Optogenetic Control of Motor Coordination by Gi/o Protein-coupled Vertebrate Rhodopsin in Cerebellar Purkinje Cells

Abstract

The G protein-mediated signaling pathway provides a pivotal module for the adjustment of neuronal networks against physiological or behavioral tasks on a second to minute time scale (1). Among G proteins, the G i/o -mediated signaling pathway is the primary role in which GPCRs 2 mediate their inhibitory action on neuronal excitability (2). The processes and importance of such modulation in cellular and network functions has mainly been investigated with the application of drugs, activating or inhibiting more or less specifically a certain GPCR pathway. Recently, we demonstrated that light-activated vertebrate rhodopsin (vRh) is a suitable alternative to control ion conductances such as G protein-coupled inward rectifying K ϩ channel and voltage-gated Ca 2ϩ channels via pertussis toxin-sensitive G i/o protein-mediated signaling (3). Therefore, vRh may allow for the precise spatiotemporal control of G i/o -mediated pathway in vivo, leading to an investigation that focuses on the function of this pathway in animal behavior or brain functions such as motor coordination.The cerebellum plays a central role in overall motor coordination and motor learning. An extensive array of GPCRs is expressed throughout the brain and is believed to be involved in the modulation of network activity and synaptic plasticity. It has been recognized that the code for motor coordination and balance lies within the firing cadence and output pattern of cerebellar PCs, which are the sole-output neurons from the cerebellar cortex (4, 5). PCs integrate a range of cortical, vestibular, and sensory information via excitatory synaptic input from parallel and climbing fiber pathways and inhibitory synaptic input originating from neighboring interneurons. The PC firing pattern is determined by several factors that include the interplay between excitatory and inhibitory synaptic inputs, several ion channel conductances that support intrinsic firing properties, and modulation by postsynaptic GPCRs like the GABA B receptor (GABA B R) (6 -8). GABA B R activation by application of the selective agonist baclofen leads to a reduction in PC firing most likely due to membrane hyperpolarization induced by G protein-coupled inward rectifying K ϩ channel activation (9 -12). The exact mechanism in which G i/o -mediated GPCR modulation may occur within PCs and how such modulation may influence the single spike pattern and motor coordination has been difficult to address in vivo, as GABA B Rs and other G i/o -coupled receptors are expressed in various cell types in the cerebellum and can only be activated by slowly diffusing drugs.To overcome the kinetic and spatial issues that the pharmacological approach presents and to investigate the functional impact of G i/o protein-mediated modulation on cerebellar function via spike modulation in cerebellar PCs, we created an optogenetic mouse model for the cell type-specific expression of vRh and demonstrated that spike modulation of PCs affects motor coordination. EXPERIMENTAL PROCEDURES Generation and Screening of Tra...

Optically triggering spatiotemporally confined GPCR activity in a cell and programming neurite initiation and extension

“…In addition, nonrhodopsin opsins selectively activate different G-protein types, suggesting that they can be individually used to regulate distinct second messengers (4,5). Rhodopsin or its chimeric forms have been valuable for globally activating G proteins (6)(7)(8). However, we found that these receptors do not allow spatial control over G-protein activation in a single cell.…”

FEF inactivation with improved optogenetic methods

“…Specifically, we report absorption coefficients two-to threefold larger than the largest reported in a recent study of fresh and frozen brain slices (30) and about 10-fold larger than the values that Yaroslavsky et al (29) reported based on measurements taken in postmortem human tissue. Unfortunately, the ex vivo values of Yaroslavsky et al (29) have been used widely in the optogenetics literature (26,28,53,(74)(75)(76)(77)(78) because, before the present study, techniques were not available for in vivo measurements of light propagation in living brain tissue across the full spectrum of visible light. The absorption coefficient that we determined for red light falls squarely within the range of reported values from extracted tissue and one in vivo study (i.e., μ a = 0.20-4.5 cm −1 ) (29, 30, 79-83).…”

Vision Science: Can Rhodopsin Cure Blindness?

“…This work also raises some fascinating physiology questions. While rhodopsin has been known to be a promiscuous G-protein coupled receptor [14,15], its efficiency in signal transduction in the bipolar and other inner retinal cells is remarkable. What G-protein is mediating these effects, and will there be compensatory changes in its signaling with chronic expression of the receptor?…”