eleganshas been used to develop several models that allowed us to identify, quantify, and analyze many signaling components that regulate neuromuscular behaviors (68). still poorly understood. The soil-dwelling nematodeCaenorhabditis elegansuses 1000 GPCRs (5% of its genome; ref.1) expressed in its neurons to respond to environmental chemical, mechanical, and thermal stimuli, mediate synaptic function, reshape neural circuits, and modulate muscle activity, all of which affect its motor behavior. Components of the anciently evolved heterotrimeric G-protein-signaling pathways are highly conserved inC. eleganswith respect to their protein sequences, functions, and signaling mechanisms. For example, there are 21 G, 2 G, and 2 Gsubunits in G proteins ofC. elegans. Among the 21 subunits, GSA-1, GOA-1, EGL-30, and GPA-12 are orthologs of the corresponding mammalian Gfamilies Gs, Gi/o, Gqand G12, respectively (2,3). Together with its fully described shape, position, and connectivity of >300 neurons,C. elegansprovides a unique model Rabbit polyclonal to RAB1A to study the cellular and molecular mechanisms of G-protein signaling (4,5). Because of its relatively simple genetics,C. eleganshas been used to develop several models that allowed us to identify, quantify, and analyze many Pergolide Mesylate signaling components that regulate neuromuscular behaviors (68). In recent years, bacterial photoactivated channels (channel rhodopsin or ChR2; refs.912), ion pumps (halorhodopsin or Halo/NpHR; refs.1316), and enzymes (photoactivated adenylyl cyclase or PAC; refs.17,18) were introduced intoC. elegansto exert spatiotemporal control over excitation and inhibition of neurons or the onset of intra- and intercellular processes affecting specific behaviors. Approaches that introduced engineered protein chimeras of mammalian Rho and GPCRs (optoXRs; ref.19), vertebrate rhodopsin (vRh; ref.20), and a synthetic optogenetic transcription device (21) into mice were also used to control GPCR-mediated physiological processes. These optogenetic tools provide additional convenient experimental means and unparalleled opportunities to dissect cellular and molecular mechanisms regulating such behaviors (for review, see ref.22). However, some intrinsic properties of native or engineered photoreceptive proteins can also limit their applications. For example, ChR2 and NpHR directly depolarize or hyperpolarize host neurons rather than indirectly affecting neuronal activity through other cellular processes. Whether optogenetic studies of PAC-induced increase in cytosolic concentrations of cyclic adenosine monophosphate (cAMP)- and OptoXR-stimulated Gsor Gqsignaling apply to endogenous signaling pathways in live host organisms has yet to be shown. Therefore, establishing optogenetic approaches that can directly monitor heterogeneous GPCRs that functionally couple to specificC. elegansG proteins to affect downstream motor behavior would be highly desirable. The mammalian opsin family members rhodopsin (Rho) and melanopsin (Mo) are photoreceptive GPCRs found in specialized rod cells or photoreceptive ganglion cells (ipGCs), respectively (23). On photoactivation, Rho couples to transducin (Gt) for visual signal transductionin vivo(24) and also to Gi/oin vitro(25), both of which belong to the Gi/osubfamily. In contrast, Mo is believed to couple to Gqfor signaling that regulates circadian rhythms (23). AlthoughC. elegansavoids lethal exposure to short-wavelength light (26,27), this soil-inhabiting nematode does not possess vision. There are no known orthologs of Rho, Mo, or Gtin the genome ofC. elegans. Because Gi/oand Gqare conserved in the transparent body ofC. elegans, it is possible to heterologously express photoreceptors and directly activate endogenous Gi/oand Gqpathways in this live organism, thus identifying G protein signaling pathway components with high spatial and temporal precision. Here, we expressed bovine (b)opsin and human (h)Mo in the nervous system ofC. elegansand used optogenetic tools to directly monitor (b)Rho and (h)Mo coupling to Gi/oand Gqsignalingin vivo. We found that exposure to a pulse of low-dose visible light sufficed to trigger a sudden and transient loss of motility in (b)opsin-expressing animals, or initiate increased locomotion of (h)Mo-expressing worms. Both light-mediated motor behaviors depended on added 9-cis-retinal, an active chromophore of Rho, and required the presence of endogenous worm Gi/oand Gq-signaling components. == MATERIALS AND METHODS == == C. elegansstrains and maintenance == Bristol N2 strainC. elegansworms were used for this study. The loss-of-function mutantsgoa-1(sa734),egl-8(md1971),egl-30(md186),gsa-1(pk75),gpa-12(pk322),gpa-3(pk35),pde-6(ok3410),tax-2(ok3403),tax-4(p678),cng-1(jh111), andcng-3(jh113), the triple mutantcng-1(jh111);cng-3(jh113);tax-4(p678), and the quadruple mutantpde-1(nj57);pde-2(tm3098);pde-3(nj59);pde-5(nj49) were obtained from theCaenorhabditisGenetics Center (CGC; University of Minnesota, Minneapolis, MN, USA).Pde-4(nj60) was generated by crossing wild-type (WT) worms with the double-mutantpde-4(nj60);pde-6(ok3410) (provided by Dr. X. Z. Xu, University of Michigan, Ann Arbor, Pergolide Mesylate MI, USA). The quadruple mutantcng-1(jh111);cng-3(jh113);tax-2(ok3403);tax-4(p678) was generated by crossing thetax-2(ok3403) mutant with the triple Pergolide Mesylate mutantcng-1(jh111);cng-3(jh113);tax-4(p678). Each mutant worm Pergolide Mesylate line was crossed 3 times with WT if it had not been reported to be outcrossed extensively by the CGC. Primers used in mutation screens and the.