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In this note, I want to introduce some of the famous research methods in Neuroscience. Of course, it is not possible to introduce all the different research method and it is also out of my capability to know about all of them, but based on my personal experience it would be helpful to have some understandings about at least some well known (historic) research method. As you may know (at least as far as I observed), most of area in science does not evolve continuous/linear fashion. I think they are evolved by some big jump made by some critical findings, experiments and theories etc. In some cases those findings was not intentional (logically planned) and happened by chance and in some other cases those findings are obtained by well planned experiments or theory by some great minds. Regardless of whether those findings happened by chance or by plan, once those findings occurred a huge amount of subsequent research would be done around those original findings using the similar methods that are used in the original findings. So if you have some understandings about those historic findings / methods, it would be much easier to read written documents or watching lecture video because you already know of the background context about them. Another reason on the importance of understanding research method is as follows : Each type of Research Method produces similar types of data. For example, some method produce a data as an image (like fMRI) and some method produces the data as various different types of graphs (e.g, intra/extra cell recording, EEG etc). If you have some basic understandings (actually the more the better) on those research method and how to interpret those results would greatly help you to understand various textbook, lectures, article, papers without putting much efforts comparing to the case where you don't have any knowledge on those research methods.
Learn from DamageOne of the biggest difficulties in Neuroscience has been around the difficulties of designing and implementing experiments. Sometimes those difficulties came from technical limitation, sometimes from lack of knowledge on what to do or sometimes from ethical reasons. I think we have difficulties from every possible reason just mentioned for human research. Some easy way for research if everything is allowed would be to remove (or make a damage) to a certain part of nervous system and observe what happen after that. But it is rarely possible to do this kind of experiment even for higher animals not to mention human. So many of important findings about human neurosience came from the patients who got damages (lesions) by accidents or some unknown reason. In this section, I would introduce some of the famous / well-known patients with lesions that lead to critical findings in human neurosicience. Phineas Gage (1823-1860)I came to know of Phinea Gage when I was taking a biology course about 35 years ago (sometime late 1980s) which was dealing with topics about basic brain functions. For me, it was the first time for me to hear of any patients with this kind of damage in brain.
Image Source : Wikipedia He was a railroad construction worker and got an iron bar peneterating through forehead part of his head as shown below. I was shocked by the tragedy and at the same time by the fact that he (fortunately) survived.
Image Source : NPR Health News After this accident, he suffered from various personalilty issues and the observation on his behavioral changes led to the findings that specific behavior or some aspect of personality associated with a specific regions of brain. And this finding had been a trigger for a lot of subsequent researches about association between various other parts of brain and corresponding changes in terms of behavior, emotion, cognitive processes etc. Henry Gustav Molaison (February 26, 1926 December 2, 2008)He was an American and is widely known as H.M and may be the most famous and well-known patients. If you have been interested in neuroscience and read some of documents (books, megazines or articles in the internet) or watch lectures, I am pretty sure you might have heard of him, at least the name H.M. Since he has been mentioned in so many documents and lectures, it would be good if you have some background information about him to follow along with those documents and lectures. He had been a patient who greatly contributed to nuerosience, especially learning and memory process, but it was not intentional at all. He just got a brain surgery just to reduce the symptom of epilepsy, but a serious memory related problem is observed after the surgery. This unintended problem led to findings where Hipocampus plays key roles in long term memory formation and memory may be localized (i.e, associated with a specific part of brain not associated with the all across the brain). Watch this video for the importance of findings from H.M
Image Source : Wikipedia He had a bilateral medial temporal lobectomy in 1953. With this surgery, he got about two thirds of anterior hippocampus removed. The removed area were parahippocampal cortices, entorhinal cortices, piriform cortices, and amygdalae. The purpose of this surgery was to cure his epilepsy. More detailed information of the brain lesion (damage) found from the autopsy were as follows : See this if you are interested in further details.
What happened after this surgery ?
I was not able to find YouTube video showing his symptom directly (he passed away before sharing video over internet is so common), but I found some videos explaining about him like this, this Clive Wearing (May 1938 ~ )Clive Wearing is a British former musicologist, conductor, tenor and keyboardist. He had similar brain lesion as H.M (i.e, lesion in Hipocampus) but he got this from his illness not by any physical surgery or accidents. He lost the functionality of large portions of Hippocampus and temporal lobe which plays crtical function in memory process.
Image Source : Alchetron After the illness, he has been suffering serious Amnesial.
Personally Clive Wearing is more familiar to me than H.M because I got several YouTubes (watch this , this) showing / describing details of the problem he is suffering, whereas I was not able to find any video about H.M showing the live behavior of H.M. Electro recordingThe nervous system, whether in a human, a mouse, or even a fruit fly and squid, is a symphony of electrical impulses, a complex orchestra of neurons firing in harmony to create thoughts, behaviors, and physiological responses. Electrophysiological recording serves as our conductor's baton, allowing us to eavesdrop on this intricate ballet of electrical activity across diverse species and scales. From the precise measurements of individual neurons within a petri dish or within a specific body part to the sweeping brainwaves captured by electroencephalography (EEG) in freely moving animals, electrophysiological techniques offer an unparalleled window into the inner workings of the nervous system. In this techblog series, we will embark on a comprehensive exploration of electrophysiological recordings, showcasing their versatility and far-reaching applications in both basic research and clinical settings. We'll delve into the diverse methods used to capture these electrical signals, from intracellular and extracellular recordings in isolated cells to multi-electrode arrays (MEAs) that monitor the activity of entire neural networks. Intra Cell Recording/MeasurementI think the origin of the single cell measurement in electrical way would be the test with a super big nerve cell (neurson) from a squid shown below. Especially thanks to this, we could learn such a detailed mechanism of Action Potential. As shown below, the size of a nerve cell in this squid is so huge that you can see the single nerve cell with naked eyes and put an electrode directly inside of the cell with the technology decades ago. This technique measuring the electric potential (voltage) of inside of single neuron called Voltage Clamp / Patch Clamp technique. With the continuous evolution since this research, both electronics and equipment manufacturing technology has been developed drastically, we would do the same type of measurement for higher animals which has much thiner / smaller nerve cells (If you are seriously interested in the intra cell recording with modern technology, I would recommend this article (Whole-Cell Recording of Neuronal Membrane Potential during Behavior).
Source : YouTube Extra Cell Recording/MeasurementExtra Cell Recording is also measuring the electrical activity of neurons like intro cell recording. Extra cellular recording measures the electrical activities outside of nerve cells (i.e, the measurement elelctrode is placed outside of the cell) whereas the measurement electrode is placed inside of the cell in Intra cell recording. Since the measurement electrode placed outside of a cell, it is not guaranteed that the measured activity is from a single cell. It can be the measurement of a single cell or multiple cells around the electrode. Advantages of Extra Cell Recording comparing to Intra Cell Recording would be that it does not require such a sophisticated setup as in intra cellular recording, meaning that it is much easier to set up the test. Micro Electrode Array (MEA)Micro Electrode Array(MEA) is a special type of electrode (bar or thread like electrode) in which has multiple electrodes embedded as illustraded below. I think the biggest advantage of MEA is that it can record the neuron activities of multiple layers of a cortex region simultaneously as shown below.
Image Source : Progress Towards Biocompatible Intracortical Microelectrodes for Neural Interfacing Applications One of the most common / famous type of MEA would be Utah Electrode Array (UEA). It is made up of basic components called shank(a bar like structure). The shank is a bar like silicon wafer on which multiple electrodes are deposited as shown below. It was developed at the University of Utah in the late 1970s and early 1980s. It was developed as part of a research project aimed at developing a brain-machine interface that could be used to control prosthetic devices, such as a robotic arm or a computer cursor, using the activity of neurons in the brain. The UEA has since been widely used in research and clinical settings, and has been instrumental in advancing our understanding of the brain and the development of treatments for neurological disorders.
Image Source : 3D high-density microelectrode array with optical stimulation and drug delivery for investigating neural circuit dynamics If you make a linear array(a vector) of shanks, you can record the neural activities of mutiple layers across a certain span of the cortex simultaneously.
Image Source : Electrode Technology with Dr. Rio Vetter | Webinar If you make a 2D array(a matrix) of shanks, you can record the neural activities of mutiple layers across a certain area of the cortex simultaneously.
Image Source : Electrode Technology with Dr. Rio Vetter | Webinar I think the most recent technology of MEA (as of writhing this note : Jan 2023) would be MEA being used in Neuralink. This electrode is a flexible structure in which a linear array of electrodes are embedded. Eac of the thread can be planted into the cortex of the brain by a special machine (a medical robot).
Image Source : Neuralink Show and Tell, Fall 2022 EEGEEG stands for ElectroEncephaloGram. This is non-invasive technique to measure the activity of brain. As shown in the picture below, it does not require the opening of the brain... just put a lot of measurement electrode on the skull. Obvious it is much easier to setup and perform the measurement comparing to cellular recording, but it is not possible to associate the measured brain activity to a specfic nerve cells.
fMRI'f' in fMRI means 'Functional' MRI. This technology was developed early 1990 and have been one of the most important contributors in brain science even until now. This equipment(technology) can take picture of brain image highlighting the specific area of the brain which are 'functioning' (called 'Active') at the time of the shot. Usually it indicates the active region as redish color (the more redish the more active) and indicates the less active area in bluish or other darker colors as shown below. Thanks to this techniq, scientist has been able to figure out which part of the brain is associated with what kind of mental activity without relying on patients with specific lesions or physical operation etc.
Image Source : PsychCentral Even though the technology is complicated, the logic of the research is pretty straight forward. Let the persion in the equipment perform a certain mental activity and see which part of the brain become active at the time of the activity. For example, it goes like this
Image Source : VISUALLY EVOKED POTENTIALS BY DONNELL J. CREEL - WEBVISION DTIDTI stands for Diffusion Tensor Imaging. Technically the term 'Diffusion Tensor' itself explains a lot but for most people like me, it doesn't make much sense. For for the layman like me in this area, you may just think of this as a technology that can measure the activity of water molecules mostly along with axon of neurson. Since the axon is the part of the neuron connecting a part of nerve system to another part of the system, this image can visualize the complex pathways in nerves system (e.g, in brain) as shown below.
Image Source : Imagilys and eBrain Transcranial Magnetic Stimulation (TMS)Transcranial Magnetic Stimulation is a technique that can generate a strong magentic field at a spefici location in the brain without any surgery (i.e, it is non-invasive technique). It would be the biggest advantage of TMS that the technology can stimulate a specific part of the brain without surgery. As you know neruon activity is performed by electric current changes. This electric current changes would induce magnetic field. This magentic field can be interfered/interfacted by the magnetic field applied by TMS. This is a short story on how TMS works.
Volum Electron MicroscopyBy far the best technology that shows the most detailed structure of cellular structure would be the electron microscope. That is, electron microscope is the technology that has be the best magnifying power. But a problem of this technique is that it requires a lot of effort to make preparation for the observation. It would be the best if CT or MRI has such a resolution as electron microsoft, but unfortunately it is only in our wish list. One of the best alternative would be to use electron microscope like CT. In short, it takes out small volume of brain tissue and slices it into a lot of layers. Each of the layers are phographed under the electron microscope. If you color a specific neurons apeared on every slices and reconstruct a 3D image, you can get some images as shown below. If you are interested in the further details of this technology, I think this is the best introductary video : Connections in a Cube/ Cell, July 30, 2015 (Vol. 162, Issue 3) and How brains see
CLARITY/Tissue ClearingI think this is very intriguing technique to me. In terms of resolution, I think Electron microscopy shown above would be the best but it would be extremely difficult to visualize the scale of whole brain (even for the small brain like rat brain). This technique is designed to make the whole brain (or large chunks of brain tissue) transparent so that we can look insde of the brain without cutting it open or slicing it out. The technique originally named as CLARITY and then with variations of the technique the types of techniques that can make the tissues/organ transparent called 'Tissue Clearing Technique'. First let's think of why most of tissue / organ (like brain) is not transparent as shown in [A] and we cannot see through it without cutting it open. It is mainly because of lipid layer of cell membrane. So the idea of making the tissue/organ transparent is to wash out the lipid. But if you just wash out the lipid layer, it will break all the cells and internal structure of the cell. It is not making it transparent, it is making a porridge :). The smart trick in this technology of making them transparent is to form gelish mesh through the whole organ/tissue and then wash out the lipid layer. Then the mesh will keep all the non-lipid biomolecules (e.g, protain) as it is located before as shown in [B].
Image Source : See-through brains Even though the technology is fancy, just making the tissue transparenet would not do much. To get more meaningful data out of the transparent tissue, you would need various additional techniques like staining and microscopy technologies. If you combine all those technologies properly, you woult get such a fancy and detailed images as shown below.
Image Source : Compiled from various documents listed in Reference Staining TechnologiesI think one of the most important technology in neuroscience (actually not only in neuroscience, but also biology in general) would be staining (dying) technology. Like most of other cells in our body, nerve cell (neuron) would just look transparent or translucent when you just observe it under microscope. It is the staining technology that make those cells look clear and outstanding. Golgi StainingGolgi staining would be the oldest and most widely used staining technology for neuron observation. Just a few examples shown below would tell you what it is like.
Image Source : Webvision: The Organization of the Retina and Visual System Fluorescent StainingSimply put, Fluorescent staining is a technology to let a special fluorescent chemicals (called fluorescent probe) stick to specific intracellular structures (e.g, protein, neucleotides) and observe it with a specially designed microscope. There are several different ways to let fluoresent chemicals stick to nerve cells. Some of the typical ways are :
Image source : here, here and here BrainbowBrainbow is a genetic technique developed to visualize individual neurons and their connections in the brain with multiple fluorescent colors. As you may easily guess the term "Brainbow" is derived from the combination of "brain" and "rainbow". it allows neurons to be labeled in a variety of distinct colors, making it easier to distinguish between them and trace their connections. The Brainbow method involves genetically modifying neurons to express different combinations of fluorescent proteins derived from jellyfish and coral. The proteins include variants of green, red, and blue fluorescent proteins, which can be combined to generate a wide array of colors. Appying this to a mouse brain as an example, Researchers make neurons in a mouse's brain light up in different colors. They used a genetic trick called Cre-Lox recombination to insert special genes for colorful proteins into the mouse's DNA. This way, when the neurons become active, they produce these colorful proteins and glow in various colors. Since the color combinations are random, each neuron gets its own unique shade, making it easy to tell them apart. This colorful brain helps scientists study how neurons connect and interact in different parts of the brain. The Brainbow method has been super helpful not only for studying mouse brains, but also for looking at other animals and even non-brain tissues.
Molecular Biology/Bio Engineering
Sea SnailThis (a sea snail) would one of the most famous animal that lead to the opening of research on learning and memory at synaptic level and molecular level. This animal has very simple architecture of nervous system with much less number of neurons comparing to higher animal (this animal has only 20,000 neurons whereas a human has 100,000,000,000 neurons), but still capable of 'learning' and 'remembering'. The size of a nerve cell is huge (in mm scale in diameter) that you can see it with naked eyes. Only small numbers of neurons (only a few hundreds) get involved in a certain behavior of the animal (Watch @11:25 of this lecture if you want further details). Scientists was able to train this animial with a certain behavior and direct observe the changes of synaptic networks and molecular composition changes before and after the learning. You can get a pretty clear idea on how the research has been done from this short video. If you are willing to tackle the far more details on this research, check out this lecture : Memories are Made of This. For more recent researches especially for gene expression, check out this lecture : Memories that Last: Genes Neurons and Synapses
Image Souce : Wiley Online Library
Image Source : (@13:27 of Memories are Made of This ) Common Molecular Aproaches are :
OptogeneticsOptogenetics is a groundbreaking research technique that combines genetics and optics to control the activity of specific neurons or other cells in living tissue using light. Thanks to this technique, researches can control the activity of specific brain cells using light. It's like a remote control for neurons!
Image Sources : In vivo Optogenetic Stimulation of the Rodent Central Nervous System / Light-Based Brain Treatments May Soon Be Used On Humans To make this work, researchers use genes from light-sensitive proteins called opsins, which are found in some algae and bacteria. They introduce these genes into the brain cells they want to control. Once the genes are inside the cells, they produce the opsins, which get incorporated into the cell membranes. Now comes the fun part! When researchers shine light on the cells with the opsins, the opsins respond by either opening or closing ion channels in the cell membrane. This changes the electrical activity of the cells, allowing scientists to turn them "on" or "off" like a switch. General procedure of applying this technolgy goes as follows :
Calcium ImagingCalcium imaging is a research technique that helps scientists visualize and study the activity of neurons (brain cells) by monitoring changes in calcium levels inside these cells. As you know, Calcium ions are crucial for many cellular processes, including how neurons send electrical signals. When a neuron becomes active, its calcium levels change, and calcium imaging detects these changes. Calcium imaging has gained popularity in neuroscience because it allows researchers to study the activity of many neurons simultaneously, even in live animals. With this technology, researches can gain a better understanding of how brain cells work together to process information and control behavior.
Image Source : In vivo Calcium Imaging to Illuminate Neurocircuit Activity Dynamics Underlying Naturalistic Behavior
Image Source : Characterizing Cortex-Wide Dynamics with Wide-Field Calcium Imaging To apply this techniuqe, researchers use special molecules that stick to calcium and light up when they're bound together. These molecules are called calcium indicators and they can be dyes or even glow-in-the-dark proteins made by the cells themselves. Here's a quick and casual rundown of calcium imaging:
C. elegansObviously C. elegans is not a technology or research method. It is an organism (animal). But I put this as a separate research method item because this can be utilized across such a wide spectrum. Through research on C. elegans, scientists have gained critical insights into neural development, synaptic function, sensory perception, and the genetic and molecular bases of behavior. Moreover, studies in C. elegans have contributed to understanding the mechanisms underlying neurodegenerative diseases, such as Alzheimer's and Parkinson's disease, making it an invaluable tool in neuroscience research.
Source : C. elegans nervous system Why so popular ?Caenorhabditis elegans (C. elegans) plays a pivotal role in neuroscience research due to several unique and advantageous features it possesses :
Experimental TechniquesExperimental techniques used with Caenorhabditis elegans (C. elegans) span a wide range, leveraging its unique biological features. Here's an overview of common methods: These techniques, often used in combination, allow researchers to dissect the molecular, genetic, and neural circuit mechanisms underlying development, behavior, and disease models in C. elegans. This integrative approach has made C. elegans a powerful model organism for studying complex biological processes
Application to AI/Neural Network ResearchThe intersection of C. elegans research with AI and neural network methodologies not only enriches our understanding of biological neural networks but also informs the development of artificial intelligence, offering a unique platform for interdisciplinary advances in both fields.
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