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.

A comprehensive overview of the optokinetic reflex (OKR) in vertebrates is given. The main objective is to compare the asymmetry in optokinetic reactions when patterns presented to one eye move horizontally in temporo-nasal or naso-temporal directions. Different hypotheses concerning the evolution of this asymmetry or symmetry in monocular horizontal OKR are discussed.

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...

Serotonergic neurons project to virtually all regions of the central nervous system and are consequently involved in many critical physiological functions such as mood, sexual behavior, feeding, sleep/wake cycle, memory, cognition, blood pressure regulation, breathing, and reproductive success. Therefore, serotonin release and serotonergic neuronal activity have to be precisely controlled and modulated by interacting brain circuits to adapt to specific emotional and environmental states. We will review the current knowledge about G protein-coupled receptors and ion channels involved in the regulation of serotonergic system, how their regulation is modulating the intrinsic activity of serotonergic neurons and its transmitter release and will discuss the latest methods for controlling the modulation of serotonin release and intracellular signaling in serotonergic neurons in vitro and in vivo.

Narcolepsy is a sleep disorder caused by the loss of orexin (hypocretin)-producing neurons and marked by excessive daytime sleepiness and a sudden weakening of muscle tone, or cataplexy, often triggered by strong emotions. In a mouse model for narcolepsy, we previously demonstrated that serotonin neurons of the dorsal raphe nucleus (DRN) mediate the suppression of cataplexy-like episodes (CLEs) by orexin neurons. Using an optogenetic tool, in this paper we show that the acute activation of DRN serotonin neuron terminals in the amygdala, but not in nuclei involved in regulating rapid eye-movement sleep and atonia, suppressed CLEs. Not only did stimulating serotonin nerve terminals reduce amygdala activity, but the chemogenetic inhibition of the amygdala using designer receptors exclusively activated by designer drugs also drastically decreased CLEs, whereas chemogenetic activation increased them. Moreover, the optogenetic inhibition of serotonin nerve terminals in the amygdala blocked the anticataplectic effects of orexin signaling in DRN serotonin neurons. Taken together, the results suggest that DRN serotonin neurons, as a downstream target of orexin neurons, inhibit cataplexy by reducing the activity of amygdala as a center for emotional processing.

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...

Although optogenetics has revolutionized rodent neuroscience, it is still rarely used in other model organisms as the efficiencies of viral gene transfer differ between species and comprehensive viral transduction studies are rare. However, for comparative research, birds offer valuable model organisms as they have excellent visual and cognitive capabilities. Therefore, the following study establishes optogenetics in pigeons on histological, physiological, and behavioral levels. We show that AAV1 is the most efficient viral vector in various brain regions and leads to extensive anterograde and retrograde ChR2 expression when combined with the CAG promoter. Furthermore, transient optical stimulation of ChR2 expressing cells in the entopallium decreases pigeons’ contrast sensitivity during a grayscale discrimination task. This finding demonstrates causal evidence for the involvement of the entopallium in contrast perception as well as a proof of principle for optogenetics in pigeons and provides the groundwork for various other methods that rely on viral gene transfer in birds.