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Tools of the future

Optogenetics is a relatively new technique that enables scientists to manipulate nature with light. Light-sensitive proteins coupled to enzymes or channelrhodopsins embedded in membranes that guide ions across plasma membranes – all of these can be used to modulate cell behaviour. Researchers around the world are working on the emerging technique in order to refine and optimise it for application in their research projects. Optogenetics seems to be a popular toy and tool of neurobiologists, cell biologists and synthetic biologists alike. Prof. Dr. Ulrich Egert, neuroscientist and professor of biomicrotechnology in the Department of Microsystems Engineering at the University of Freiburg uses optogenetics to examine interactions between neural networks. Egert likens the current interest in optogenetics to a gold rush.

Prof. Dr. Ulrich Egert from the University of Freiburg believes that the rapid rate at which optogenetics papers are currently being published is, amongst other things, due to the fact that the discoverers of the tool have been very generous in passing on knowledge, thereby enabling the rapid application of the technique. “Everyone can try it out and use it for solving their specific research issues,” Egert said. “It is suitable for everyone, including neurobiologists.” Optogenetics uses light-gated transmembrane channels to trigger the flow of ions across the membrane, resulting in a change in a cell’s behaviour. Although it has taken quite a while for Egert and his team of researchers to get all the problems under control, the effort was worthwhile, as the benefits of optogenetics show.

While electrical stimulation can be used to excite neurons or neural networks in the brain, it is unable to inhibit or suppress neural activity. “Optogenetics is far better as it eliminates this drawback. When we use optogenetic methods to incorporate a light-sensitive channel protein into an inhibitory cell, we can evoke activity in neurons, and hence acute inhibition, simply by illumination with light,” said Egert. The method also enables researchers to target and influence certain cell groups and analyse the effect of cells that play a role in specific diseases, to name but one example.

Transfection with viruses

Light-sensitive ion pumps such as channelrhodopsins (CHR2) can be activated with blue light, Natromonas pharaonis halorhodopsins (NpHL) can be activated with yellow light. The neurons can be activated or inhibited depending on the type of light-sensitive channel protein used. © Modified from Marina Corral, Nature Methods 8, 24-25 (2011)
So how do I get a channelrhodopsin (CHR2) into a specific cell? “I will make the cell produce the molecule itself,” said the neuroscientist. “The advantage of this is that the expression of protein-coding genes and the optical sensor can be coupled to the expression of another molecule.” The required genetic material can be brought into the cell with viral particles whose genome has been removed and replaced with specific genetic information. The transgenic cell transcribes the foreign DNA, produces a protein and incorporates it into its membrane. Illumination only activates the cells carrying the construct. There is none of the background noise that is sometimes associated with the use of extracellular fluorescent labels. Light-sensitive proteins that are suitable for optogenetics include channel proteins such as channelrhodopsin and halorhodopsin as well as enzymes and signalling molecules that can be coupled to other proteins. Egert also uses chimeras like melanopsin that consist of channel- and signalling molecules. He is particularly interested in the dynamic of neural networks in the brain. “Our brain is not just a conglomeration of individual neurons that do whatever they want. Instead, it is the interaction between neurons that enable people to think, move, feel emotions, etc. Egert and his team hope to solve the question as to how the networks interact with each other and what happens when the network is interfered with.

Cell cultures provide better insights

Egert would like to find out the following: what does the interaction between the neural subnetworks mean, how can it be recognised and what does it depend on? “We have no models that give us a clear picture of the interaction between such networks. Optogenetics is an excellent tool for elucidating the issues we are trying to solve. Optogenetics can be used to manipulate neurons in subnetworks; the networks in the human brain are a series of independent neural networks that are moderated by some intermediary. “This enables us to identify the contribution of subpopulations to neural networks to which we would otherwise not have access. And this is done physiologically. When I activate or suppress the activity of these cells, the next cell does not see how I did it.”

Egert uses cell cultures derived from the murine neocortex for his optogenetic investigations. On this primitive level, the exact complex architecture of the brain does not play a big role. Therefore, it is much easier to study general parameters such as the probability of individual cells connecting with each other and the bundling of fibres. “The nice thing about using cell cultures is that it clearly enables us to pharmacologically, electrically and optically stimulate and study cells that research involving animals would not allow,” said Egert highlighting the advantage of using cell cultures.

Epilepsy and glial cells

Genetically manipulated astrocytes can produce a fluorescent dye. © Prof. Dr. Ulrich Egert, University of Freiburg

One scientific focus of Egert and his colleagues is to find out what happens in epileptic patients on the neuronal level. Subpopulations of cells that are normally embedded in larger networks gradually die in the epileptic focus, a small portion of the brain that drives the eptileptic response. Such network loops are found in the hippocampus; their interaction is disturbed in epileptic patients. The scientists are trying to understand how partly autonomous networks, including those that have many connections with each other, function. Egert and his team have found that glial cells not only surround neurons and hold them in place, but that they also remove excess neurotransmitters. 

In addition to neural networks, there are glial cell networks that seem to have their own dynamic. This dynamic can be effectively studied with optogenetic methods. The researchers assume that glial cells attenuate the activity of the much faster neurons, thereby acting as a kind of adjustment station that breaks down in epileptic brains. “Optogenetics enables us to use genes that are only transcribed in specific glial cells, namely astrocytes, as switches,” Egert said. “We can thus manipulate their function, and achieve considerable changes in network activity.”

Strength and weaknesses of optogenetics

Every tool has its pitfalls. Working with optogenetic tools requires light-dependent processes that are completely interference-free. The simultaneous measurement of calcium with fluorescent markers can lead to the unwanted activation of channelrhodopsins. In addition, optogenetic tools only produce their maximum effect when the right concentration of light sensor is present in the cell. Too high a concentration causes the molecules to agglutinate and become ineffective. Moreover, the exact number of transfected neurons is not known; it might be 5 percent and it might on occasions be 50 percent. This is a big difference and has an effect on the results. Despite these pitfalls, Egert is nevertheless optimistic: “Optogenetics will soon become a standard neuroscience tool, even though everything is not yet working 100% as it should.”

Further information:
Prof. Dr. Ulrich Egert
Department of Microsystems Engineering (IMTEK)
University of Freiburg
Georges-Köhler-Allee 102
79110 Freiburg
Tel.: +49 (0)761 / 203 - 7524
Fax.: +49 (0)761/ 203 - 97759
E-mail: egert(at)imtek.uni-freiburg.de

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/tools-of-the-future