Selected publications
BioRxiv 2024
PinkyCaMP a mScarlet-based calcium sensor with exceptional brightness, photostability, and multiplexing capabilities
Here, we present PinkyCaMP, the first mScarlet-based Ca2+ sensor that outperforms current red fluorescent sensors in brightness, photostability, signal-to-noise ratio, and compatibility with optogenetics and neurotransmitter imaging. PinkyCaMP is well-tolerated by neurons, showing no toxicity or aggregation, both in vitro and in vivo. All imaging approaches, including single-photon excitation methods such as fiber photometry, widefield imaging, miniscope imaging, as well as two-photon imaging in awake mice, are fully compatible with PinkyCaMP.
Fluorescence lifetime imaging of sDarken as a tool for the evaluation of serotonin levels
Recent advances in the development of genetically encoded biosensors have resulted in a variety of different neurotransmitter sensors for the precise measurement of the dynamics of neurotransmitters, neuromodulators, peptides and hormones in real time. However, intensity-based measurements of fluorescent biosensors are limited by their dependence on the expression level of the sensor, the intensity of the excitation light, and photobleaching overtime. Here, we show that the FLIM of sDarken (a GPCR-based genetically encoded sensor for serotonin) decreases with increasing serotonin concentrations. Different members of the sDarken family, with different affinities for serotonin, show concentration-dependent changes in fluorescence lifetime according to their dynamic range. We believe that this feature of sDarken is a value-adding complement to intensity-based information and may lead to a better understanding of serotonin dynamics in health and disease.
BioRxiv 2024
Nature Communications
2022
Next generation genetically encoded fluorescent sensors for serotonin
We developed a family of genetically encoded serotonin (5-HT) sensors (sDarken) on the basis of the native 5-HT1A receptor and circularly permuted GFP. sDarken 5-HT sensors are bright in the unbound state and diminish their fluorescence upon binding of 5-HT. Sensor variants with different affinities for serotonin were engineered to increase the versatility in imaging of serotonin dynamics. Experiments in vitro and in vivo showed the feasibility of imaging serotonin dynamics with high temporal and spatial resolution. As demonstrated here, the designed sensors show excellent membrane expression, have high specificity and a superior signal-to-noise ratio, detect the endogenous release of serotonin and are suitable for two-photon in vivo imaging.
Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice
Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of single neurons and region-specific transmitter release, up to whole neuronal circuitries in the central nervous system, and allows the direct measurement of behavioral outcomes. Neurons express optogenetic tools via an injection of viral vectors carrying the DNA of choice, such as Channelrhodopsin2 (ChR2). The plasticity of neuronal networks can also be investigated in great detail through long-term stimulation or behavioral observations after optical stimulation. Optogenetics will help to enlighten neuronal signaling in several kinds of neurological diseases.
JoVE
2020
Curr. Biol 2016
Melanopsin Variants as Intrinsic Optogenetic On and Off Switches for Transient versus Sustained Activation of G Protein Pathways
G-protein-coupled receptors (GPCRs) represent the major protein family for cellular modulation in mammals. However, a tool that combines precise control of the activation and deactivation of GPCR pathways and/or neuronal firing with limited phototoxicity is still missing. We compared the biophysical properties and optogenetic application of a human and a mouse melanopsin variant (hOpn4L and mOpn4L) on the control of Gi/o and Gq pathways in heterologous expression systems and mouse brain. We found that GPCR pathways can be switched on/off by blue/yellow light. The proteins differ in their kinetics and wavelength dependence to activate and deactivate G protein pathways. Whereas mOpn4L is maximally activated by very short light pulses, leading to sustained G protein activation, G protein responses of hOpn4L need longer light pulses to be activated and decline in amplitude. Based on the biophysical properties, hOpn4L and mOpn4L represent the first GPCR optogenetic tools, which can be used to switch GPCR pathways/neuronal firing on an off with temporal precision and limited phototoxicity. We suggest to name these tools moMo and huMo for future optogenetic applications.
Vertebrate cone opsins enable sustained and highly sensitive rapid control of Gi/o signaling in anxiety circuitry
G protein-coupled receptors (GPCRs) coupling to Gi/o signaling pathways are involved in the control of important physiological functions, which are difficult to investigate because of the limitation of tools to control the signaling pathway with precise kinetics and specificity. We established two vertebrate cone opsins, short- and long-wavelength opsin, for long-lasting and repetitive activation of Gi/o signaling pathways in vitro and in vivo. We demonstrate for both opsins the repetitive fast, membrane-delimited, ultra light-sensitive, and wavelength-dependent activation of the Gi/o pathway in HEK cells. We also show repetitive control of Gi/o pathway activation in 5-HT1A receptor domains in the dorsal raphe nucleus (DRN) in brain slices and in vivo, which is sufficient to modulate anxiety behavior in mice. Thus, vertebrate cone opsins represent a class of tools for understanding the role of Gi/o-coupled GPCRs in health and disease.
Neuron
2014
Complete list of publications
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