[
J Neurochem,
2000]
We have previously identified two G protein-linked acetylcholine receptors (GARs), GAR-1 and GAR-3, in the nematode Caenorhabditis elegans. Whereas GAR-3 is a homologue of muscarinic acetylcholine receptors (mAChRs), GAR-1 is similar to but pharmacologically distinct from mAChRs. In the current work we isolated a new type of GAR using C. elegans genome sequence information. This receptor, named GAR-2, consists of 614 amino acid residues and has seven putative transmembrane domains. Database searches indicate that GAR-2 is most similar to GAR-1 and closely related to GAR-3/mAChRs. The overall amino acid sequence identities to GAR-1 and GAR-3 are approximately 32 and approximately 23%, respectively. When GAR-2 was coexpressed with the G protein-activated inwardly rectifying K(+) (GIRK1) channel in XENOPUS: oocytes, acetylcholine was able to evoke the GIRK current in a dose-dependent fashion. Oxotremorine, a classical muscarinic agonist, had little effect on the receptor, indicating that GAR-2 is pharmacologically different from mAChRs but rather similar to GAR-1. GAR-2 differs from GAR-1, however, in that it showed virtually no response to muscarinic antagonists such as atropine, scopolamine, and pirenzepine. Expression studies using green fluorescent protein reporter gene fusion revealed that GAR-2 is expressed in a subset of C. elegans neurons, distinct from those expressing GAR-1. Together with our previous reports, this study demonstrates that diverse types of GARs are present in C. elegans.
[
Methods Mol Biol,
2015]
Optogenetics was introduced as a new technology in the neurosciences about a decade ago (Zemelman et al., Neuron 33:15-22, 2002; Boyden et al., Nat Neurosci 8:1263-1268, 2005; Nagel et al., Curr Biol 15:2279-2284, 2005; Zemelman et al., Proc Natl Acad Sci USA 100:1352-1357, 2003). It combines optics, genetics, and bioengineering to render neurons sensitive to light, in order to achieve a precise, exogenous, and noninvasive control of membrane potential, intracellular signaling, network activity, or behavior (Rein and Deussing, Mol Genet Genomics 287:95-109, 2012; Yizhar et al., Neuron 71:9-34, 2011). As C. elegans is transparent, genetically amenable, has a small nervous system mapped with synapse resolution, and exhibits a rich behavioral repertoire, it is especially open to optogenetic methods (White et al., Philos Trans R Soc Lond B Biol Sci 314:1-340, 1986; De Bono et al., Optogenetic actuation, inhibition, modulation and readout for neuronal networks generating behavior in the nematode Caenorhabditis elegans, In: Hegemann P, Sigrist SJ (eds) Optogenetics, De Gruyter, Berlin, 2013; Husson et al., Biol Cell 105:235-250, 2013; Xu and Kim, Nat Rev Genet 12:793-801, 2011). Optogenetics, by now an "exploding" field, comprises a repertoire of different tools ranging from transgenically expressed photo-sensor proteins (Boyden et al., Nat Neurosci 8:1263-1268, 2005; Nagel et al., Curr Biol 15:2279-2284, 2005) or cascades (Zemelman et al., Neuron 33:15-22, 2002) to chemical biology approaches, using photochromic ligands of endogenous channels (Szobota et al., Neuron 54:535-545, 2007). Here, we will focus only on optogenetics utilizing microbial rhodopsins, as these are most easily and most widely applied in C. elegans. For other optogenetic tools, for example the photoactivated adenylyl cyclases (PACs, that drive neuronal activity by increasing synaptic vesicle priming, thus exaggerating rather than overriding the intrinsic activity of a neuron, as occurs with rhodopsins), we refer to other literature (Weissenberger et al., J Neurochem 116:616-625, 2011; Steuer Costa et al., Photoactivated adenylyl cyclases as optogenetic modulators of neuronal activity, In: Cambridge S (ed) Photswitching proteins, Springer, New York, 2014). In this chapter, we will give an overview of rhodopsin-based optogenetic tools, their properties and function, as well as their combination with genetically encoded indicators of neuronal activity. As there is not "the" single optogenetic experiment we could describe here, we will focus more on general concepts and "dos and don'ts" when designing an optogenetic experiment. We will also give some guidelines on which hardware to use, and then describe a typical example of an optogenetic experiment to analyze the function of the neuromuscular junction, and another application, which is Ca(2+) imaging in body wall muscle, with upstream neuronal excitation using optogenetic stimulation. To obtain a more general overview of optogenetics and optogenetic tools, we refer the reader to an extensive collection of review articles, and in particular to volume 1148 of this book series, "Photoswitching Proteins."