Optogenetic activation of mGluR1 signaling in the cerebellum induces synaptic plasticity.

Neuronal plasticity underlying cerebellar learning behavior is strongly associated with type 1 metabotropic glutamate receptor (mGluR1) signaling. Activation of mGluR1 leads to activation of the Gq/11 pathway, which is involved in inducing synaptic plasticity at the parallel fiber-Purkinje cell synapse (PF-PC) in form of long-term depression (LTD). To optogenetically modulate mGluR1 signaling we fused mouse melanopsin (OPN4) that activates the Gq/11 pathway to the C-termini of mGluR1 splice variants (OPN4-mGluR1a and OPN4-mGluR1b). Activation of both OPN4-mGluR1 variants showed robust Ca2+ increase in HEK cells and PCs of cerebellar slices. We provide the prove-of-concept approach to modulate synaptic plasticity via optogenetic activation of OPN4-mGluR1a inducing LTD at the PF-PC synapse in vitro. Moreover, we demonstrate that light activation of mGluR1a signaling pathway by OPN4-mGluR1a in PCs leads to an increase in intrinsic activity of PCs in vivo and improved cerebellum driven learning behavior.

Reverse optogenetics of G protein signaling by zebrafish non-visual opsin Opn7b for synchronization of neuronal networks.

Opn7b is a non-visual G protein-coupled receptor expressed in zebrafish. Here we find that Opn7b expressed in HEK cells constitutively activates the Gi/o pathway and illumination with blue/green light inactivates G protein-coupled inwardly rectifying potassium channels. This suggests that light acts as an inverse agonist for Opn7b and can be used as an optogenetic tool to inhibit neuronal networks in the dark and interrupt constitutive inhibition in the light. Consistent with this prediction, illumination of recombinant expressed Opn7b in cortical pyramidal cells results in increased neuronal activity. In awake mice, light stimulation of Opn7b expressed in pyramidal cells of somatosensory cortex reliably induces generalized epileptiform activity within a short (<10 s) delay after onset of stimulation. Our study demonstrates a reversed mechanism for G protein-coupled receptor control and Opn7b as a tool for controlling neural circuit properties.

Design of an Ultrafast G Protein Switch Based on a Mouse Melanopsin Variant.

The primary goal of optogenetics is the light-controlled noninvasive and specific manipulation of various cellular processes. Herein, we present a hybrid strategy for targeted protein engineering combining computational techniques with electrophysiological and UV/visible spectroscopic experiments. We validated our concept for channelrhodopsin-2 and applied it to modify the less-well-studied vertebrate opsin melanopsin. Melanopsin is a promising optogenetic tool that functions as a selective molecular light switch for G protein-coupled receptor pathways. Thus, we constructed a model of the melanopsin Gq protein complex and predicted an absorption maximum shift of the Y211F variant. This variant displays a narrow blue-shifted action spectrum and twofold faster deactivation kinetics compared to wild-type melanopsin on G protein-coupled inward rectifying K+ (GIRK) channels in HEK293 cells. Furthermore, we verified the in vivo activity and optogenetic potential for the variant in mice. Thus, we propose that our developed concept will be generally applicable to designing optogenetic tools.

CaMello-XR enables visualization and optogenetic control of Gq/11 signals and receptor trafficking in GPCR-specific domains.

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.