Synthetic Biology Research Group

The synthetic biology group uses the mouse as a model system. Due to the similarities of the mammalian brain, the questions addressed in connection with the serotonin balance also provide important information on the function of serotonin in other mammalian species, including humans. Mouse and human neurons have a very similar structure and mammalian brains are highly similar. More than 80 percent of the genes of humans are also found in mice, and there are hardly any differences, especially at the molecular and cellular levels. Therefore, the findings can be transferred well to the human organism. In the course of a year, researchers use about 100 to 200 mice in animal experiments, depending on the number of studies currently being conducted.

The mouse animal model is particularly suitable for addressing these questions, since a large volume of data from experiments in vivo (with mice) and in vitro (with cell culture techniques) is available as a basis - also from our own preliminary studies. In addition, a large number of genetically modified mouse lines (i.e. mice with altered genetic information) are available. They allow a targeted methodical treatment of the research question. Mice are particularly suitable for research because they are easy to keep and can be bred in the laboratory by the scientists themselves.

Good husbandry is a basic requirement for conducting animal experiments, because only animals that are not stressed can be used for meaningful animal experiments. The release of stress hormones would have a negative effect on the entire organism and lead to a falsification of the data collected.

Currently, there are about 300 mice in the mouse husbandry of the Synthetic Biology group. The animals are always housed in holding systems in groups - so-called "isolated ventilated cages" (IVC). This system ensures that no germs are carried into the cages via the air, thus ensuring optimal ventilation, humidity, and temperature in each individual cage. All cages are equipped with a variety of nesting material and houses for hiding. The general condition of all animals is checked daily by experienced staff and keepers. For mice that are currently in a trial, the stress level is determined and body weight is measured on a weekly or, if necessary, daily basis.

In the Synthetic Biology group, various techniques are used to study the functions of serotonin. These include molecular biological, electrophysiological, and animal experimental methods. Whenever possible, researchers conduct their experiments without using animals and try to use alternative methods such as studies with cell culture techniques (in vitro) or on brain slices (ex vivo).

To specifically study neuronal circuits, the lab uses optogenetics - a relatively new neuroscience method that allows electrical signals, action potentials (AP), in neurons to be specifically turned on or off using light. By incorporating certain light-sensitive proteins, such as channelrhodopsin (ChR2), into mouse neurons, they then respond with an AP upon exposure to light. In recent years, the laboratory and its collaborators have also developed the possibility of switching on certain serotonergic signaling pathways in nerve cells via light. With the help of optogenetics, the researchers can specifically switch on serotonergic signaling pathways in nerve cells and thus investigate exactly what role these signaling pathways play in certain brain regions in various behaviors and diseases.

To deliver the genetic information of these light-activatable proteins into neurons, the researchers are using adeno-associated viruses (AAVs) as a delivery system. AAVs are non-pathogenic and thus pose no threat to humans or animals. The AAV is introduced into the brain of a mouse via injection with a very thin glass needle. (On average 0.001 millimeter. For comparison, a human hair is about 0.1millimeter thick.)

During this short operation, the mouse is under anesthesia and also receives pain medication. For later light activation in the behavioral experiment, a thin glass fiber (diameter 200 micrometers) is implanted chronically into the mouse. After awakening from anesthesia and a recovery period of at least two weeks, the animals can now enter the behavioral experiment, where they can move freely and unhindered.

In some experiments, the activity of neurons in the awake animal is measured, either by calcium imaging or electrophysiological methods. In calcium imaging, similar to the optogenetic experiments, a virus is injected into the mouse brain, which leads to the expression of a calcium sensor. A mini-microscope lens is then implanted in the same brain region. Again, the animals are anesthetized and receive pain medication. After about 3 to 4 weeks, during which the animal is allowed to recover, a mini-microscope (weighing less than 2 grams) can then be used to record the activity of several hundred neurons simultaneously. This technique makes it possible to specifically analyze the activity of individual neurons during different behaviors in the behavioral test.

Open-Field Test

The open-field test is primarily a behavioral test to study the motor and fear behavior of rodents. The arena consists of a square Plexiglas box (50 centimeters x 50 centimeters) in which the mice are placed for five minutes. The distance, speed, and time spent in the different areas of the arena are measured. Since rodents usually avoid open areas to avoid being captured by predators, they stay as close as possible to the wall or in the corners of the arena. The amount of time animals stay in the center of the box can be used as a parameter for fear behavior. Particularly brave animals stay in the center more than particularly fearful animals.

However, the open-field arena also finds application in experiments in which memory or learning performance is studied. For example, the novel-object-recognition task examines the extent to which an animal remembers a familiar object that was already in the arena the day before.

Elevated-Plus-Maze Test

The Elevated-Plus-Maze test is another fear test. The maze consists of two open arms and two closed arms. Since rodents hide from predators by instinct, they stay more in the closed arms. Here, the time animals spend on the open arms can be measured as a parameter for fear behavior. Brave animals spend more time on the open arms than fearful ones.

Novelty-Supressed-Feeding

A behavioral test to examine exploration and fear behavior is the Novelty-Supressed-Feeding test. To keep the mice motivated, they are given no food for one day. However, water is always available to them without limitation. In the open-field arena, a food pellet is placed in the center during performance. This serves to bring the animals into conflict: The food is in the dangerous part of the arena, the center. This test measures, among other things, the time it takes the mice to actually start eating. The less fearful the mice are, the faster they start eating.

Barnes Maze

The Barnes Maze test tests spatial memory and learning. The maze consists of a circular platform with 20 holes of equal size distributed around its perimeter. Underneath one hole is an escape box. Due to the natural instinct to hide from predators, the mouse tries to find the box as quickly as possible. For four consecutive days, the mice are trained to find this box. On the fifth day, the box is removed and the mouse's behavior is studied: The faster the mouse finds the place where the box was located and the longer the mouse stays at this place, the better its spatial memory.

Depression models

In order to get as comprehensive a picture as possible of depressive symptomatology in relation to serotonin, two established depression models are used in the research group: The Chronic Mild Stress Test and the Chronic Social Defeat Test. The combination of both models offers the possibility to investigate both the consequences of stress due to physical environmental influences and the consequences of social stress and its effect on depressive symptomatology and the influence of serotonin on these symptoms.

In the chronic mild stress test, the experimental animals are exposed to different mild physical stressors, such as wet, or dirty litter, crooked cages, or noise, on a daily basis for a fixed period of time (six weeks). This chronic stress leads to long-term depression-associated symptoms in the experimental mice, such as anhedonia or poorer grooming. Anhedonia occurs as a core symptom of depression in both mice and depressed humans. To experimentally determine anhedonia, the so-called sugar preference test is performed. Since anhedonia means the loss of pleasure, an anhedonic mouse no longer feels pleasure from eating something sweet. If a mouse with anhedonia is now offered both a drinking bottle with water and a drinking bottle with a sugar solution, this mouse no longer has a preference for the sugar water.

In addition to sugar preference, body weight and coat grooming, i.e., grooming behavior and coat condition, are examined, as these are also indicators of depressive symptoms.

The Chronic Social Defeat Test, on the other hand, is a behavioral test suitable for examining the social component of depressive symptomatology. People suffering from depression often withdraw from their social environment. This behavior also occurs after chronic social stress in mice and is considered a depressive symptom. In this experimental model, the experimental animal is exposed to social stress for ten days. Daily, the experimental mouse is confronted with a foreign mouse of an aggressive mouse strain (aggressor mouse). Contact is physical for ten minutes and sensory only for the rest of the day. The two mice can continue to interact and smell each other during this time, and the cages are separated only by a Plexiglas wall that has holes in it. This is repeated for ten consecutive days. On the 11th day, the social behavior of the experimental mouse is examined in the Social Interaction (SI) test. This test lasts five minutes and takes place in the Open Field Arena. Here, we measure how long the experimental mouse interacts with a mouse it does not know (interaction mouse). In this test, the interaction mouse is separated from the Open Field Arena by a Plexiglas wall that has holes in it. A depressed mouse would spend less time overall in social interaction compared to a healthy control animal. In this depression model, there are also resilient animals that do not show depressive symptomatology. Using this model, researchers can look very closely at what distinguishes resilient mice from mice that are prone to depression.

Mouse Gambling Task (MGT).

The Mouse Gambling Task (MGT) is a behavioral test in which the decision making of mice is analyzed. The starting concept for this test is the so-called Iowa Gambling Task, an experimental paradigm for humans in which subjects have to make decisions with high uncertainty on positive and negative outcomes.

For mice, this is implemented with different reward levels. As a reward, the researcher:s use a strawberry milkshake, which the mice are very happy to eat.

The mouse has a choice of four options. The choices differ in the amount of reward and the probability of receiving a reward. There are always two advantageous choices where the amount of reward is less but the probability of being rewarded is higher. The other two choices are unfavorable, where the amount of reward is higher but the probability of being rewarded is much lower. Based on the percentage of advantageous choices, the mice undergoing this test can be divided into different groups. Using this behavioral test, researchers can investigate how addictive behaviors (repeated risky choices) arise, for example, and how they might be influenced by serotonin.

Alternative and replacement methods for certain animal experiments already exist, such as cell cultures, organoids, multi-organ chips, stem cell therapies, and many more. Wherever possible, these alternative methods are used by researchers (Replace). The German Animal Welfare Act even stipulates that animal experiments may only be carried out if no alternative methods are available. Many of the studies on molecular and cellular mechanisms of the serotonergic system are already carried out by the scientists in cell cultures. However, to study complex behaviors, such as decision making, social behavior, or anxiety and depression, it is necessary to conduct behavioral experiments in a living, behaving animal. Our brain and nervous system are among the most complex structures we know. The number of connections that nerve cells have with each other is greater than the number of all the stars in the universe. It will not be possible to simulate this circuitry and complexity until we have a far better understanding of how the brain works. For this, science currently still needs animal experimental research. So it is not yet possible to copy the brain and all its complexity in a petri dish or to simulate it on a computer.

Thus, researchers are always trying to keep the number of animals used as low as possible (Reduce). For example, they achieve this by analyzing as many parameters as possible in one animal. They also reduce the stress on the animals to a minimum (Refine). The use of anesthetics and painkillers effectively counteracts the development of pain and, as a matter of principle, those procedures are selected that represent the least stress for the animals for the investigation of the corresponding issue.