Microbial Rhodopsin Optogenetic Tools: Application for Analyses of Synaptic Transmission and of Neuronal Network Activity in Behavior.

Over the past 15 years, optogenetic methods have revolutionized neuroscientific and cell biological research, also in the nematode Caenorhabditis elegans. In this chapter, we give an update about current optogenetic tools and methods to address neuronal activity and inhibition, as well as second messenger signaling, based on microbial rhodopsins. We address channelrhodopsins and variants thereof, which conduct cations or anions, for depolarization and hyperpolarization of the membrane potential. Also, we cover ion pumping rhodopsins, like halorhodopsin, Mac, and Arch. A recent addition to rhodopsin-based optogenetics is voltage imaging tools that allow fluorescent readout of membrane voltage (directly, via fluorescence of the rhodopsin chromophore retinal, or indirectly, via electrochromic FRET). Last, we report on a new addition to the optogenetic toolbox, which is rhodopsin guanylyl cyclases, as well as mutated variants with specificity for cyclic AMP. These can be used to regulate intracellular levels of cGMP and cAMP, which are important second messengers in sensory and other neurons. We further show how they can be combined with cyclic nucleotide-gated channels in two-component optogenetics, for depolarization or hyperpolarization of membrane potential. For all tools, we present protocols for straightforward experimentation to address neuronal activation and inhibition, particularly at the neuromuscular junction, and for combined optogenetic actuation and Ca2+ imaging. We also provide protocols for usage of rhodopsin guanylyl and adenylyl cyclases. Finally, we list a number of points to consider when designing and conducting rhodopsin-based optogenetic experiments.

Photoactivated Adenylyl Cyclases as Optogenetic Modulators of Neuronal Activity.

In the past 15 years, optogenetic methods became invaluable tools in neurobiological research but also in general cell biology. Most prominently, optogenetic methods utilize microbial rhodopsins to elicit neuronal de- or hyperpolarization. However, other optogenetic tools have emerged that allow influencing neuronal function by different approaches. In this chapter we describe the use of photoactivated adenylyl cyclases (PACs) as modulators of neuronal activity. Using Caenorhabditis elegans as a model organism, this chapter shows how to measure the effect of PAC photoactivation by behavioral assays in different tissues (neurons and muscles), as well as their significance to neurobiology. Further, this chapter describes in vitro cyclic nucleoside-3',5'-monophosphate measurements (cNMP) to characterize new PACs in C. elegans.

Optogenetic tools for manipulation of cyclic nucleotides functionally coupled to cyclic nucleotide-gated channels.

BACKGROUND AND PURPOSE: The cyclic nucleotides cAMP and cGMP are ubiquitous second messengers regulating numerous biological processes. Malfunctional cNMP signalling is linked to diseases and thus is an important target in pharmaceutical research. The existing optogenetic toolbox in Caenorhabditis elegans is restricted to soluble adenylyl cyclases, the membrane-bound Blastocladiella emersonii CyclOp and hyperpolarizing rhodopsins; yet missing are membrane-bound photoactivatable adenylyl cyclases and hyperpolarizers based on K+ currents. EXPERIMENTAL APPROACH: For the characterization of photoactivatable nucleotidyl cyclases, we expressed the proteins alone or in combination with cyclic nucleotide-gated channels in muscle cells and cholinergic motor neurons. To investigate the extent of optogenetic cNMP production and the ability of the systems to depolarize or hyperpolarize cells, we performed behavioural analyses, measured cNMP content in vitro, and compared in vivo expression levels. KEY RESULTS: We implemented Catenaria CyclOp as a new tool for cGMP production, allowing fine-control of cGMP levels. We established photoactivatable membrane-bound adenylyl cyclases, based on mutated versions ("A-2x") of Blastocladiella and Catenaria ("Be," "Ca") CyclOp, as N-terminal YFP fusions, enabling more efficient and specific cAMP signalling compared to soluble bPAC, despite lower overall cAMP production. For hyperpolarization of excitable cells by two-component optogenetics, we introduced the cAMP-gated K+ -channel SthK from Spirochaeta thermophila and combined it with bPAC, BeCyclOp(A-2x), or YFP-BeCyclOp(A-2x). As an alternative, we implemented the B. emersonii cGMP-gated K+ -channel BeCNG1 together with BeCyclOp. CONCLUSION AND IMPLICATIONS: We established a comprehensive suite of optogenetic tools for cNMP manipulation, applicable in many cell types, including sensory neurons, and for potent hyperpolarization. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.