let there be light

Fascinating developments in various and distant fields of research matured at the beginning of the 21st century to the rule of synthesis, which enables the control of brain activity through light. Ofer Yizhar describes the historical process that culminated in the activation of animal brain cells using light rays

Dr. Ofer Yizhar, Odyssey

In an announcement that shocked the world, Francis Crick and James Watson announced in 1953 that they had deciphered the structure of DNA and introduced the double helix. The discovery, which changed the life sciences in many ways, opened a window to the world of science to a new and exciting field - molecular biology.

Crick realized that the proteins, the "molecular machines" that perform most of the biological functions of the living creature, are produced according to the "instructions" written in the genetic material. The genes, long sequences of DNA bases, encode the sequence of proteins and constitute the "instruction book" for the creation of all body proteins.

During the decade after this breakthrough, Crick formulated the "central dogma" of molecular biology. Then, when he decided that the central questions of molecular biology had been resolved, Crick turned to deal with an equally difficult, intriguing and exciting question. He wanted to know what the biological foundations of human consciousness are.

Crick moved from England to the United States and served as a visiting scientist at the Salk Institute in San Diego. There, surrounded by the best young minds of the world of science, he began to deal with the question of consciousness. As a distinct rationalist, Crick believed that our thoughts, feelings, self-concept - everything that makes us human - are a direct result of the electrical and chemical activity of the tens of millions of nerve cells (neurons) of the human brain.

He believed (contrary to the opinion of many in the field), that the question of consciousness should not be left as an area of ​​philosophical discussion only. It is possible to obtain and explain consciousness, he claimed, using scientific tools and the fundamental scientific method. This is, of course, provided that the right questions are asked.

Following Crick's work and the discovery of the genetic code, molecular biology recognized the basic building blocks (the bases of DNA). As early as the late 70s, it was possible to read the genetic code, change it with relatively simple means, and study the results of such genetic changes in order to understand the role and effect of specific genes. In contrast, the brain, the organ responsible for the development of consciousness according to modern scientific concepts, was almost a "black box".

Over a minimal volume of one cubic millimeter in the brain are scattered dozens of different types of neurons, which perform different actions and are connected to each other by tens of thousands of connections (synapses). Two neurons that are connected to each other and participate in the performance of a certain action, or represent one item of information, may be, physically, very far from each other, and in most cases it is not possible to isolate functional units based on the location of the neurons involved in them.

For example, in almost all parts of the brain you can find two types of neurons: excitatory and "inhibitory". The excitatory neurons increase the activity of their neighbors, while the inhibitory ones do the complete opposite - weaken it.

However, all over the brain, and especially in the cerebral cortex (cortex), neurons of both types are mixed together, so that they can only be separated according to their genetic characteristics. This branched network, for all its complexity, is also highly dynamic, and synapses are able to strengthen, weaken, form and disappear in response to changes in network function.

The incredible complexity of this system is also the main reason for the inherent difficulty in brain research. In order to isolate the effect of a single neuron, of a group of neurons or of an entire region of the brain, tools are needed that can deal with this complexity. In an article published by Crick in 1979 in the "Scientific American" newspaper, he wrote that "the great challenge facing brain research is the need to control with great precision the operation of a single neuron without affecting its neighbors."

Crick believed that only a multidisciplinary approach, which combines molecular biology, new techniques for the precise activation of functional units in the brain and innovative methods for data analysis, can bring us closer to a deeper understanding of the functioning of the brain and consciousness.

Salt-loving bacteria "see the light"

In 1973, six years before Crick formulated his hypothesis about the brain and indicated the need for new methods, two scientists from the University of San Francisco were working on microbial proteins (proteins found in microorganisms) in salt ponds in southern San Francisco Bay. In the study, any connection between it and brain research seems purely coincidental, Dieter Osterhalt and Walter Stockenius tried to find out how an ancient species of Archaea bacteria managed to survive in the almost impossible conditions of high salinity and low oxygen concentration, which prevailed in the salt ponds.

Osterhalt and Stockenius found that in order to cope with the impossible living conditions, these single-celled creatures are able to push hydrogen ions out of the intracellular environment, thus creating a situation where, due to their low concentration inside the cell, the hydrogen ions "aspire" to return to it to create a balance electrochemical. While entering back into the cell, the hydrogen ions activate an energy production system, capable, in the absence of oxygen, of providing the cell with all its needs.

It also became clear in the research that in order for this "backup" system to be operated successfully and succeed in producing energy, one thing is needed - light. Osterhalt and Stockenius found that the "ion pump" is responsible for one protein, which uses light energy to pump the hydrogen ions out of the cell.

They called this protein Bacteriorhodopsin, a name made up of two words - bacterium and rhodopsin, the name of the protein found in the retinal cells of all seeing animals. In the latter, the protein rhodopsin converts the light energy hitting the retina into chemical/biological signals, thus enabling vision. Bacteriorhodopsin, then, was a sort of bacterial equivalent of the familiar vision protein.

The discovery of bacteriorhodopsin made waves, and over time the understanding of the mechanism of action of this miraculous protein deepened. The studies described, step by step, the molecular processes that take place in the protein from the moment it is hit by a single photon (light particle) until the transfer of one hydrogen ion. The world of biophysics continued to find new proteins, members of the bacteriorhodopsin family, that convert light energy, and the characterization of these unique proteins while deepening their structure and function.

Thus, for example, the protein Halorhodopsin (halorhodopsin), which was discovered in the late seventies, is used, similar to bacteriorhodopsin, as an ion pump, but unlike bacteriorhodopsin, halorhodopsin draws chlorine ions (having a negative charge) from the extracellular environment into the cell. During that entire period of experiments and successes, this branch of research was part of biophysics and microbiology, and there was no connection, apparently, between this world and the study of the brain.

The age of the genome, the discovery of channelrhodopsin

Towards the end of the XNUMXs, the technology matured that made it possible to read, base by base, the genomic sequence - the DNA base sequence - in the genetic material of whole organisms. What began with the "Human Genome Project", expanded to include an effort to read and understand the genomes of all species that were of interest to the world of science in some way. Academic, government, and private research groups began to publish complete genetic sequences from single-celled organisms, which could be assumed to be useful to the scientific community and industry.

The US Department of Energy established the Joint Genome Institute, and it began publishing complete DNA sequences of various organisms, which could be used in research in various fields, such as agriculture, alternative energy sources, and medical research. In Japan, a research group called Kazusa began publishing genetic sequences of algae, plants, bacteria and viruses. The world of science was flooded with a tremendous abundance of genetic sequences, most of them unknown, which were available to everyone and were a vast breeding ground for new discoveries.

Among the first organisms selected by Kazosa were the unicellular algae Chlamydomonas reinhardtii. These are creatures one hundredth of a millimeter in size, capable of moving towards light sources using a primitive "vision" system. This system allows them to operate two biological engines, thus navigating towards areas where sunlight can be used to generate energy.

The algae C. reinhardtii has been the object of research in many fields, starting with the working mechanisms of biological engines and ending with innovative ways to produce energy (these algae are known for their ability to produce hydrogen as an alternative energy source to oxygen).

In 2001, two young scientists, Peter Hagmann and Georg Nagel, found within the C. reinhardtii sequence collection of the Kazusa group, a gene that is remarkably similar to the gene responsible for the production of the bacteriorhodopsin protein, mentioned above. They assumed that its product (the protein) might be the molecular "eye" of the algae, which allows it to navigate towards the light sources. They isolated the new gene and produced the protein encoded by its DNA sequence. The new protein was indeed similar to bacteriorhodopsin, but different enough to pique Hagman and Nagel's curiosity.

Hagman and Nagel used genetic techniques to induce living cells to produce the new protein. When the cells containing the protein were illuminated with blue light, a kind of "channel" opened on the surface of the cell's membrane: positively charged ions (sodium, potassium and calcium) began to move into the cell, thus changing the electrical balance on the surface of the membrane.

They named the new protein Channelrhodopsin, because they concluded that unlike biological "pumps" (such as bacteriorhodopsin and hallorhodopsin), channelrhodopsin allows ions to flow freely into the cell - of course, only while it is illuminated.

An unexpected synthesis

Hagman and Nagel knew that neurons are extremely sensitive to changes in their electrical charge. As soon as a sufficient amount of positive charges penetrates into the cell, the neuron produces an electrical signal, which causes the release of substances called neurotransmitters and the activation of all the neurons to which it is connected through synaptic connections. It was therefore clear to them that if they succeeded in causing the formation of these new channels within the neurons, it would be possible to control their electrical activity through illumination with blue light.

In this way, the old vision of Francis Crick will be fulfilled - a neuron that contains the channelrhodopsin will be immediately activated by blue light, but a neuron that does not contain the gene necessary for its production, will remain indifferent to light. This will enable the necessary selectivity to begin and deal with the enormous complexity of the brain.

In order to make the neuron produce the channelrhodopsin protein and bring it to the cell membrane, where it can perform its action in response to light, genetic tools were required that would allow the gene (derived from the algae reinhardtii C.) to be inserted into the neurons. The findings from Nagel's and Hagman's work were published in two articles, in 2001 and 2002, and upon publication, the two began looking for a connection with brain scientists who could realize the promise inherent in channelrhodopsin.

Carl Deiserot, a young American scientist who at the time completed his training as a psychiatrist and also completed a doctorate in brain research at Stanford University, decided to try and test how realistic the "crazy" idea was - to take a gene encoding a protein derived from unicellular algae, inject it into brain neurons using methods of Genetic engineering, resulting in the ability to control the operation of the "engineered" neurons using only light.

Daisrot, along with a number of students who joined his laboratory that was established that year, grew brain cells in a Petri dish, and using genetic methods inserted into them the gene responsible for the production of channelrhodopsin. Many thought that such an experiment was hopeless. The channelrhodopsin protein, which comes from algae, differs in its properties from the neuron proteins. In many cases, proteins from "lower" organisms are not well expressed in mammalian cells.

In order to function, the channelrhodopsin proteins need a molecule called retinal (retinal) - a derivative of vitamin A (which is also needed for the activity of the retina), and it was not clear whether neurons outside the retina and deep inside the brain contain retinal in sufficient quantity, in order to allow the activity of these proteins The channelrhodopsin.

Daisroth and his colleagues took the petri dish containing the channelrhodopsin-loaded neurons under the microscope, and recorded their electrical activity. A powerful lamp was attached to the microscope which can be activated quickly, with an accuracy of a thousandth of a second. With each flash of blue light delivered to the cells through the microscope system, the researchers recorded an immediate electrical response of the illuminated cells.

The first findings of this research were published in 2005. The new method for activating neurons using light was called optogenetics - a combination of genetic tools with light for the purpose of controlling the electrical activity of cells.

In a follow-up article, published in 2007, the group presented a new method for "silencing" the activity of neurons using light. This time the researchers chose to inject the protein halorhodopsin ("chlorine" pump) into the neurons. It was found that yellow light "silenced" the electrical activity of the cells into which the negatively charged chlorine ions were injected, preventing them from communicating with the neurons connected to them.

Now it was possible, therefore, to use light of two colors (blue and yellow) in order to bidirectionally control the electrical activity of neurons. The huge advantage of these new tools was the ability to control, by genetic means, the identity of the groups of cells that would express the proteins. Channelrhodopsin and halorhodopsin react to light almost immediately (within a few milliseconds), a time range that is remarkably suitable for the activity rates of neurons.

These publications created tremendous interest in the field of brain research. Researchers from all over the world began to use these innovative tools to activate or silence different types of cells in the brain and to examine, for the first time, the causal relationships between the activity of certain neurons and between physiological and behavioral phenomena related to their activity.

The first indication that there is a chance that this method will work not only in the laboratory, but in-vivo, in the living brain (in animals or, in the future, in humans) was received, when researchers from Duke University in North Carolina managed to produce a genetically modified mouse (transgenic mouse), which contained the The gene that codes for channelrhodopsin - the same gene that was inserted at Stanford into a certain group of cells in the cerebral cortex (cortex) and caused them to respond to light.

The members of Daisrot's group at Stanford received these mice, attached an optical fiber connected to a laser that produces blue light to the mouse's head, and used it to illuminate an area of ​​the brain responsible for movement (the motor cortex). As soon as the laser illuminated the area in question through the optical fiber, the mouse began to walk in circles according to the intensity and frequency of the laser flashes.

This simple experiment is noteworthy because it made it clear that the new technology, which combines genetic engineering, new molecular tools and optical and engineering enabling technology, will lead to a breakthrough in the ability of scientists to precisely control brain activity.

In the last few years, my colleague and I in the group of Daisro at Stanford University focused on expanding the field through the development of new optogenetic tools and the development of new applications for the existing tools. We made changes to the channelrhodopsin protein through molecular engineering and created new channels that are highly sensitive to light of different wavelengths (colors), so that different groups of neurons in the same location in the brain can be activated by different colors of light.

By changing the rates of action of the channelrhodopsin, we created tools capable of operating over extremely long periods of time (up to tens of minutes), which may be used in the future for chronic activation or silencing of neurons, both in research and in clinical applications. Using these new tools, which we developed in collaboration with Peter Hagman's group in Berlin, we are now investigating the physiological and behavioral phenomena resulting from changes in the relationship between the activity of excitatory and inhibitory neurons in the frontal cortex.

The front brain is involved in higher thought processes and integration between different systems in the brain. Many evidences also indicate that damage to the normal functioning of this area is associated with psychiatric and developmental diseases, such as schizophrenia and autism. One of the central hypotheses regarding the mechanism underlying these disorders is changes in the interrelationship between excitation and inhibition in the forebrain region.

Using the optogenetic tools we are able, for the first time, to directly and in a controlled manner control the activity of the excitatory and inhibitory cells in the forebrain, thus directly examining the physiological and behavioral changes resulting from the manipulation of these neuron populations.

Why "control"?

The development of the research indicates that, through the encoded genes and the light projected on them, it will be possible to control and direct the brain's actions in the fields
many. Why do we need to "control" the activity of neurons? The explained reader must now be imagining a wide variety of science fiction films that dealt with the subject, and wondering about the practical and philosophical implications of such an "ability", if and when it will be realized. Beyond future science fiction (or not), the need to control the system we are investigating is a basic need in science. It makes it possible to ask precise questions about the contribution of defined elements in the system to its overall functioning, and in addition it contributes to precise research and experimentation.

Today there are many methods that allow scientists to study brain activity even without control, starting with electrical recordings of "brain waves" (EEG), through imaging methods such as functional MRI and ending with electrical recordings from electrodes implanted inside the brain.

These methods allow researchers to understand how the brain represents information, when and how it responds to sensory stimuli, and even how the brain's structure and activity change as a result of diseases affecting it. But from the information obtained from these methods, although it is sometimes fascinating and allows us to understand more about brain activity, it is difficult to learn about the causal relationships between the activity of certain neurons, or groups of neurons, and the final product of brain activity - behavior.

In order to learn about the role of a certain gene and how important it is to the functioning of the entire organism, it is customary in genetics to carry out a manipulation called knock-out - canceling the gene by "deleting" it from the DNA. From the results of the knock-out it is possible to learn about the role of the normal gene. Indeed, through systematic work and the elimination of the action of individual genes, one by one, scientists succeed in insisting on the role of defined genes and proteins in the development and action of the living being.

The brain, which many claim is the most complex organ in the human body, is also formed in a way dictated by the action of genes. But unlike other organs (and perhaps similar to the immune system, which also has the "ability to learn"), the brain is constantly undergoing a process of design, defined by the individual events that make up the "experience of the individual".

In many cases, the part of genetics in shaping the functioning of the adult brain is smaller than the part of the plastic processes that continuously shape the brain, so that the final product is far from being "predefined" and is in a constant state of change.

It has been known for a long time that damage to different areas of the brain can cause mental disorders of different types. In the book - and this is just one example - "The Man Who Thought His Wife Was a Hat", neurologist Oliver Sacks describes a wide variety of patients he treated, each of whom developed, as a result of different types of brain injuries, a unique behavior pattern.

On the other hand, innovative treatments for diseases such as Parkinson's and clinical depression today allow patients who do not respond to drug treatment to get up and walk, or alternatively, to get out of black bile that lasts for years, and this by means of electrical stimulations transmitted through electrodes implanted in their brains.

Despite the successes in treatment, electrodes are an extremely crude tool. An electric current transmitted into the brain activates all types of cells that are around the electrode. The fact that certain diseases can nevertheless be treated with this tool teaches us that if we can better control the nature of the manipulations we perform, we may be able to expand the range of clinical conditions that can be treated.

In the last decade, the percentage of people suffering from psychiatric illnesses in the general population has increased to 25%, and this rate is expected to increase as the society in which we live continues to advance technologically and its average life expectancy will be higher. Even if the benefit inherent in extending life expectancy is a matter that can be debated, there is no dispute about the fact that the brain injuries that lead to diseases such as schizophrenia, clinical depression, Alzheimer's and Parkinson's are a heavy burden on the individual and society.

New methods for brain research, including optogenetics, will make it possible to better understand this complex system, and perhaps even form the basis for innovative treatments that will be more adapted to the unique characteristics of the brain, its complexity, diversity and branching systems of connections.

If we put aside for a moment the scientific breakthroughs that have already occurred, and the medical applications that will, without a doubt, arise in the future from the introduction of optogenetics into the scientific world and brain research, the story of the birth of this technology is first and foremost a story of the success of the basic science method.

The synthesis that occurred in the XNUMXs between microbial proteins such as bacteriorhodopsin, genomic information available to all, advanced genetic engineering and optical and engineering technology would not have been possible if these fields had not progressed independently, separately and independently, and created the broad knowledge base that made this wonderful synthesis possible.

The full article was published in issue number 11 of the journal "Odysseus - a journey between ideas".