Electrophysiology Measurements for Studying Neural Interfaces

By Mohammad M. Aria

Electrophysiology Measurements for Studying Neural Interfaces helps readers to choose a proper cell line and set-up for studying different bio-electronic interfaces before delving into the electrophysiology techniques available. Therefore, this book details the materials and devices needed for different types of neural stimulation such as photoelectrical and photothermal stimulations. Also, modern techniques like optical electrophysiology and calcium imaging in this book provides readers with more available approaches to monitor neural activities in addition to whole-cell patch-clamp technology.

• Details steps of an electrophysiology project from start to finish for graduate students employing the technique in their research
• Includes sample electrophysiological studies with multiple cell lines (PC12, N2a, NG108, SHSY, and embryonic stem cell lines) to facilitate research
• Features data analysis of electrophysiology results from various relevant experiments and cell culture tips

• Language: English
• Publisher: Academic Press
• Release date: May 15, 2020
• ISBN: 9780128170717

Mohammad M. Aria

Mohammad Mohammadi Aria MSc, graduated in the field of Micro-Nano Electronics from Electrical department of Sharif University Of Technology. He is currently a research assistant at Koç University in Istanbul, Turkey, focusing on electrical and electronic manufacturing and its applications in biomedical engineering.

Book preview

Electrophysiology Measurements for Studying Neural Interfaces

Mohammad M. Aria, PhD candidate in Biomedical engineering

Research Assistant, Koc University, Istanbul, Turkey

Table of Contents

Cover image

Title page

Copyright

Dedication

Preface

Chapter 1. Bioelectricity and excitable membranes

1.1. Introduction

1.3. Cell structure

1.4. Superfamily of voltage-gated ion channels

1.5. Hodgkin-Huxley model

1.6. The Hodgkin-Huxley model predicts action potential shape

1.7. Ionic currents and action potential shape

1.8. Summary

Chapter 2. Principle of whole-cell patch-clamp and its applications in neural interface studies

2.1. Introduction

2.2. Electrophysiology setup

2.3. Capillary glass electrodes

2.4. Measurement principle

2.5. Charge transfer mechanism

2.6. General protocol to patch cells

2.7. Voltage clamp

2.8. Current clamp

2.9. Whole-cell recordings

2.10. Extraction of H-H model parameters

2.11. Faradaic stimulation of neurons

2.12. Capacitive stimulation of neurons

2.13. Anode break excitation

2.14. Photocapacitive stimulation of cells by photoswitch (Ziapin2)

2.15. Photovoltaic stimulation

2.16. Photothermal stimulation

2.17. Conclusion

Chapter 3. Electrophysiological characteristics of neuron-like cancer cells and their applications for studying neural interfaces

3.1. Introduction

3.2. PC12 cells and differentiation to neuronal-like cells

3.3. NG108-15 cells

3.4. SHSY-5Y cells

3.5. Neuro-2a

3.6. Conclusion

Chapter 4. In vivo electrophysiology

4.1. Introduction

4.2. Spike sorting

4.3. Animal studies

4.4. Conclusion

Chapter 5. Calcium imaging and optical electrophysiology

5.1. Introduction

5.2. Neuronal calcium imaging

5.3. Fluorescent imaging

5.4. Calcium indicators

5.5. Dye-loading approaches

5.6. Analysis of calcium images and videos

5.7. Optogenetics

5.8. Optical electrophysiology (Optopatch)

5.9. Two-photon manipulation and imaging

5.10. Calcium imaging in neuron cell death

5.11. Conclusion

Chapter 6. Electronic circuits in patch-clamp system

6.1. Introduction

6.2. Power supply

6.3. Signal generators

6.4. Head stage

6.5. Bioamplifier

6.6. Voltage-clamp mode circuit

6.7. Current-clamp mode circuit

6.8. Capacitance compensation circuit

6.9. Output nulling circuit

6.10. Analog to digital convertor unit

6.11. Model cell

6.12. Calibration and operation tests

6.13. Extracellular amplifier circuit

6.14. Conclusion

Appendix 1: Operational amplifier circuits

Index

Copyright

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Dedication

This book is dedicated to my family: My mother, for all your supports and care, I love you!

My father, for all your supports and encouragements, you are always in my heart.

My beautiful sisters Sara and Samira who encouraged me to finish this book!

Preface

The multidisciplinary field of bioelectronics and neuroprosthetics demand wide technical knowledge from a variety of scientific backgrounds, including electrical engineering, material sciences, biology, and neuroscience. Furthermore, recent advances in neural interfaces with bionanomaterials require precise investigation and understanding of the underlying neural stimulation mechanisms. For this important goal, all the elements necessary for such a project including the development of a neural interface, the design of experimentation with neuron-like cells or neurons, and the examination of the performance of neural interfaces with the aforementioned cells have been discussed in this book.

This book has six chapters including the fundamentals of bioelectricity and excitable membranes (Chapter 1), exploring neural stimulation techniques such as the photoelectrical and photothermal stimulation of neurons by introducing whole-cell patch-clamp electrophysiology (Chapter 2), electrophysiological studies of neuron-like cell lines (such as PC12, Neuro-2a, NG108, and SHSY) with data analysis of patch-clamp results from various relevant experiments and cell culture tips (Chapter 3), extracellular recording and spike sorting (Chapter 4), optical monitoring and the control of neuronal activities through fluorescent indicators and optogenetic technology (Chapter 5), and the operation of electronic circuits in the patch-clamp system (Chapter 6).

Whole-cell patch-clamp electrophysiology has been widely used to measure the electrophysiological properties (membrane potential and ionic conductances) of cell membranes that control neuronal functions such as firing of action potential or neural silencing. Therefore, it is a very useful technology for investigating the performance of neural interfaces. For example, photovoltaic interfaces that are embedded with organic polymers—such as poly(3-hexylthiophene)—organic pigment photocapacitors, and inorganic quantum dots for photoelectrical stimulation of cells have recently attracted attention for neuroprosthtics application, such as retinal prothesis. However, the study of the efficacy and the mechanism behind neural stimulation requires an analysis and understanding of cells' neurophysiological behaviors that have been realized by the patch-clamp technique. Hence, in Chapter 2, basic functions of the patch-clamp setup and its applications for studying various types of interfaces have been reviewed and working mechanisms of different types of photoelectrical and photothermal neural stimulation have been discussed. Although whole-cell patch-clamp and extracellular recording mainly used in studying neural stimulation methods, optical techniques provide remote neural modulation and monitoring based on optogenetics and fluorescent imaging. In this regard, I have reviewed the basics of calcium imaging and optical electrophysiology with some practical examples from recent studies in Chapter 5.

I tried my best to explain my experiences and recent studies in bioelectronic interfaces for neural stimulation in this book. And I hope that this book will be practical for new researchers to use its contents in their projects and studies. Enjoy it!

Mohammad Mohammadi Aria

April 2020

Chapter 1

Bioelectricity and excitable membranes

Abstract

Bioelectricity in the form of ion fluxes is used for electrical communication by cells and tissues. Ionic gradients could be generated by the activity of voltage-gated ion channels. These ionic gradients are propagated as electrical signals among neurons and by the spread of excitation in heart and skeletal muscle. In this chapter, the structure of a neuron cell, including cell body and membrane, is explained to clarify the role of voltage-gated ion channels in neuronal activities. Retinal cells have been chosen for this neuron cell model, and their function has been described to illustrate a neuronal circuit and how their elements work together and perform neuronal functions. In addition, the superfamily of voltage-gated ion channels includes sodium, potassium, calcium voltage-gated ion channels, and others, including the large and diverse transient receptor potential (TRP) channel family, whose members respond when temperature, irritants, and other sensory inputs are introduced. Finally, based on a remarkable quantitative model of action potential introduced by Hodgkin-Huxley, the role of different ion channels in action potential shape has been explained.

Keywords

Action potential; Electrical excitability; Hodgkin-Huxley model; Neurons; Retinal cells; Voltage-gated ion channels

1.1. Introduction

Electrical signals are generated, propagated, and processed in the brain. To implement different functions, the brain uses different neuronal circuits including different types of neurons and connections. The author begins with retinal cells as some of the most interesting neurons and their functions, together with photoreceptors that enable vision. The visual system includes retinal cells, photoreceptors, and visual cortex neurons. In fact, photoreceptors play the role of sensors that receive visual information from the environment. This information will be transmitted in a complex network of different types of neurons to the visual cortex with optic nerve. The visual cortex also has many different types of neurons, and they form different circuits to implement different functions. To begin with the basis of a neuronal circuit, one needs to understand elements of a neuronal circuits and how they work together to implement a function. More importantly, each neuron has voltage-gated ion channels or excitable membranes that enable them to receive and send electrical signals. The basic concept of excitable membranes and voltage-gated ion channels in cell physiology is the central focus of this chapter.

Decoding the content of neuronal signals is a major goal of neurobiological research. As different neuronal circuits have different roles, the meaning of the signals depends on their origin and where they are transmitted, as well as signal parameters such as the frequency and duration of activation. Each individual nerve cell can receive thousands of inputs from other neurons. In this way, with integrating this input information, the cell generates a new output message that can send a new complex meaning, such as the presence of light with different colors in one's field of vision.

This chapter explains how neuronal activities from a big picture (function of a group of cells in a circuit) to a small picture of a single neuron enable specific functions. In the following, basic components of a neuron including cell body, axons, and dendrites are discussed in detail. As neuronal signals and functions are transmitted and controlled by electrical signals, the concept of excitable membranes and a superfamily of voltage-gated ion channels has been explained to prepare audiences for the needs of neuronal stimulation and inhibition for neuronal prosthesis. The high importance of ion channels is because of their role in membrane excitability, encoding neuronal activity, their diversity, and their effects on cellular functions which need close attention for studying neuronal prosthesis. A second goal in this chapter is to understand the electrical circuit of a single neuron based on the Hodgkin-Huxley (H-H) quantitative description. The landmark work of the H-H model describes a quantitative and an electrical circuit to understand neuronal activities. This model correlates different elements of ionic conductance, cell membrane capacitance, and excitation amplitude to action potential shape in time.

There are two types of electrical signals in the brain: local graded potentials, which are localized over short distances, and action potentials, which are transmitted over long distances. For instances, in synapses (junctions of one neuron to another), upon release of chemical transmitter molecules that bind to specific chemoreceptor molecules in the target cell membrane, a local graded potential activates or inhibits the cell depending on the transmitter and the corresponding receptors. The efficacy of synaptic transmission may be affected by molecules, hormones, and drugs. Action potentials are also generated when the membrane potential is increased and passes a threshold level of −45   mV. In addition, action potentials could be like a train of spikes transmitted through myelinated axons.

1.2.1. Neuron cell

Each neuron cell consists of a cell body, an axon, and dendrites. Fig. 1.1 shows a ganglion neuronal cell structure. The cell body consists of the cell membrane, nucleus, and organelles like other type of cells. Axons are connections that go from one cell body to another targeted cell. Dendrites act as receivers for excitation and inhibition through incoming fibers. Not all neurons have similar structure, as shown in Fig. 1.1. Certain nerve cells without an axon can communicate through other neurons that have an axon. In this case, having dendrites enables neurons conducting impulses to target cells. While ganglion cells include dendrites, a cell body, and an axon, other cells such as photoreceptors do not have an axon or dendrites. Because the stimulation or in other words the input signal in photoreceptors comes through light, not through input from another neuron, they do not need to have axons or dendrites.

1.2.2. Signaling in nerve cells

The first biopotential to be discussed is resting membrane potential, which arises as a result of ionic charge balance across the cell membrane. Resting membrane potential is a negative value about −69   mV for neurons. It is measured from inside of the cell in comparison to the extracellular medium. The signals that increase the cell membrane potential to a more positive value lead to cell depolarization, making the inside less negative, while the signals that hyperpolarize it make the intracellular medium more negative. As mentioned, there are two different electrical signals. The first includes local graded potentials such as receptor potentials and subthreshold membrane potential oscillations. For example, light falling on a photoreceptor in the eye leads to a hyperpolarization of the photoreceptor cell, which causes bipolar cell depolarization. This is because of the inhibitory mechanism of the synapse between them. This depolarization consequently excites the following cell, a ganglion cell, causing it to generate action potentials at a higher frequency. Local potentials vary in amplitude, depending on the strength of the activating signal. They usually spread only a short distance from their site of origin because they depend on passive electrical properties of the nerve cells. Action potentials are the second category of electrical signals that play an important role in signal communication and processing in neuronal networks if localized graded potentials are large enough to depolarize the cell membrane beyond a threshold level (called the threshold). Once action potentials are initiated, myelinated axons transmit them rapidly over long distances. For example, generated action potentials from ganglion cells are transmitted through axons to the optic nerve from the eye to the lateral geniculate nucleus, and then to the cortex. Action potentials have a constant amplitude and duration. The only properties that could be modulated are duration of activation, silencing, and refractory period of frequency.

Figure 1.1 Structure of a typical neuron including cell body and membrane, nucleus, axon, myelin sheath, and dendrites.

1.2.3. Retinal structure

The different types of neuronal cells in the retinal layer include bipolar cells, ganglion cells, horizontal cells, retina amacrine cells, and rod and cone photoreceptors. Fig. 1.2 shows simply the structure of the vertebrate retina. Retinal structure includes photoreceptors as light sensors. A wide spectrum of light enters the eye and reaches the photoreceptors. These photoreceptors generate graded potentials, and bipolar cells transduce these graded potentials to the proper stimulation signals for triggering