Synthetic biology: Next generation genetically encoded fluorescent sensors for serotonin

Synthetic Biology: Serotonin Enters, Light Exits
Characterization in HEK cells. a Design principle of sDarken. b Screening result of linker variants. Bars indicate fluorescence changes upon application of serotonin. Blue bar represents mutant M34. c Expression of mutant M34 (sDarken) in HEK cells. Scale bar 20 µm. Fluorescence before and after the application of 800 nM 5-HT. Representative fluorescence trace of sensor variant M34 expressing HEK cell during the application of 800 nM 5-HT. Experiments were at least repeated three times. d (i) Representative image sequence of repetitive 5-HT application via wash in. (ii) Example fluorescence measurement, repetitive application of 5 µM 5-HT in 9 example cells in two dishes. Application of 5-HT leads to a reversible reduction of fluorescence. No baseline corrections were performed. (iii) Heatmap of all analyzed ROIs. Scale bar in the first frame applies to all following frames. Scale bar 20 µm. e Example trace of 5-HT uncaging (405 nm, laser power: 90%, bleaching period: 90 ms, interval: 50 s) evoked sDarken responses. Blue circle indicates uncaging) area. White line indicates the part of the membrane that was used for analysis. Fluorescence signal of sDarken. ΔF/F values of part of membrane (white line) over time (exemplary trial). Blue bars indicate uncaging (i.e., bleaching) intervals. Repetitive uncaging of 5-HT resulted in decreasing ΔF/F values of sDarken. f Fast 5-HT application to an outside-out patch. Representative image from an outside-out patch containing sDarken 48 h post transfection (left). Fluorescence intensity changes in the patch. The fluorescence recovery after 5-HT wash out is significantly slower than the activation. No background/baseline corrections were performed. g Dose response curve measured in response to different 5-HT concentrations. Group data n = 20 from at least 2 replicates, mean and ±SEM are shown. h Fluorescence changes after application of serotonin, related substances or neurotransmitters (if not mentioned differently, concentration 10 µM (n = 10). Box represents the 25% percentile to the 75% percentile. The line in the middle of the box represents the median. One-Way ANOVA Multiple Comparison, ***p < 0.001, 5-HT serotonin, 8-OH 8-OHDPAT, L-Tryp tryptophan, IAA 5-hydroxyindoleacetic acid, Dopa dopamine, His histidine, NE norepinephrine, GABA Glu glutamate, Ace acetylcholine, PBS phosphate buffer. Credit: Nature Communications (2022). DOI: 10.1038/s41467-022-35200-w

Serotonin is an important chemical messenger in the central nervous system. It falls under the category of so-called neuromodulators and is responsible for transmitting information between nerve cells in the brain and the entire body.

In particular, the assumption that anxiety and depression are caused by a deficiency in the brain regularly puts this chemical messenger in the spotlight in science. However, causal relationships between and serotonin deficiency have not yet been sufficiently described. Suitable methods have been lacking so far.

Better understanding of signal exchange between nerve cells

"Various genetically encoded sensors have been developed in recent years in order to monitor the release of neurotransmitters more closely," says lead author Martin Kubitschke from the Synthetic Biology working group at the University of Bremen.

"While other teams focused on the chemical messengers glutamate, dopamine, norepinephrine, and acetylcholine, we wanted to understand exactly what functions serotonin has in the brain. When we started developing the sensors, there was no way to see serotonin live in the brain."

The researchers use a naturally occurring human 5-HT1A receptor as the framework for the new "sDarken" sensors. The receptor was genetically modified with the help of a green fluorescent protein to develop fluorescent properties and glow green. The decisive mechanism behind this: As soon as serotonin docks at the sensors, its luminosity decreases.

Darkening thus gives a direct indication of the activity of serotonin. Since it is a genetically encoded sensor, any type of nerve cell or brain region can be equipped with this sensor to visualize serotonin dynamics.

Fast reaction speed, good position, high luminosity

What was remarkable was the very good resolution, which was achieved both temporally and spatially during measurements. "With the help of patch-clamp fluorometry, we were able to determine that 'sDarken' reacts to changes in serotonin concentration in much less than a second," says Professor Andreas Reiner from the Faculty of Biology and Biotechnology at Ruhr-Universität Bochum.

"The fact that 'sDarken' is excellently positioned in the membrane of helps us to determine the spatial activity of chemical messengers," adds Professor Dr. Olivia Masseck, corresponding author and head of the Synthetic Biology working group at the University of Bremen. "The 'sDarken' sensors work so well that we expect to be able to visualize activities on individual nerve cell spines in the future."

The sensors are extremely sensitive and specific: even the tiniest concentrations of serotonin led to an optical change in the sensors and were therefore able to be measured by the scientists. "sDarken" did not react when other neurotransmitters or similar substances were administered. In addition, "sDarken" proved to be extremely robust, photo- and pH-stable and thus fulfilled important requirements for long-term imaging.

"By means of targeted mutation, we have developed three different variants of 'sDarken,' which react differently to serotonin," continues Martin Kubitschke. "It is advantageous to use a sensor with a very high affinity to detect so-called volume transfer in neurotransmitters. If, on the other hand, the focus is on serotonin secretions via individual synapses, a sensor with a lower affinity is advantageous. All these characteristics are now covered by the 'sDarken' sensors."

Ideal supplement to existing serotonin sensors

Its properties make "sDarken" an ideal addition to existing serotonin sensors and expand the range of instruments for imaging serotonin dynamics. In cooperation with the research teams of the Ruhr-Universität Bochum (Prof Andreas Reiner), the DZNE in Bonn (Dr. Martin Fuhrmann) and the ZNMH in Hamburg (Prof Simon Wiegert), it has already been shown that even fine details of serotonin release can be observed with "sDarken" in active animals.

Olivia Masseck is therefore convinced: "The new will make it possible to better understand the role and function of serotonin in the brain in the long term."

The research is published in the journal Nature Communications.

More information: Martin Kubitschke et al, Next generation genetically encoded fluorescent sensors for serotonin, Nature Communications (2022). DOI: 10.1038/s41467-022-35200-w

Journal information: Nature Communications

Provided by Universität Bremen
Citation: Synthetic biology: Next generation genetically encoded fluorescent sensors for serotonin (2022, December 16) retrieved 28 March 2023 from
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