using the stimulation models and algorithms proposed in Chapter 2 Utilizingevaluation methods and analyzing the obtained results is the basis forevaluating the stimulation algorithm model and the system for stimulation andrecording the electrical activity of the built neuron

6 Scientific and practical signification

From the understanding of electrical activity of neurons, the thesis hasinvestigated the frequency and amplitude parameters of stimulation pulsesthrough modeling electronic circuit of neurons This is the basis for assessingthe response of neurons to DC stimulation parameters through intracranialself-stimulation (ICSS) From there, to suggest the suitable stimulationparameters for the study subject

From the signification and widely role of electrical stimulation inmedicine, the dissertation has proposed the construction of a system forstimulation and recording the electrical activity of neurons along with 4algorithms of electrical stimulation of neurons in 4 experimental tests onanimals In addition to the proposed research facilities, these four tests help toassess the spatial response of the "reward" system in the brain and neurons in

a given environment These results contribute to the electrical functionevaluation of neurons, which is the basis for assessing the physiologicalactivity of the central nervous system

The thesis also addresses the need to synchronously built and developthe system and program to stimulate and record neuronal electrical activity tosolve the current problem in functional research of the central nervous system

CHAPTER 1 OVERVIEW ABOUT ELECTRICAL ACTIVITY OF NEURONS

The study of characteristics, especially the electrical properties of cellmembranes and the effect of electrical stimulating parameters on neuronsserves as a basis for building an algorithmic model and a neuron stimulationsystem The successful combination of a neuronal stimulation system with therecording of electrical activity of neurons into a complete system is important

to evaluate the activity of each neuron in relation to the environment and thewhole organism Research in building neuron stimulation system andrecording the electrical activity of hippocampal nerve cells will help medicalresearchers to evaluate the operational characteristics of the hippocampalplace cells under the influence of several stimuli in the environment

1.1 Membrane potential of neurons

1.1.1 Structure of nerve cells membrane

Neurons are analogous to other cells, which have structural components

of cell membranes, nuclei and organelles The electrical activity of normalcells as well as neurons is highly related to the structure and characteristics ofthe cell membrane [1]

Nerve cells (also called neurons) are composed of three main components,the cell body, dendrites and axons, which are visualized in Figure 1.1 [10]

The cell body (also called the soma) is the largest part of the neuron,containing the nucleus and the majority of the cytoplasm (the physical spacebetween the nucleus and the cell membrane) Most of the cellular metabolismtakes place here, including the production of Adenosine Triphosphate (ATP)and the synthesis of proteins The neuron body processes and makes decisionsabout the flow of information going to and from here

Dendrite are short tentacles that develop from the cell body This iswhere the signal pulse from other nerve cells is transmitted (afferent signals).The action of these impulses may cause excitation or inhibition at thereceiving neuron A nerve cell in the brain cortex can receive afferentimpulses from tens or even hundreds of thousands of neurons.

Figure 1.1 Basic structure of nerve cell.

Axon is the only long extension that develops from the cell body Axonscarry the processed signal pulse from the cell body to another cell such asneuron or myocyte, adenocyte, The diameter of the axon in a mammal inthe range of 1 - 20µm In some animals, the axon can be several meters long.The axon may be wrapped by an insulating layer called a myelin sheath, made

by Schwann cells The myelin sheath is not seamless but is divided intosegments Between Schwann cells are the nodes of Ranvier The structuralcharacteristics of the Myelin sheath and the nodes of Ranvier have a greatinfluence on the speed of impulse conduction on nerve fibers

Similar to other cells in the organism, neurons are surrounded by a 7.5–10nm cell membranes The cell membrane plays a very important role inestablishing the resting properties and electric activity of the cell whenstimulated by regulating the movement of ions between extracellular andintracellular spaces Some ions such as HCO3-, Cl- could move to both sidesthrough cell membrane easily due to the difference in concentration gradient Butsome of the ions, especially Na+ and K+ must follow selective transportmechanism to move through cell membrane This leads to a potential gradientbetween the two sides of the membrane and creates a potential field This fieldexerts force on ions across the cell membrane Therefore, the movement ofmembrane ions is influenced by both the electric and diffusion forces.

The existence of a cell membrane depends on the permeability of thenecessary substances from the external environment into the cell and theexcretion of metabolites and debris from within the cell The permeability ortransportation of substances through the cell membrane is carried out in theforms of direct transport, phagocytosis, pinocytosis and exocytosis Directtransportation of substances through the membrane can be divided into threecategories: diffusion, passive transport and active transport [1]

The resting potential of ions in a cell is described in Figure 1.2 [52] Themain ions are potassium (K+), sodium (Na+), chlorine (Cl-) and calcium (Ca2+)

In particular, the electrical activity of the cell is mainly determined by K+ and

Na+ ions The activity of K+ and Na+ ions respectively determines the restingpotential and the action potential of the cell membrane The potential equilibrium

is obtained when the diffuse force is equal to the electric field force of all ions.For membrane with selective permeability of only one type of ion, equilibriumcondition is when the electric field creates a force equal to and in oppositedirection to the diffuse force The steady-state values of the membrane

potentials when there are some forms of ions in the intracellular andextracellular environments, as they cross the cell membrane, are specificallydescribed in the Goldman - Hodgkin - Katz voltage equation [26], [61].

Figure 1.2 Concentration and potential of ions at rest.

1.1.2 Resting and action potential

The membrane potential of a cell is defined as the difference in potentialbetween the internal and external side of the membrane due to the differencebetween ions on either side At rest, the ions distributed on both sides of themembrane are in equilibrium and depend on two forces - diffusion andelectrostatic forces The ions diffused out in the resting cell state are mainly K+,

so the diffusion force is calculated by the work needed to pass 1 mole of K+ ionsacross the membrane The electrostatic force at rest is calculated by the workneeded to resist the repulsion of ions with the same sign and the attraction of theopposite ions, in order to transfer a mole of K+ ions across the membrane

[1] Thus, in order to pass a mole of ions across the membrane, a total force(called electrochemical potential) is needed, which is equal to the sum of thediffuse and electrostatic forces and when the ions are in equilibrium, the twoforces are equal (but opposite) In the resting state (polarized state), theresting voltage Ek, determined by the K+ ion, is calculated using the Nernstequation and usually fluctuates between -70mV and -90mV and the cellmembrane is now in a polar state.

- n: valence of ion; with K+, n = 1

- F: Faraday constant (electric charge per mole of electrons).

When the cell is excited, the membrane potential is changed by changingthe permeability of the membrane with Na+ ions The Na+ channel is opened, andthe Na+ ions on the outside of the membrane rush into the cell to redistribute ions

on either side of the membrane: the number of positively charged ions on theinside of the membrane is greater than on the outside At this time, the membrane

is polarized from the polar state to the depolarized state and the excited or actionpotential EA appears This potential originates from the stem cell along the axonand is conducted to other cells The operating potential value can reach 120mVbut because at the starting point the membrane potential

(in the polarized state) has a value of -90mV, the actual voltage is about + 30mV.

Figure 1.3 Direction of potential field lines around a neuron.

When depolarizing neuron membrane generates action potentials, theyincrease the electrical conductivity of the excitation-sensitive areas of themembrane such as at the axon hillock or the soma The potential current entersthe cell through these locations into the core of the cell and then out to themembrane located at nearby inactive positions and returns to the place where thepotential current enters through many different ways This process forms apotential electric field around the neuron and its properties depend on the size

and shape of the cell, as well as the position and time on which themembrane’s conductivity is enhance [42], as described in Figure 1.3.

The term EF (extracellular field) or SPF (spike potential field) mentionsthe potential field around an active neuron when producing potential pulse

The membrane voltage of an excitable cell is defined as the potential on the inside surface of the membrane compared to the outside surface potential 0:

= − 0 ( )

(1.3)

This definition is independent of the cause of the potential and whetherthe membrane voltage is constant, periodic or non-periodic in operation.Fluctuations in the membrane potential can be classified according to theirproperties in a variety of ways According to Bullock [15], the transmembranepotential transmissions can consist of resting and changing potentials due toactivity When there is a series of stimuli to the cell membrane, a certaindegree of response potential is induced If the amplitude of the responsepotential is small and does not exceed the threshold, the response is notpropagated (electric tone) If the response is strong enough, a nerve impulse(action potential impulse) will be produced according to the "all or none" rule

1.2 Electrical nerve stimulation and medical significance

The basic theory of cells in general and neurons in particular is the basisfor assessing the stimulation and response of neurons to stimuli, in which thestimulation with an electric impulse plays an extremely important role in theintensive study of neurons

When a neuron is stimulated, the membrane potential of the cell changes.After response to stimulation, the membrane potential returns to its initialresting value If the membrane stimulation is insufficient to induce atransmembrane potential that reach the threshold, the membrane will not be

activated The response of the membrane to this type of stimulation isinactivation.

The model of stimulating and recording the action potential of a neuron

is illustrated in Figure 1.4a, the electrical impulse generation system Is withset parameters stimulate the neuron membrane via stimulating electrode Theresponse potential of the cell is recorded via the electrode and the recordingsystem With sufficient stimulation, the transmembrane potential reaches thethreshold and the membrane produces a special electrical impulse called anerve impulse This potential response follows a characteristic pulseregardless of the stimulus threshold strength Or the action potential of a cellmembrane obeys the "all or none" law This respond potential is call theaction potential in Figure 1.4a [10] which represents the stimulus model by

the I s current and measurement of the membrane voltage V m.

c

Figure 1.4 Changes in membrane potential under the effect of stimulation pulses.

Figure 1.4c shows the pulse shape and the intensity of the stimulation pulse.The change in membrane voltage is shown in Figure 1.4b under the effect ofexcitation pulses with different parameters: depression-induced stimulation

(1) and excitation-induced stimulation (2, 3, 4) In which the stimulating pulse(2) has not reached the stimulating threshold yet, it will only cause a passiveresponse Stimulation pulse (3) reaches the stimulus threshold can either cause

an action potential (3b) or not (3a) With stimulation pulse (4) exceed thethreshold, action potential is always created

Described in 1954 when two Canadian scientists, James Olds and PeterMilner, found that animals implanted with electro-stimulating electrodes inthe septal region would quickly return to the previously stimulated area inorder to receive more reinforcing stimuli Thus, electric stimulation acts as areward through which it is possible to train the animal to press the leverappropriately designed to receive stimulation [94] Modeling a behavioralevaluation model whereby the animal learns to manipulate itself to receivestimulating impulses into its own specific brain region that this brain region isthought to belong to in the pathway involves processing the brain's reward,involved in regulating both natural reward and BSR

Figure 1.5 Dopamine transmission pathways of mesolimbic

and mesocortical systems.

Studies show that it is possible to induce ICSS in many animals rangingfrom fish to humans; by creating BSR into brain regions belong to the

"reward" mechanism such as the medial forebrain bundle (MFB), or thedopaminergic mesolimbic brain regions such as substantia nigra, ventraltegmental area, the central nuclei of the amygdala, septal nuclei, nucleusaccumbens (NAcc); or the superior area of hippocampus, locus ceruleus,nucleus caudatus, olfactory tubercle, cerebellum In addition, several otherbrain regions also participate in the reward system such as the middle septalnucleus and the prefrontal cortex [9], [17], [94]

Moreover, BSR has outstanding features in the study of memory andmotivation that other studies which use rewards as food, sex or substances [8],

[53], [70], [85] does not have such as not affected by emotions, anxiety orrewards "saturation" … [17], [32], [83], [69] BSR is the type of stimulationthat directly targets the reward system of the brain ICSS-trained animals cancontinually perform tests to receive rewards for hours or even all day untilexhaustion, or in life-threatening condition the animal will still choose BSRover food or heating in condition of very low temperatures [94] Moreover, in

a certain limit, the increment of stimulation intensity (increase dose) inducesthe increment of ICSS response; on the other hand, other type of reward such

as food, water, sex or substances will decrease the response and create aanxiety state or “saturation” of rewards

Parameters affecting ICSS such as frequency, intensity of stimulation orduration of a single or cluster of stimulation pulse can be easily controlled andcustomized according to the purpose and model of the research; in which themost common are the first two parameters through which the stimulus thresholdand the maximum response level are determined In this study, electrodes areimplanted in the posterioral lateral hypothalamic area, under the influence of

the dopaminergic system (figure 1.5) BSR stimulation pulses are cathodepositive pulses and vary in intensity or frequency.

1.3 The response of cell membranes to electrical stimulation

When a stimulating pulse with set values depolarizes the membrane atrest or exceeds the threshold voltage, the membrane responds with an actionpotential Described in Figure 1.4b in item 3b and section 4 "action potentialresponses" corresponding to stimuli that reached threshold 3b and beyondthreshold 4 The response is characterized by a rapid increase in initialtransmembrane potential, to positive peak potential, and then slowly recover

to resting potential This phase reaction determines the action potential

Figure 1.6 Cell membrane’s response to stimulus signals

The response of the cell membrane to stimuli of varying intensity (a) follow the intensity-time curve (b) The minimum level of stimulus induced

response is called the rheobase threshold The minimum time required for astimulating pulse is twice the intensity of the baseline to initiate thedepolarization process called chronaxy [10].

Quantitative analysis of the characteristics of the action potentials wasinvestigated by Hodgkin et al [38] on the large squid axon fiber - a species ofnerve fiber with a large diameter (about 0.5mm), which is sufficient forplugging the two electrodes into the intracellular space In addition, thedevelopment of feedback control device called voltage clamp, which can keepthe transmembrane voltage at any value The action potential is a very fastchanging resting potential at the membrane, from polarization to polarization,polarity reversal and repolarization

There are questions to ask when studying cell membrane performance.Should an electric stimulating cell be activated depending on whichparameters: intensity, frequency of electrical impulse and duration ofstimulation? Can the membrane voltage be reached by a short, strong stimulus

or a long, weak stimulus? The curve illustrates the relationship betweenintensity and time in Figure 1.6 The smallest current to initiate activation iscalled the rheobase or current threshold Theoretically, the baseline thresholdtakes an infinite amount of time to trigger activation

The time required to stimulate the cell with twice the current baselinethreshold is called the chronaxy The changing and adaptive process of thecell which is repeated or continuous stimulated indicates an increase incellular excitation so there is a decrease in the threshold The latencyrepresents a delay between two events The time between applying a stimuluspulse and starting the activation is important parameters in the study

Once stimulation has begun, the membrane is not sensitive to new stimuli,regardless of the magnitude of the stimulus intensity This stage is called the

absolute allergy stage Near the end of the stimulating pulse, the cell can beactivated, but with only a stronger stimulus than usual, this stage is called therelative inert phase The activation process involves current, potential,conductivity, density, and ionic current When activated in nerve cells,electrical impulses are called nerve impulses The bioelectric measurementfocuses on the potential difference across the cell membrane so the electricalmeasurement of the action pulse is called the action potential - describing theelectrical response of the membrane during activation In biomagneticmeasurements, electric current is the source of the magnetic field Therefore,the term active current is corresponding to the source of the biomagneticsignal during the action pulse (Figure 1.6).

- The source of the action impulse is the nerve corresponding to it and being called a nerve impulse

- The electrical signal measured from the action pulse can be a potential or a current, the corresponding recording is called an active potential or an active current.

1.4 The recording methods of the neuronal action potential

Neuroelectrophysiology has been studied from the eighteenth centurythrough the report of Gavani on frog's thigh muscles during electricalstimulation Neuronal potential recording techniques has been in developmentsince 1940, originating from Renshaw et al's report on recording the actionpotentials of cortical and hippocampal neurons that helped to understand theoperating characteristics of structures in the the central nervous system [80] Thistechnique has been applied in many research directions such as usingextracellular microelectrodes to determine the potential characteristics of aneuron, thereby solving the fundamental problem that is the excitement property

of neurons [31], [71] and till recently the research on the response of neurons inrelation to behavior and stimulating factors in awakened and moving

animals [66] or determine connections between neurons in different regions ofthe brain At the same time, technology and equipment have also beenimproved to allow for a more accurate and convenient examination andanalysis of neuron activity [42].

Currently used extracellular potential recording techniques include twobasic methods: (a) using micropipettes containing the appropriate electrolytesand connecting the input amplifier with the Ag/AgCl electrode or platinumwire; (b) Use highly conductive metal electrodes with micron-sized head andinsulated body In which, the micropipette electrode is usually a glasselectrode and is used in the acute recording of cell potentials for the brainregions proximal to the cortex as shown in Figure 1.7 [20]

Figure 1.7 Demonstration of extracellular potential recording technique and the

data form.

Left: glass pipette electrode, filled with AgCl and attached with silver wireand approaching a neuron The amplifier amplifies the input potential from theextracellular potential field Upper right: Action potential is identified on the

signal noise when the electrode is close enough to the cell Lower right: Fromthe moment of the pulse, a histogram (not show) or a raster can be developedfor neuron response to stimulation.

Single metal microelectrode (monotrode) is widely used, developedincreasingly sophisticated and described through scientific publications [40],

[95] for electrodes made of glass-coated tungsten, stainless steel orplatinum/iridium alloy Recent developments include extracellular potentialrecording techniques using dual microelectrodes (steriotrode) or quadruplexmicroelectrodes (tetrode)

Recording extracellular potentials using metal microelectrodes has manyadvantages, such as having little resistance, therefore the signal is low in noise,

or relatively durable and easy to experiment in large quantity [42] A remarkablefeature in the use of metal electrodes is that it could continuously record thebehavior of a cell when the animal is awake, functioning normally or during tests

[93]. These behaviour characteristics may include impulse thresholds, latencytimes, pulse frequency (spontaneous or stimulated) or activities characteristics

by the time [20]

On the other hand, in the study of neurophysiology, especially themechanism of memory, the informative entry to the brain is thought to beencoded and stored in the form of synaptic connection levels in the neuronnetwork [46] A classic report on the influence of environmental propertiesthrough activity related to the location of hippocampal pyramidal cells [24]

has suggested the hypothesis that the map of the environment is encodedthrough the synaptic connection between pyramidal cells in the hippocampus.Thus, recording the activity of the hippocampal pyramidal cells in relation tospatial stimuli can help elucidate the hippocampus's memory mechanism forthe characteristics of the environment in which animals live

1.5 Hippocampus and hippocampal place cells

1.5.1 Structural characteristics

In humans and primates, Hippocampus (HPC) is a structure of thetemporal lobes in the sagittal (a) and coronal (b) [69] diagrams depicted inFigure 1.8 Hippocampus is the main component and along with otherstructures of the midbrain and cortex such as the olfactory cortex, mammillarybody, septum to form the Limbic system Studies show that HPC has a role

in storing information, converting short-term memory to form long-termmemory and the ability to navigate in space [74]

a

Figure 1.8 Diagram of rodent brain and the location of the hippocampus.

1.5.2 The function of the Hippocampus

The human hippocampus plays an important role in the process of memoryand learning, in humans and in primates is important for segmented memory

[25], [36], [59], [79] In mice, hippocampal injuries or interventions causedmany disabilities in learning and spatial tests [33], [86], [90], [92] At the sametime, recording of the hippocampal neuron potential also shows that thepyramidal cells in this region emit pulses in connection with a certain place(place - field) when the animal is moving in a specific environment Because theactivity characteristics of the hippocampal pyramidal cells is in relation with

place, these cells are called place cells [73], [75], [93] The place - field area

is affected by internal and external signals [48], [54 - 56], [67], [91], [97] or incertain information contexts [28], [34], [62] The above affirms the importantrole of the hippocampus in spatial memory [54 - 55], [93]

Besides many other functions, it seems that the main function of thehippocampus is providing the brain's neuronal material that reflects theexternal physical environment [25], [59] Based on this ability of thehippocampal place cell, it is thought to be the basis for studying or performingspatial cognitive exercises [19], [24], [25], [47], [48], [63], [81] In addition,many studies also suggest that an important function of hippocampalpyramidal cells is the ability to remember the order of events [5], [76]

In mice, hippocampal pyramidal cells generate pulses at specificlocations in the environment and are influenced by environmental factors For

a given environment, place cells have different place-field regions, and theseresponse areas can be influenced by reference points located near or far fromthe animal's position or even by other intrinsic factors

Therefore, hippocampal place cells in the brain have been discovered tohelp explain why we remember where we've been and can find our way back towhere we were before Maguire et al [60] compared the size of hippocampus oftaxi drivers (who need to memorize many streets, roads and places) and busdrivers (less space memorized) in the city of London (UK) and reported certaindifferences in the size of the hippocampus between the two groups From there,

it further clarifies the function of the hippocampus related to spatial memory.The hippocampus is regarded as the most studied sub-cortical region inboth humans and animals mainly due to its role and function in cognitivelearning and memory When injured, hippocampal dysfunction symptoms aremanifested as impairments in memory, attention, emotions, spatial navigation

and executive function Diseases that cause degeneration or damage tohippocampal neurons can be seen in Alzheimer's disease (AD) [23], [5], [77],

(MTLE) [68]; Schizophrenia [78]… In terms of neural networks, thehippocampus is located in a central position, which acts as a bridge to manyother brain regions involved in many cognitive processes It is suggested thatthe metabolic needs of various centers make hippocampus vulnerable in manyneurological conditions [22], [35] Using hippocampus as the center, therebycreating a hippocampal network model (HNM) that can be applied in thestudy of nervous system disorders [51], [64]

This dissertation focuses on researching to build a system to verify andfrom which can clarify the role of hippocampal place cells in memory andlearning through testing the effects of stimulating factors, especially theinfluence of spatial factors on experimental animals

1.6 Fundamentals of electronic circuit model of neuron

To further study and understand the activity of membrane potential onelectric stimulation, scientists have modeled the conducting system as anelectronic circuit model

Figure 1.9 Experimental equipment for the formation of the axon cable equation.

Supposely, the axon is immersed in an electrolyte of a certain concentration (similar to the extracellular medium) and the stimulation pulse is introduced through

a pair of electrodes, one electrode determines the external axon in the extracellular medium and one determines the inner axon as illustrated in Figure 1.9 [10] The

total stimulating current (Ii) around the axis of the axon decreases with distance

from the inner axis through the membrane to the return of the outer axon current Note that the direction of positive currents is to the right with and 0 in case the

and outside of the axon, the voltage does not change with respect to the direction of the nerve and this system represents a symmetry axis Axon radius is much smaller than axon length.

In this model, each section represents the transverse component of theinner axon with the boundary of the extracellular solution chosen shorter thanthe total axon length The membrane modeled as a scattered resistor andcapacitor in parallel is shown in Figure 1.10a The intracellular andextracellular paths both have resistances, which is the basis for describingexperiments on axons

Figure 1.10 Electronic circuit model and voltage chart of neurons.

Stimulation on living cells with a sufficiently large electrical impulse will

cause a response that changes the membrane potential When the stimulatingparameter reaches a certain threshold, the action potential of the cell will begenerated After this response, the membrane potential will gradually return toits initial resting potential value If the excitation pulse is not large enough,the cell will not be activated The response of the membrane to this kind ofstimulus is passive If the stimulating impulse is strong enough, the membranepotential reaches a threshold and the membrane produces a characteristicelectrical impulse called a nerve impulse.

If is the excitation current per unit area, from the circuit theory applied toparallel RC circuits shown in Figure 1.10b [10], we have:

′ = (1 − ⁄ )

(1.4)

In which:

- V': membrane charge [mV].

- I s : stimulation currents per unit of time [μA/cm2]

- R m : membrane resistance per unit area [kΩ.cm2]

- t: stimulation time [ms].

- τ: membrane time constant = R m C m [ms].

- C m : membrane capacitance per unit plane [μF/cm2]

In prolonged studies in live animals, usually using about 70 - 80% of thethreshold value should satisfy the requirements [88] but in case exceeding thethreshold, the response is also non-linear and can even cause the death ofanimals Potential time, the immobile threshold are relative concepts In atypical state, a timing chart may be based on an average of a specific number

of experiments In this case, the threshold (rheobase) and the valence(chronaxie) are measured experimentally by the stimulus response Generally,the faster the physiological system's expectations are met, the smaller thechronaxie value [10]

In the study of electrical stimulation of nerve cells, the parameters ofelectrical stimulation signals are very important: first, make sure not to causedamage to the stimulating object and it is also important to receiveinformation Needs study of neurons The relationship between parameters ofelectric impulsive stimulus, for each given intensity, there is thecorresponding frequency interval or duration of suitable stimulus announced

in the study [81]; The relationship between intensity and duration ofexcitation in nerve stimulation: the greater the intensity (certain value), thesmaller the stimulus time and vice versa [13 - 14], [39]

1.7 Related research to this dissertation

as the electrical signal transmission in the circuit Laying the groundwork forelectronic circuitry model of neurons are Hodgkin AL and Huxley AF [38],

describe the action potential pulse using linear differential equations given byFitzHugh [27], [43] is an additional and evolving step for the neuron electronicmodel of Hodgkin and Huxley After that, many scientists

studied electronic circuit modeling for neurons by making various electroniccircuit models to evaluate the stimulation of neurons as well as describe therespond by operating pulse form with the value of potential close to reality [57],[58]. In parallel with the study of parameters affecting electrical stimulation ofnerve cells [13 - 14], [39], neuronal potential recording technology has beendeveloped since the 1940s Originating from the recording of the actionpotential of a single neuron has helped to understand the operatingcharacteristics of central nervous system structures Later on, this techniquewas applied in many research directions such as using extracellularmicroelectrodes to locate potential characteristics of a neuron in in-vitroresearch, thereby helping to solve the fundamental problem which is theexcitability of dendrites of neurons [31], [71] Recently, researchs focus onthe response of neurons in relation to awakening and motor behaviors, oridentifying connections between neurons in different centers [66] At the sametime, technology and equipment have also been improved to allow for moreaccurate and convenient nerve cell survey and analysis [42] The technique torecord the activity of nerve cell voltage has also been studied and published

by many scientists [20], [31], [42], [71] The system that stimulates andrecords nerve cell activity as well as the animal's behavioral response tostimulation has also been studied and tested Numerous studies of stimulusand recording systems in response to the act of pressing the lever andreceiving rewards were also reported [17], [32]; or the spatial response of thehippocampal place cell in the maze [3], [75], [84] In animals, hippocampallesions or dopamine receptor deficiencies will affect the animal's ability toremember with spatial response [86 - 90]

Moreover, depending on the purpose of the research, scientists use devicesfor stimulating and recording the electrical activity of neurons as well as