A bright future

Optogenetics for dummies

Polina Loseva, "The Attic"

15 years ago, scientists came close to creating a laboratory "light weapon": it turns out that it is possible to teach cells to react to light and control them with a flashlight. Let's try to figure out how optogenetic methods are convenient and how bacteria helped scientists treat diabetic mice using a smartphone.

Precision is the courtesy of Kings

The further medicine develops, the more it strives for an accurate and purposeful effect on the body. The good old methods like bloodletting or mercury remain in the past, in the modern world the task is to act selectively on specific groups of cells. For example, to make the pancreatic cells secrete insulin (and only them, and not cells, say, eyes or bones). Or stimulate a certain part of the brain. The same questions are faced by researchers studying specific processes in the body or in complex cell cultures. The more precisely we learn to control the physiology of selected groups of cells, the clearer it is how these cells work and which of them is involved in the development of diseases.

For quite a long time, these tasks have been solved either with the help of substances acting only on certain types of cells (but not in every case they can be selected), or by genetic engineering. You can "feed" cells with additional genetic information and force them to produce proteins that are atypical for them, or, conversely, turn off genes already working in cells. However, none of the methods allows you to control what is happening. For example, it is not possible to abruptly stop the effect on cells if something went wrong; it is not always possible to dose the signal, vary it in time and space and exclude side effects. And I would like to be able to embed some kind of switch into the cells and activate it with the help of a stimulus that is safe both for the cells themselves and for their environment. Such an incentive was invented in 2002 – it turned out to be light.

I see the light!

In the human body, only specialized photoreceptor cells – rods and cones located on the retina of the eye - can respond to light. The rest of the cells do not react directly to light, so you have to turn to genetic engineering for help and embed additional light-sensitive proteins in them. Let's try to figure out how these proteins work.

In our photoreceptors, opsin proteins are responsible for the reaction to light. The retinal aldehyde of vitamin A is associated with them. Catching a photon, it receives additional energy, rearranges chemical bonds in its long "tail" and changes shape. Because of this, the structure of the entire molecule changes, and it gets the opportunity to interact with other molecules. With the help of several intermediaries, the opsin sends a signal to the ion channel on the cell membrane. Normally, the channel is open, after receiving the signal, the channel closes, and sodium ions stop flowing inside.

Changes in the structure of the retinal under the influence of light and when returning to darkness. The bond between the 11th and 12th carbon atoms changes from cis conformation to trans and vice versa. Image: P. Mahmoudi et al., 2017 / CC BY-NC-SA 3.0

Rods and cones are modified neurons, therefore they have excitability and conductivity. In the language of the nervous system, excitation is a change in the number of ions (i.e. charged particles) around the membrane of a neuron. When retinol catches a photon and the ion channel closes, positively charged sodium ions stop entering the cell, their number inside decreases. Consequently, the charge around the membrane changes and there is a signal that photoreceptors transmit to other cells, and those in turn forward it further to the visual centers of the brain.

Microbes rush to the rescue

However, for optogenetic tasks, our own opsins are generally useless. Rod opsins capture light from the entire visible spectrum (and scientists usually want to have more specialized proteins, for example, to activate different cells with different light), and cone proteins are not sensitive enough. In addition, not every cell in the body can be activated by closing the ion channel. This works if we are dealing with neurons, but if, for example, we want a cell to release a protein on a signal or trigger the work of a specific gene, then we need other types of light-sensitive proteins that are not in our body.

Our small (or even microscopic) and incredibly distant relatives come to the rescue. Analogues of rhodopsins have been found in drosophila, as well as bacteria and unicellular algae. In addition, photosensitive proteins with various functions are also found in fungi and higher plants. Some of them are more selective than our proteins, for example, they react only to blue light or to ultraviolet (which we do not see at all). Others perceive red light, which is less absorbed by ordinary cells, so it penetrates through them into the deeper layers of tissue more easily (this is important if we want to control cells deep in the brain). Finally, some proteins are much more sensitive than ours and are able to capture minimal amounts of light, which avoids overheating of the surrounding tissues.

Generous donors of photosensitive proteins: volvox, Leptospaheria maculans fungus, Chlamydomonas, oats, arabidopsis, halobacteria. Images: Frank Fox, Ralph Lange, H. Zell, Alberto Salguero Quiles, NASA

This is one of the characteristic cases when it becomes clear who needs fundamental science. Why, it would seem, study the photosensitive eye of chlamydomonas, when there are so many more pressing problems around? But it is chlamydomonas that turns out to be the owner of a unique protein that once became necessary for a lot of applied research.

Antennas on the head and a light in the window

In general, optogenetic technology looks like this: scientists select the photosensitive protein they need, use genetic engineering methods to deliver it to a certain group of cells, and then a wire with a diode at the end is brought to the right place, which can be turned on and off, observing what is happening with cells or animals as a whole (modern technologies allow implanting wire so that it does not interfere with the animal's movement and does not cause discomfort).

Stimulation of the central nucleus of the amygdala of the mouse
makes her hunt for inanimate objects

With the help of this technology, you can, for example:

• Selectively stimulate groups of nerve cells and obtain data on their functions. So, recently "Attic" wrote about mice that "drank" blue light. Photosensitive proteins susceptible to the blue region of the spectrum were embedded in the cells of their receptors responsible for the perception of sour taste. First, the mice were taught to drink ordinary water from the spout of the drinker, and then a blue diode was inserted into the spout, and the water was removed. The light activated sour taste receptors, and thirsty mice licked the spout of the drinker as actively as if there was real water there. This allowed us to conclude that they feel water with the help of "acidic receptors".

• Treat brain diseases. Scientists have managed to achieve a significant improvement in the condition of mice who have suffered a stroke. It has long been shown that electrical stimulation of the brain contributes to its recovery, but it remained unknown due to which cells and interactions. It turned out that it is possible to selectively include cells of the lateral nucleus of the cerebellum, which send excitatory impulses to the motor and sensitive areas of the forebrain. After such point stimulation, the mice began to pass behavioral tests much better, and these results persisted even after the end of therapy.

• Identify the area responsible for cocaine addiction. Mice were given multiple doses of cocaine to form a habit. And also embedded photosensitive proteins in the neurons of the prefrontal cortex. When these neurons were activated, the mice were less willing to use cocaine, and when they were suppressed, on the contrary, they attacked it greedily. Apparently, cocaine acts on this group of cells, and its activation leads to concomitant pleasure.

• Create fake memories. Scientists from England and the USA placed mice in different rooms and acted on areas of the hippocampus, which is associated with memory formation. For example, the first time they were placed in a room, and they noticed which areas of the hippocampus were active. The next time they were placed in room B (differently colored) and acted as a stress factor (electric shock), but at the same time they shone on the neurons that had previously been active in room A. The neurons were activated, a memory was formed. When the mice were then placed in Room A, they behaved fearfully, although they were not really subjected to stress there.

However, with larger mammals, the situation is more complicated. For example, a group of American scientists who worked with macaques faced the following problem: the primate brain is surrounded by three shells, while the outer one of them – a hard shell, or dura mater – is quite strong and opaque. When injecting a virus with a photosensitive protein and implanting a wire, attempts to penetrate it often lead to brain damage. In addition, due to the thickness of the shell and the complex three-dimensional architecture of the brain, it can be difficult to accurately direct a beam of light to the area of interest. The solution to this problem was a real window into the brain: part of the hard shell was replaced with a transparent nylon film that does not disrupt the work of the brain, and finally it was possible to literally watch how optogenetics works live.

Wireless Mice

In all of the above experiments, we were talking about neurons, and for the most part lying close to the surface. But what if we are interested in the cells inside the body? For example, we want to build a system that generates insulin by a light signal. It is clear that it is pointless to isolate it under the skin, so the activated cells should be located much deeper. But in this case, the question arises about what to shine on them and how to control the light. Scientists from China and Switzerland have developed a clever wireless system that avoids constant manipulation of the body.

They took a cell culture that was "trained" to secrete insulin under the influence of a distant red light. These were not neurons, but kidney cells with an integrated complex genetic engineering design. The modified cells were placed in capsules made of biopolymers and together with them "packed" small red diodes activated remotely by a special controller. Capsules were implanted into the abdominal cavity of mice with diabetes. Thus, the light source was immured in the immediate vicinity of the cells.

The whole system works as follows. The researcher uses a blood glucose meter to determine the level of sugar in the animal's blood. The blood glucose meter automatically transmits this information to the smartphone, where a specially designed application is installed, and to the controller. When the sugar concentration is high, the controller automatically starts the diodes. At the same time, the application on the smartphone allows you to adjust the individual dose and adjust the process more precisely by sending signals to the controller via the Internet. As a result, the diodes emit red light, it falls on the cells, and they release insulin in response, which penetrates through the pores in the capsule shell and enters the blood. The result is that the blood sugar level of diabetic mice drops.

All these small experiments are gradually bringing us closer to personalized remote medicine. In an ideal scenario, a person should learn by himself or with the help of simple devices to measure various indicators of the state of his own body. The devices will send the measurement results to the doctor (or a special application, if the doctor's decision is not required), and the doctor (or the application) will be able to send a light signal to individual groups of cells and change their activity. This is how, without unnecessary operations, consultations and queues, the present bright future should look like.

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