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.

G-protein-coupled receptors (GPCRs) represent the major protein family for cellular modulation in mammals. Therefore, various strategies have been developed to analyze the function of GPCRs involving pharmaco- and optogenetic approaches [1, 2]. 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 different biophysical properties, brief light activation of mOpn4L is sufficient to induce sustained neuronal firing in cerebellar Purkinje cells (PC), whereas brief light activation of hOpn4L induces AP firing, which declines in frequency over time. Most importantly, mOpn4L-induced sustained firing can be switched off by yellow light. 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.

Serotonin 2c receptors (5-HT 2c -Rs) are drug targets for certain mental disorders, including schizophrenia, depression, and anxiety. 5-HT 2c -Rs are expressed throughout the brain, making it difficult to link behavioral changes to circuit specific receptor expression. Various 5-HT-Rs, including 5-HT 2c -Rs, are found in the dorsal raphe nucleus (DRN); however, the function of 5-HT 2c -Rs and their influence on the serotonergic signals mediating mood disorders remain unclear. To investigate the role of 5-HT 2c -Rs in the DRN in mice, we developed a melanopsin-based optogenetic probe for activation of Gq signals in cellular domains, where 5-HT 2c -Rs are localized. Our results demonstrate that precise temporal control of Gq signals in 5-HT 2c -R domains in GABAergic neurons upstream of 5-HT neurons provides negative feedback regulation of serotonergic firing to modulate anxiety-like behavior in mice.S erotonin (5-hydroxytryptamine, or 5-HT) is an important modulator of anxiety circuits (1). The diverse effects of serotonin are mediated through various 5-HT receptors (5-HT-Rs), including 5-HT 1-7 -Rs (2). Recent pharmacologic and genetic studies have highlighted an important role of 5-HT 2c -Rs in anxiety disorders; however, the interpretation of physiological and behavioral data remains difficult owing to a lack of selective pharmacologic ligands (3).5-HT 2c -Rs are expressed in various cell types and brain regions of the anxiety circuit, including the amygdala and the dorsal raphe nucleus (DRN), a midbrain region containing high concentrations of 5-HT neurons. It has been suggested that 5-HT 2c -Rs are expressed in GABAergic neurons, and that 5-HT 2c -R activation may contribute to an inhibitory feedback control of 5-HT cell firing (4). The functional and behavioral consequences of such a possible inhibitory feedback mechanism for 5-HT firing have not yet been investigated, however.Unfortunately, current techniques for identifying the functions of 5-HT 2c -Rs in vertebrate brains are of limited value. For example, agonists and antagonists of 5-HT 2c -Rs are often unspecific, and their action is not restricted to a specific cell type. Complete and conditional knockouts of the receptor gene have limited control of developmental and compensation effects by other G-protein-coupled receptors (GPCRs), and none of the current techniques allows for the physiological control of the 5-HT 2c -R activation on a millisecond to second time scale.To overcome the limitations of pharmacologic and genetic approaches, we have developed a new light-activated GPCR based on vertebrate melanopsin (vMo). Both 5-HT 2c -Rs and vMo couple to the Gq signaling pathway (5, 6). To investigate the functional consequence of Gq signal activation in the cell types and cellular structures where 5-HT 2c -Rs are located, we virally expressed vMo carrying the C terminus (CT) of the 5-HT 2c -R in GABAergic neurons in the DRN. We found that light activation of vMo-CT 5-HT2c decreases the firing of 5-HT neurons and modulates anxiety behaviors in mice. O...

Controlling gain of cortical activity is essential to modulate weights between internal ongoing communication and external sensory drive. Here, we show that serotonergic input has separable suppressive effects on the gain of ongoing and evoked visual activity. We combined optogenetic stimulation of the dorsal raphe nucleus (DRN) with wide-field calcium imaging, extracellular recordings, and iontophoresis of serotonin (5-HT) receptor antagonists in the mouse visual cortex. 5-HT1A receptors promote divisive suppression of spontaneous activity, while 5-HT2A receptors act divisively on visual response gain and largely account for normalization of population responses over a range of visual contrasts in awake and anesthetized states. Thus, 5-HT input provides balanced but distinct suppressive effects on ongoing and evoked activity components across neuronal populations. Imbalanced 5-HT1A/2A activation, either through receptor-specific drug intake, genetically predisposed irregular 5-HT receptor density, or change in sensory bombardment may enhance internal broadcasts and reduce sensory drive and vice versa.

The signal specificity of G protein-coupled receptors (GPCRs) including serotonin receptors (5-HT-R) depends on the trafficking and localization of the GPCR within its subcellular signaling domain. Visualizing traffic-dependent GPCR signals in neurons is difficult, but important to understand the contribution of GPCRs to synaptic plasticity. We engineered CaMello (Ca2+-melanopsin-local-sensor) and CaMello-5HT2A for visualization of traffic-dependent Ca2+ signals in 5-HT2A-R domains. These constructs consist of the light-activated Gq/11 coupled melanopsin, mCherry and GCaMP6m for visualization of Ca2+ signals and receptor trafficking, and the 5-HT2A C-terminus for targeting into 5-HT2A-R domains. We show that the specific localization of the GPCR to its receptor domain drastically alters the dynamics and localization of the intracellular Ca2+ signals in different neuronal populations in vitro and in vivo. The CaMello method may be extended to every GPCR coupling to the Gq/11 pathway to help unravel new receptor-specific functions in respect to synaptic plasticity and GPCR localization.