Abstract
Cannabis sativa, or marijuana, a popular recreational drug, alters sensory perception and exerts a range of potential medicinal benefits. The present study demonstrates that the endogenous cannabinoid receptor agonists 2-arachidonoylglycerol (2-AG) and anandamide (AEA) activate a canonical cannabinoid receptor in Caenorhabditis elegans and also modulate monoaminergic signaling at multiple levels. 2-AG or AEA inhibit nociception and feeding through a pathway requiring the cannabinoid-like receptor NPR-19. 2-AG or AEA activate NPR-19 directly and cannabinoid-dependent inhibition can be rescued in npr-19-null animals by the expression of a human cannabinoid receptor, CB1, highlighting the orthology of the receptors. Cannabinoids also modulate nociception and locomotion through an NPR-19-independent pathway requiring an α2A-adrenergic-like octopamine (OA) receptor, OCTR-1, and a 5-HT1A-like serotonin (5-HT) receptor, SER-4, that involves a complex interaction among cannabinoid, octopaminergic, and serotonergic signaling. 2-AG activates OCTR-1 directly. In contrast, 2-AG does not activate SER-4 directly, but appears to enhance SER-4-dependent serotonergic signaling by increasing endogenous 5-HT. This study defines a conserved cannabinoid signaling system in C. elegans, demonstrates the cannabinoid-dependent activation of monoaminergic signaling, and highlights the advantages of studying cannabinoid signaling in a genetically tractable whole-animal model.
SIGNIFICANCE STATEMENT Cannabis sativa, or marijuana, causes euphoria and exerts a wide range of medicinal benefits. For years, cannabinoids have been studied at the cellular level using tissue explants with conflicting results. To better understand cannabinoid signaling, we have used the Caenorhabditis elegans model to examine the effects of cannabinoids on behavior. The present study demonstrates that mammalian cannabinoid receptor ligands activate a conserved cannabinoid signaling system in C. elegans and also modulate monoaminergic signaling, potentially affecting an array of disorders, including anxiety and depression. This study highlights the potential role of cannabinoids in modulating monoaminergic signaling and the advantages of studying cannabinoid signaling in a genetically tractable, whole-animal model.
Introduction
Cannabis sativa, or marijuana, has long been a popular recreational drug because of its unique ability to alter sensory perception and cause euphoria. More importantly, marijuana also has been reported to exert a wide range of medicinal effects (Pacher et al., 2006). Cannabis contains >60 bioactive compounds, or phytocannabinoids, the two most common being Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). In addition, the endogenous cannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide or AEA) are synthesized within the brain and CNS. Cannabinoids primarily activate Gαo-coupled cannabinoid receptors 1 and 2 (CB1 and CB2). CB1 is localized primarily to the brain and CNS (Herkenham et al., 1990; Glass et al., 1997; Martin et al., 1998), whereas CB2 is restricted to the periphery and certain leukocytes (Munro et al., 1993). Endocannabinoids and phytocannabinoids activate the same receptors and elicit similar cellular responses despite their structural differences. Both 2-AG and AEA mediate retrograde inhibition of synaptic neurotransmission via activation of CB1 on presynaptic membranes (Ohno-Shosaku and Kano, 2014). 2-AG or AEA inhibition is terminated by monoacylglycerol lipase (MAGL) or fatty acid amide hydroxylase (FAAH), respectively, with inhibition of either, eliciting analgesic and antinociceptive behavior (Piomelli et al., 2006; Long et al., 2009a; Long et al., 2009b).
Thus far, the majority of studies on cannabinoids have been conducted at the cellular level, sometimes using mammalian tissue explants to observe receptor activation. In contrast, our goal was to examine the role of cannabinoids on whole-animal behavior and to dissect the role of cannabinoid signaling in the modulation of sensory integration and downstream decision making. Therefore, the present study was designed to examine the effects of cannabinoid receptor agonists on nociceptive behaviors in the nematode (C. elegans) model system because CB1 appears to suppress pain in mammals (Sofia et al., 1973; Yaksh and Reddy, 1981; Tsou et al., 1995; Walker and Huang, 2002).
Our results demonstrate that mammalian cannabinoid receptor ligands activate a conserved cannabinoid signaling system in C. elegans and also modulate monoaminergic signaling, potentially affecting an array of disorders, including anxiety and depression. In contrast to published reports, C. elegans contains an endogenous canonical cannabinoid signaling system (McPartland and Glass, 2001; Pastuhov et al., 2016). Inhibiting the breakdown of endogenous 2-AG or AEA mimics 2-AG or AEA addition and inhibits nociception and feeding through a pathway that requires the cannabinoid-like receptor NPR-19. Cannabinoids activate NPR-19 directly and npr-19-null animals can be rescued by the expression of human CB1, confirming the orthology of the two receptors. In addition, higher exogenous cannabinoid levels also activate an α2A-adrenergic-like receptor (OCTR-1) and a 5-HT1A-like receptor (SER-4) to modulate both nociception and locomotion through NPR-19-independent pathways. Cannabinoids activate OCTR-1 directly when expressed heterologously in Xenopus laevis oocytes. In contrast, 2-AG does not activate SER-4 directly and cannabinoids appear to enhance SER-4-dependent serotonergic signaling by increasing endogenous serotonin (5-HT). This study highlights the potential role of cannabinoids in modulating monoaminergic signaling and the advantages of studying cannabinoid signaling in a genetically tractable, whole-animal model.
Materials and Methods
Nematode strains and construction of C. elegans transgenes.
Strains were maintained as described in Brenner (1974). The following strains were used: N2 (Bristol), ckr-2 (tm3082), dop-1 (ok298), mod-5 (n3314), npr-3 (tm1583), npr-5 (ok1583), npr-19 (ok2068), npr-24 (ok3192), octr-1 (ok371), ser-2 (pk1357), ser-4 (ok512), and tph-1 (n4622). RNAi transgenes were generated by PCR fusion as described in Esposito et al. (2007) and coinjected with f25b3.3::gfp (to 100 ng). The octr-1 (+), npr-19 (+) full-length genomic and npr-19::npr-19::gfp transcriptional transgenes were generated by PCR fusion and coinjected with f25b3.3::gfp (to 50 ng). The npr-19::gfp transcriptional transgene was constructed by PCR fusion of 1.5 kb npr-19 promoter including the first intron fused to gfp::unc-54 3′′-UTR and coinjected with unc-122::rfp (to 50 ng). The npr-19::CNR1::gfp transgene was generated by 3-piece PCR fusion of the npr-19 promoter including the first intron, full-length human CNR1 cDNA, and gfp::unc-54 3′′-UTR and were coinjected with unc-122::rfp (to 50 ng). unc-17β-driven transgenes were generated by PCR fusion of the unc-17β promoter (562 bp) to GPCR cDNA and gfp::unc-54 3′′-UTR and coinjected with unc-122::rfp (to 50 ng). npr-9::ser-4::gfp transgene was generated by PCR fusion using native npr-9 promoter and coinjected with unc-122::rfp (to 50 ng). PCR fusions were performed as described in Hobert (2002).
Octanol avoidance assays.
Octanol avoidance assays were performed as described in Chao et al. (2004) and as modified by Harris et al. (2011). For all behavioral assays, L4 stage animals were picked 24 h before assaying. 2-AG and AEA plates were prepared 10 min before assay by spreading 60 μl of 2-AG or AEA (in H2O) on fresh NGM plates. To measure aversive responses to 1-octanol, the blunt end of a hair was briefly dipped in 1-octanol and placed in front of a forward-moving worm and the time taken to initiate backward locomotion was recorded. Animals were first transferred to intermediate (nonseeded) plates, left for 30 s, transferred to assay plates, and tested after 10 min. In all assays, 20–25 worms were examined for each strain and condition and each assay was performed at least three times. Statistical analysis was performed using mean ± SE and Student's t test.
Heterologous expression and electrophysiology in X. laevis oocytes.
The human CB1 (CNR1), α2A-adrenergic (ADRA2A), 5-HT1A (HTR1A), GIRK1, GIRK2, and C. elegans npr-19, octr-1, and ser-4 cDNAs were cloned between Not1 and Age1 restriction enzyme sites into a Xenopus expression vector containing a T7 promoter and the Xenopus 5′ and 3′ β-globin UTRs to generate pxGIRK1, pxGIRK2, pxCNR1, pxADRA2A, pxHTR1A, pxser-4, pxoctr-1, and pxnpr-19, respectively. Linearized plasmids were transcribed using an Ambion mMessage mMachine T7 kit (Applied Biosystems). ADRA2A, CNR1, GIRK1, and GIRK2 cDNAs were from Addgene and HTR1A cDNA was from GE Healthcare. X. laevis oocytes were from Xenopus One and Nasco. Oocytes were separated mechanically before incubation in ND-96 (Ca2+ free) medium (96 mm NaCl, 2 mm KCl, 1 mm MgCl2, 5 mm HEPES, pH 7.6) containing 1 mg/ml collagenase type 1A (Sigma Aldrich) for 30 min. Defolliculated oocytes were separated and incubated in modified Barth's medium with 1 mm Na pyruvate, 0.01 mg/ml gentamicin, and 1× antibiotic–antimycotic (Invitrogen) at 16°C overnight. Receptor cRNAs were injected at 50 ng/50 nl and GIRK1 and GIRK2 channel cRNAs were injected at 0.5 ng/50 nl. Oocytes were incubated at 16°C for 48–72 h after injection and then transferred to 4°C. Two-electrode voltage-clamp (TEVC) recordings were performed 72 h after injection using an Axon Gene Clamp 500 Amplifier (Molecular Devices) as described previously (Stühmer, 1998; Bamber et al., 2003). For TEVC recordings, standard low K+ Ringer's solution (115 mm NaCl, 2.5 mm KCl, 1.8 mm CaCl2, 10 mm HEPES, pH 7.2) and a high K+ Ringer's solution (96 mm KCl, 2 mm NaCl, 1.8 mm CaCl2, 10 mm HEPES, pH 7.2) were applied by gravity perfusion. Ligands were applied by gravity perfusion initially at 1 μm. Oocytes coexpressing GIRK1/2 and GPCRs were perfused with intervals of increasing concentrations of 2-AG and AEA to determine ligand specificity and EC50. 2-AG and AEA dose–response curves were fitted with the equation: I − Imax/(1 + 10(log EC50−[agonist]) × n), where I is the current at a given 2-AG or AEA concentration, Imax is current at saturation, EC50 is the 2-AG and AEA concentration required to elicit half-maximal current, and n is the slope coefficient. Curve fitting was performed using GraphPad Prism software.
Confocal imaging.
To localize NPR-19, a transcriptional npr-19::gfp transgene was generated using 1.5 kb upstream of the predicted npr-19 start site, including the first intron. To identify a subset of amphid sensory neurons, animals were incubated in 5 μm 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD; Invitrogen) for 1 h and then transferred to a standard NGM plate seeded with OP50 for 1 h to destain. For neuronal identification, npr-19::gfp was coinjected with tph-1::rfp, tdc-1::rfp, flp-8::rfp, flp-18::rfp, or ceh-36::rfp. All imaging was performed on an Olympus IX81 inverted confocal microscope. Animals expressing the npr-19::gfp transgene were immobilized on agarose pads with 20 mm sodium azide and imaged for GFP/RFP/DiD fluorescence.
Pharyngeal pumping assay.
Pharyngeal pumping was assayed on NGM plates. 2-AG plates were prepared 10 min before assay by spreading 60 μl of 320 μm 2-AG (in H2O) on fresh, predried NGM plates. For all pumping assays, L4 animals were picked 24 h before assay. Animals were moved from food plates to either a nonseeded NGM plate for control or 2-AG plates and incubated for 10 min. During assay, locomotion was recorded using a Sony Exwave HAD color-video digital camera for 2 min. Videos were played back in slow motion and the number of pharyngeal pumps per minutes was counted. Statistical analysis was performed using mean ± SE and Student's t test.
Feeding assay.
Uptake of fluorescently labeled latex beads was performed as described in Kiyama et al. (2012). Fluoresbrite YG Microspheres were from Polysciences (1.00 μm; catalog #17154-10), diluted in ethanol, and stored at 4°C. Feeding plates were made by spreading 150 μl of M9 bead solution (1 × 108 microspheres/plate) and drying for 30 min. Wild-type and npr-19-null animals were incubated for 10 min on plates containing 2-AG, AEA, or no drug. Animals were transferred to bead plates ± 2-AG or AEA, allowed to feed for 30 min at room temperature, and then removed, washed with M9 to remove excess beads, and immobilized on agarose pads with 20 mm Na azide for imaging using an Olympus IX81 inverted confocal microscope. Images were analyzed using ImageJ. Statistical analysis was performed using mean ± SE and Student's t test.
Locomotory (body bend) assay.
Freshly poured agar plates (non-NGM) containing either 320 μm 2AG/AEA were used for assay. Well-fed, young adult hermaphrodite animals are picked before assay and maintained on NGM plates with E. coli OP50. During assay, seven animals were transferred to the assay plate. Motility was assessed as number of body bend/20 s at 5 min intervals for 30 min starting as soon as animals were transferred. Each strain was assayed at least three times with seven animals per assay. Statistical analysis was performed using mean ± SE and Student's t test.
Endocannabinoid compounds.
2-AG, AEA, JZL184, and URB597 were all from Tocris Bioscience and stock solutions are in DMSO or ethanol at 100 mm and are stored at −80°C.
Results
Endocannabinoids 2-AG and AEA inhibit aversive behavior
2-AG and AEA have been identified recently in C. elegans extracts by mass spectrometry (Lehtonen et al., 2011), but a simple BLAST search using the human cannabinoid receptor CB1 failed to identify any C. elegans receptors with significant identity to CB1, consistent with previous reports that C. elegans lacks clear mammalian cannabinoid receptor orthologs (McPartland and Glass, 2001; Pastuhov et al., 2016). In mammals, 2-AG and AEA exert antinociceptive action in models of acute inflammatory and neuropathic pain; therefore, we examined their effects on aversive responses to 1-octanol in C. elegans (Iskedjian et al., 2007; Clapper et al., 2010). This aversive decision-making circuit is mediated primarily by the two ASH sensory neurons and has been characterized extensively (Wragg et al., 2007; Harris et al., 2011; Mills et al., 2012). 2-AG and AEA inhibited the more rapid initiation of aversive responses to 100% 1-octanol in C. elegans (2-AG: t = 17.5, df = 16, p < 0.0001; AEA: t = 7.8, df = 8, p < 0.0001), with 2-AG exhibiting an EC50 of ∼1 μm (Fig. 1A,B). These relatively high concentrations of ligands were probably necessary to overcome the relative impermeability of the nematode cuticle.
C. elegans contains an endogenous cannabinoid signaling system requiring the cannabinoid receptor NPR-19. The initiation of aversive responses to 1-octanol was examined as described by Harris et al. (2009). A, 2-Arachidonoylglycerol (2-AG), anandamide (AEA), JZL184 (JZL), or URB697 (URB) inhibition of aversive responses to 1-octanol. B, 2-AG dose–response curve for wild-type animals. C, Screen for receptor-null animals resistant to 2-AG-dependent inhibition of aversive responses. D, Screen for receptor-null animals resistant to JZL184-dependent inhibition of aversive responses. E, npr-19 or CB1 expression driven by a minimal npr-19 promoter in npr-19-null animals. *Significantly different from wild-type animals in the absence of effector (p ≤ 0.05). Data are presented as a mean ± SE (n) and were analyzed by two-tailed Student's t test.
In mammals, the degradation of 2-AG and AEA and termination of signaling are initiated by a membrane-bound MAGL and FAAH, respectively (Long et al., 2009b). The predicted C. elegans proteins, Y97E10AL.2 and FAAH-1, exhibit significant sequence identity to human MAGL (39%) and FAAH (38%), respectively, and selective inhibitors are available for both predicted mammalian orthologs (Piomelli et al., 2006; Long et al., 2009a). As anticipated, the inhibition of either MAGL with JZL184 or FAAH with URB597, predicted to inhibit the degradation of 2-AG or AEA, respectively, mimicked 2-AG or AEA addition and inhibited aversive responses to 1-octanol (JZL184: t = 10.1, df = 11, p < 0.0001; URB597: t = 20.9, df = 7, p < 0.0001; Fig. 1A). Together, these results suggest that C. elegans contains an endogenous cannabinoid signaling system.
2-AG/JZL184 inhibition of aversive responses is absent in npr-19-null animals
To identify potential C. elegans cannabinoid receptors, we reexamined protein BLAST data using human CB1 and identified a number of previously characterized C. elegans monoamine receptors and predicted neuropeptide receptors, including NPR-19, with limited identity to CB1 (McPartland and Glass, 2001). To determine whether any of these receptors were required for the cannabinoid-mediated inhibition of aversive responses, we screened the appropriate null animals for loss of JZL184 or 2-AG-dependent inhibition of aversive responses (Fig. 1).
JZL184 or 2-AG still inhibited aversive responses in ckr-2-, dop-1-, npr-3-, octr-1-, ser-2-, and ser-4-null animals. In contrast, JZL184 or 2-AG inhibition was dramatically reduced in npr-19-null animals (2-AG: t = 10.8, df = 17, p < 0.0001; JZL184: t = 10.3, df = 12, p < 0.0001; Fig. 1C,D). Similarly, JZL184 or 2-AG inhibition was absent after npr-19 RNAi knockdown (2-AG: t = 10.2, df = 13, p < 0.0001; JZL184: t = 5.1, df = 7, p < 0.0001) using a predicted 1.5 kb npr-19 promoter (Fig. 1C,D). 2-AG inhibition could be rescued in npr-19-null animals by expression of a full-length npr-19 transgene driven by the predicted 1.5 kb promoter, including 1 kb of the npr-19 3′-UTR (Fig. 1E). In addition, wild-type animals overexpressing this npr-19 transgene mimicked the addition of 2-AG and initiated aversive responses more slowly than wild-type animals in the absence of 2-AG (t = 2.8, df = 12, p < 0.001; Fig. 1E). Importantly, 2-AG sensitivity in npr-19-null animals could also be rescued by the expression of CNR1 cDNA, the human CB1-encoding gene, driven by the npr-19 promoter described above, confirming the orthology of the two receptors (t = 8.8, df = 12, p < 0.0001; Fig. 1E).
As predicted, although NPR-19 and human CB1 exhibited only 23% sequence identity, many key amino acids involved in AEA binding appear to be conserved (Fig. 2). The residues delimiting the AEA-binding pocket are largely hydrophobic, based on both modeling and site-directed mutagenesis (Reggio, 2010), and include F189, L193, F379, and S383. All four residues were conserved in NPR-19 (Fig. 2). F189 interacts with the AEA amide oxygen and an F189A mutation in CB1 decreases AEA binding sixfold (McAllister et al., 2004). The AEA amide oxygen also interacts with a charged residue at position 192 (K in CB1, D in NPR-19) and the AEA hydroxyl forms a hydrogen bond with S383 (McAllister et al., 2003).
Comparison of CB1 and NPR-19 aa sequences. CB1/NPR-19 protein alignment. Conserved key amino acid residues involved in AEA binding (F189, L193, L192, F379, and S383) are highlighted in red; identical residues are bolded and indicated with an asterisk.
These data highlight the effective coupling of a human G-protein-coupled receptor to endogenous C. elegans G-proteins and strongly support the hypothesis that NPR-19 is a mammalian cannabinoid receptor ortholog.
2-AG and AEA activate NPR-19 heterologously expressed in Xenopus oocytes directly
To demonstrate that 2-AG/AEA activate NPR-19 directly, Xenopus oocytes were coinjected with npr-19 and GIRK1/2 cRNAs. GIRK1/2 encode inwardly rectify potassium channel subunits activated by G-protein βγ subunits and were coexpressed on the assumption that NPR-19 would be Gαo-coupled, based on the observation above that the Gαo-coupled human CB1 rescued aversive phenotypes in npr-19-null animals. As expected, 2-AG and AEA had no effect on oocytes expressing GIRK1/2 alone, but initiated robust inwardly rectifying currents in oocytes expressing NPR-19 (Fig. 3A,B), with EC50s of 395 ± 5.1 nm (Fig. 3C) and 14 ± 2.4 nm (Fig. 3D), respectively. The EC50s for 2-AG and AEA are in the range of EC50s reported for human CB1, 125 nm (Luk et al., 2004) and 89 nm (McAllister et al., 1999), respectively. Together, these data demonstrate that NPR-19 is a cannabinoid receptor.
2-Arachidonoylglycerol (2-AG) and anandamide (AEA) activate NPR-19 expressed in Xenopus laevis oocytes. Two-electrode voltage-clamp (TEVC) recordings were performed on oocytes expressing NPR-19 and GIRK1/2 subunits 72 h after injection, as described previously (Stühmer, 1998; Bamber et al., 2003). Representative traces are shown for NPR-19 activation by 2-AG (A) and AEA (B). IHK represents the current induced upon a switch from low-K+ to high-K+ Ringer's solution. ILigand represents the current induced after ligand application. NPR-19 dose–response curve is shown for 2-AG (C) and AEA (D). Data are represented as a mean ± SE (n).
NPR-19 is expressed in a limited number of neurons and inhibits pharyngeal pumping and feeding
Based on fluorescence from an npr-19::gfp transgene, NPR-19 is only expressed in a limited number of neurons, including the two inhibitory, glutamatergic M3 pharyngeal motorneurons (Fig. 4A,B) and the two URX sensory neurons (Fig. 4A,C) that play key modulatory roles in regulating pharyngeal pumping and avoidance behavior, respectively (Raizen and Avery, 1994; McGrath et al., 2009). As predicted, npr-19 RNAi knockdown in the URXs, using either the URX-selective gpa-8 or flp-8 promoters, mimicked the npr-19-null phenotype and significantly decreased 2-AG-dependent inhibition of aversive responses to 100% 1-octanol (gpa-8: t = 13.6, df = 10, p < 0.0001; flp-8: t = 16.8, df = 11, p < 0.0001; Fig. 4D). The inhibitory M3s repolarize pharyngeal muscle after contraction and ablation of the M3s decreases the rate of pharyngeal pumping and feeding (Raizen and Avery, 1994). Indeed, 2-AG or AEA also inhibited pharyngeal pumping of food (2-AG: t = 5.2, df = 5, p < 0.001; AEA: t = 5.7, df = 6, p < 0.001; Fig. 4E), although at higher concentrations than those required for the inhibition of nociception (320 vs 3.2 μm). In contrast to nociception, JZL184 or URB597 had no effect on pumping (Fig. 4E), presumably because of the higher cannabinoid levels required for inhibition.
NPR-19 is expressed in a limited number of neurons, including URX and M3. A–C, A transcriptional npr-19::gfp transgene was generated using 1.5 kb upstream of the predicted npr-19 start site, including the first intron. A, DiD-stained wild-type animal expressing npr-19::gfp. B, C, For neuronal identification, npr-19::gfp was coinjected with either flp-8::rfp or ceh-36::rfp. Wild-type animals coexpressing npr-19::gfp and either ceh-2::rfp (B) or flp-8::rfp (C) were used to identify M3s and URXs, respectively. D, Aversive responses to 1-octanol after selective npr-19 RNAi knockdown in the URXs via flp-8 or gpa-8 promoters. E, Concentration-dependent 2-arachidonoylglycerol (2-AG) and anandamide (AEA) inhibition of pharyngeal pumping. F, Pharyngeal pumping of npr-19-null and rescue animals. G, Effects of 2-AG/AEA on feeding as measured by the uptake of fluorescent beads, as described in Kiyama et al. (2012). H, Pharyngeal pumping after selective npr-19 RNAi knockdown in the M3s via egl-36 or glt-1 promoters. flp-8 and gpa-8 promoters drive expression in the two URXs and a limited number of other neurons; egl-36 and glt-1 promoters drive expression in the two M3s and a limited number of other neurons (Wormbase). *Significantly different from wild-type animals in the absence of effector (p ≤ 0.05). Data are presented as a mean ± SE (n) and were analyzed by two-tailed Student's t test.
These higher cannabinoid levels also inhibited feeding, as assessed by the uptake of fluorescently labeled latex beads (2-AG: t = 6.8, df = 4, p < 0.001; AEA: t = 16.6, df = 4, p < 0.0001; Fig. 4G) in wild-type animals. The cannabinoid-dependent inhibition of both pumping and feeding were npr-19 dependent and, as predicted, could be rescued by the expression of a full-length npr-19 transgene driven by the predicted 1.5 kb promoter, including 1 kb of the npr-19 3′-UTR [pumping (2-AG): t = 9.5, df = 11, p < 0.001; pumping (AEA): t = 10.3, df = 10, p < 0.0001; feeding (2-AG): t = 8.2, df = 10, p < 0.001; feeding (AEA): t = 9.0, df = 13, p < 0.0001; Figure 4F,G]. More specifically, npr-19 RNAi knockdown in the M3s using either the M3-selective glt-1 or egl-36 promoters, mimicked the npr-19-null phenotype and significantly decreased the 2-AG-dependent inhibition of pharyngeal pumping (Fig. 4H). To ensure that the neuron-specific RNAi phenotypes did not result from transgene overexpression, we expressed a dsgfp RNAi using the same promoters. As predicted, these RNAi transgenes had no effect on nociception or feeding (Fig. 4D,H). These data demonstrate key neuron-specific roles for cannabinoids and NPR-19 in the modulation of aversive behavior, pharyngeal pumping, and feeding.
At higher exogenous cannabinoid concentrations, both serotonin and octopamine (OA) receptors are required for the cannabinoid-dependent inhibition of nociception and locomotion
Because the recreational use of cannabinoids might elevate total cannabinoid levels beyond what were normally observed endogenously, we examined the effects of elevated 2-AG and AEA levels on worm behavior. Surprisingly, at higher exogenous cannabinoid concentrations (32 vs 3.2 μm) the α2A-adrenergic-like receptor OCTR-1 and the 5-HT1-like receptor SER-4 are both required for the 2-AG inhibition of nociception in addition to NPR-19 because octr-1 and ser-4-null animals are also resistant to 2-AG inhibition (octr-1: t = 7.4, df = 49, p < 0.0001; ser-4: t = 5.8, df = 48, p < 0.0001; Fig. 5A). The monoaminergic modulation of aversive responses is complex and involves the synergistic and antagonistic interactions of multiple monoamine receptors interacting at multiple levels in the locomotory decision-making circuit modulating nociception (Wragg et al., 2007; Harris et al., 2011; Mills et al., 2012). An octr-1::gfp transgene is broadly expressed, including both the ASHs and the ventral nerve cord (Wragg et al., 2007), and 2-AG sensitivity could be restored in octr-1 animals by octr-1 expression driven by the predicted 5 kb octr-1 promoter (Fig. 5B). 2-AG activated OCTR-1 directly after heterologous expression, with an EC50 of 365 ± 24 nm (Fig. 5C,D). Interestingly, NPR-19 and OCTR-1 exhibited similar EC50s for 2-AG, although OCTR-1-dependent phenotypes were only observed at higher exogenous 2-AG concentrations. These differences could be explained in a number of ways, including differential modulation of the receptors in vivo, differential localization of the receptors relative to ligand entry, or the degree of receptor activation required for the phenotype. In contrast, ser-4 is only expressed in a limited number of neurons and 2-AG sensitivity could be restored in ser-4-null animals by ser-4 expression in the two AIB interneurons (Fig. 5E). In contrast to OCTR-1, 2-AG (5 μm) did not activate SER-4 directly and had no effect on SER-4 affinity for 5-HT (Fig. 5F).
SER-4 and OCTR-1 are required for cannabinoid-dependent inhibition of nociception and locomotion at higher exogenous cannabinoid concentrations. Aversive responses to 1-octanol were examined as described by Harris et al. (2009). A, Concentration-dependent 2-arachidonoylglycerol (2-AG) inhibition of aversive responses. B, octr-1 rescue of 2-AG inhibition of aversive responses in octr-1-null animals. C, D, F, Two-electrode voltage-clamp (TEVC) recordings performed on oocytes coexpressing OCTR-1 or SER-4 and GIRK1/2 subunits 72 h after injection, as described previously (Stühmer, 1998; Bamber et al., 2003). C, Representative trace of direct OCTR-1 activation by 2-AG. D, OCTR-1 dose–response curve for 2-AG. E, 2-AG inhibition of aversive responses to 1-octanol in ser-4-null animals. F, SER-4 dose–response curves for serotonin (5-HT) and 5-HT + 5 μm 2-AG after SER-4 expression in Xenopus oocytes. G–I, 2-AG-dependent locomotory inhibition. H, 5-HT- and 2-AG-dependent inhibition of locomotion and 2-AG-dependent locomotory inhibition in 5-HT receptor quintuple-null animals expressing ser-4 off-target in the cholinergic motorneurons (MNs). Data are represented as mean ± SE (n) and were analyzed by two-tailed Student's t test. *Significantly different from 0 min; †significantly different from N2 at 15 min (p ≤ 0.05).
Although MAGL or FAAH inhibition with JZL184 or URB597, respectively, had no effect on locomotion, increasing exogenous cannabinoid levels even further (to 320 μm) also caused animals to become sluggish and stop moving for brief periods (t = 6.7, df = 36, p < 0.0001; Fig. 5G). Both SER-4 and OCTR-1 were involved in this cannabinoid-dependent locomotory inhibition, but, in contrast to aversive responses, NPR-19 was not involved because npr-19-null animals behaved as wild-type animals and were similarly inhibited by 2-AG (t = 3.9, df = 40, p < 0.0001; Fig. 5G). In fact, this cannabinoid-dependent locomotory phenotype mimicked the 5-HT-dependent “locomotory confusion” phenotype mediated by the 5-HT activation of the Gαo-coupled 5-HT1-like receptor SER-4 in the two AIB interneurons (Law et al., 2015). Indeed, ser-4 and 5-HT receptor quintuple-null animals were both resistant to cannabinoid-dependent locomotory inhibition and, as predicted, could be rescued by ser-4 expression in the AIBs of ser-4-null animals (t = 6.2, df = 12, p < 0.0001; Fig. 5G). Interestingly, octr-1-null animals were also resistant, potentially involving the direct 2-AG activation of the inhibitory OCTR-1 in the motorneurons (Fig. 5G). Indeed, octr-1 overexpression inhibited locomotion in the absence of 2-AG compared with wild-type animals (6.5 vs 9.6 body bends/20 s; data not shown). These results demonstrate that elevated cannabinoid levels have the potential to stimulate both octopaminergic and serotonergic signaling to modulate an array of key behaviors.
Cannabinoids increase endogenous 5-HT levels
As noted above, SER-4 was only required at higher exogenous cannabinoid concentrations and 2-AG did not activate SER-4 directly. Therefore, we hypothesized that cannabinoids might increase endogenous 5-HT levels, leading to the locomotory inhibition, either by increasing 5-HT release or inhibiting reuptake. To examine this hypothesis directly, we examined tph-1-null animals that lack tryptophan hydroxylase, the rate-limiting enzyme in 5-HT biosynthesis. As anticipated, tph-1-null animals that lack endogenous 5-HT were resistant to 2-AG-dependent locomotory inhibition (t = 5.1, df = 12, p < 0.001), suggesting that 5-HT is required for 2-AG inhibition and that 2-AG may increase endogenous 5-HT (Fig. 5H). Indeed, the 2-AG and 5-HT-dependent locomotory confusion phenotypes are similar and 2-AG-dependent locomotory inhibition mimics that observed in mod-5-null animals that lack a key 5-HT reuptake transporter and also display elevated 5-HT levels and inhibited locomotion (Fig. 5H). However, 2-AG still inhibits locomotion in these already slowed mod-5-null animals (t = 8.2, df = 26, p < 0.0001; Fig. 5H), suggesting additional 2-AG targets, including direct effects on 5-HT release. In addition, 5-HT or 2-AG also inhibit locomotion in transgenic quintuple 5-HT receptor-null animals expressing SER-4 off-target in the cholinergic motorneurons (t = 3.8, df = 40, p < 0.001; Fig. 5I), supporting the observation that 2-AG stimulates global increases in 5-HT. These mutant transgenic animals have been used previously to identify SER-4 agonists for use as potential anthelmintics because the SER-4/Gαo-mediated inhibition of the cholinergic motorneurons leads to a rapid flaccid paralysis (Law et al., 2015).
The increased 5-HT levels might also explain the requirement for OCTR-1 in the inhibition of nociception at higher cannabinoid levels because 5-HT stimulates aversive responses in part by activating serotonergic signaling in an array of additional neurons that is antagonized by ASH OCTR-1. Indeed, this complex serotonergic/octopaminergic antagonism in the modulation of ASH-dependent aversive responses has been characterized previously, with at least three different 5-HT receptors, SER-1, SER-5, and MOD-1, involved in stimulating the initiation of aversive responses (Wragg et al., 2007; Harris et al., 2009; Mills et al., 2012). Together, these data highlight the complex interaction among cannabinoid, serotonergic, and octopaminergic signaling and suggest that they may also be relevant to understanding the role of exogenous cannabinoids in the modulation of human behavior because C. elegans has proven previously to be a useful model for understanding monoaminergic modulation in mammals (Komuniecki et al., 2012; Mills et al., 2012) (Fig. 6).
Model of 2-arachidonoylglycerol (2-AG)-dependent modulation of behavior. Endogenous 2-AG activates NPR-19 in the URXs to inhibit nociception. In addition, at higher 2-AG levels via exogenous application, 2-AG (italic gray) also activated NPR-19 to inhibit pharyngeal and feeding and also activated directly and indirectly a number of monoamine receptors to inhibit locomotion through an NPR-19-independent mechanism. These elevated 2-AG levels appear to increase endogenous serotonin (5-HT) and activate SER-4 in the two AIB interneurons to initiate locomotory confusion and paralysis that has been characterized previously (Law et al., 2015). Interestingly, 5-HT stimulates the initiation of aversive responses by activation of at least three additional 5-HT receptors, each operating at different levels within the ASH-dependent aversive circuit (Harris et al., 2011). However, this potential 5-HT stimulation appears to be overcome by the direct 2-AG activation of the α2A-adrenergic-like OA receptor OCTR-1 in the ASHs to inhibit 5-HT-stimulated aversive responses, as demonstrated previously (Wragg et al., 2007). MAGL degrades 2-AG and terminates signaling and JZL184, an MAGL inhibitor, increases endogenous 2-AG levels and mimics the application of low levels of 2-AG.
Discussion
The present study demonstrates that C. elegans contains an endogenous cannabinoid signaling system that modulates an array of key behaviors (Fig. 6). For example, the endocannabinoids 2-AG and AEA inhibit both aversive behavior and feeding. 2-AG and AEA have been identified previously in C. elegans extracts by GC/MS (Lehtonen et al., 2011). In contrast to previous reports suggesting that C. elegans does not contain a canonical cannabinoid receptor (McPartland et al., 2001), although a mutant screen did identify that the predicted neuropeptide receptor, NPR-19 was involved in the effects of cannabinoids on axon regeneration in C. elegans (Pastuhov et al., 2016). In the present study, we have demonstrated that NPR-19 is essential for many cannabinoid-dependent behaviors and responds directly to cannabinoid ligands with high affinity. Indeed, although Gαo-coupled NPR-19 exhibits only 23% identity to the human Gαo-coupled cannabinoid receptor CB1, many of the key amino acids involved in ligand binding are conserved in the two receptors and phenotypes in npr-19-null animals could be rescued by the expression of human CB1, confirming the orthology of the two receptors (McPartland et al., 2001).
These cannabinoid ligands also activate octopaminergic and serotonergic signaling by functioning as agonists for the α2A-adrenergic-like OA receptor OCTR-1 and increasing endogenous 5-HT. This is based on the observations that 5-HT mimics the inhibitory effects of 2-AG on locomotion and that the 2-AG inhibition of both nociception and locomotion is significantly reduced in tph-1-null animals that lack a key 5-HT biosynthetic enzyme and have dramatically reduced 5-HT levels. The monoaminergic modulation of aversive responses to 1-octanol is complex and involves multiple sensory neurons and an array of monoamine receptors. For example, 5-HT stimulates the initiation of an ASH-dependent aversive response and requires three distinct 5-HT receptors operating at different levels within the locomotory circuit (Harris et al., 2009), with 5-HT decreasing ASH calcium but increasing ASH depolarization and activity (Zahratka et al., 2015). In contrast, OA antagonizes this 5-HT-dependent stimulation via the Gαo-coupled α2-adrenergic-like OA receptor OCTR-1, inhibiting the ASHs directly and the Gαq-coupled OA receptor SER-6, which stimulates the release of an additional layer of inhibitory monoamines and neuropeptides (Mills et al., 2012). Indeed, this octopaminergic modulation of nociception, with a Gαo-coupled receptor inhibiting the primary nociceptor and a Gαq-coupled receptor stimulating the release of multiple inhibitory neuropeptides, mimics the noradrenergic modulation of chronic pain in humans (Komuniecki et al., 2012). The levels of OA appear to be critical for the inhibitory response because OA inhibition is masked at higher OA concentrations by activation of a second antagonistic Gαq-coupled OA receptor, SER-3, in the ASHs, highlighting the delicate and dynamic balance of this modulatory system (Wragg et al., 2007). At low levels of cannabinoid receptor ligands, achieved by either inhibition of their endogenous breakdown or exogenous application, behaviors appear to be modulated exclusively by NPR-19. However, at higher levels of exogenous addition, both the octopaminergic and serotonergic signaling systems are activated. Indeed, it was puzzling at first why SER-4 and OCTR-1 were only required for the inhibition of nociception at higher cannabinoid concentrations. However, we propose that these higher cannabinoid concentrations increase endogenous 5-HT and its potential stimulation of aversive responses must be antagonized by ASH OCTR-1 for the cannabinoid-dependent inhibition of nociception to be realized. C. elegans contains multiple serotonergic neurons that function independently in the modulation of key behaviors. For example, global 5-HT released by the two neurosecretory NSMs stimulates the initiation of aversive responses to 1-octanol, whereas more local 5-HT from the ADFs appears to inhibit this response (Song et al., 2013). In contrast, ADF 5-HT is responsible for the increased pharyngeal pumping associated with the presence of food, whereas the NSMs located directly above the pharynx apparently are not involved. Interestingly, 2-AG dramatically inhibited the 5-HT quintuple-null animals expressing the inhibitory 5-HT1A-like receptor SER-4 off target in the cholinergic motor neurons, suggesting that cannabinoids initiate a more global increase in 5-HT. Whether this results from the direct and/or indirect stimulation of secretion by the NSMs or the inhibition of 5-HT reuptake is unclear, but the locomotory confusion phenotype initiated by either 5-HT or 2-AG is mimicked by knockdown of the key 5-HT reuptake transporter MOD-5. Cannabinoid receptor agonists also modulate α-adrenergic and serotonergic signaling in mammals and function as agonists for the human α2A-adrenergic receptor. For example, cannabinoid receptor stimulation activates the hypothalamic–pituitary–adrenal axis because central administration of THC in rats leads to an increase in plasma adrenocorticotrophin hormone levels (Corchero et al., 1999) and in the expression of corticotrophin releasing hormone mRNA in the anterior pituitary (Corchero et al., 2001). 2-AG increases norepinephrine (NE) release (Kurihara et al., 2001) and AEA or HU-210, a synthetic CB1 agonist, significantly increases the level of circulating corticosterone (McLaughlin et al., 2009). In fact, interactions between endogenous cannabinoid and noradrenergic signaling have been observed in a number of organ systems. For example, cannabinoid receptor signaling plays a role in noradrenergic splenic contraction and interacts with adrenergic systems in the prefrontal cortex (Simkins et al., 2016). In addition, cannabinoids can block neuronal NE uptake and the phytocannabinoid cannabigerol functions as a α2-adrenoceptor agonist in isolated mouse vas deferens (Cascio et al., 2010). Cannabinoids also modulate the synthesis, release, and turnover of 5-HT and appear to inhibit 5-HT reuptake and enhance 5-HT1A signaling (Egashira et al., 2002; Sagredo et al., 2006). For example, both endogenous and synthetic cannabinoids inhibit 5-HT reuptake in rats and chronic THC administration increases endogenous 5-HT levels in the prefrontal cortex of rats. In addition, the phytocannabinoid Δ9-tetrahydrocannabivarin appears to act through 5-HT1A receptors to produce antipsychotic effects by functioning as an allosteric modulator, increasing the efficacy but not the EC50 of the potent 5-HT1A agonist 8-OH-DPAT (Cascio et al., 2015). Together, these observations highlight the similarities between the nematode and mammalian cannabinoid signaling system and the potential of the C. elegans whole-animal model for the study of cannabinoid/monoamine interactions.
Footnotes
This work was supported by the National Institutes of Health (Grant AI072644 to R.K.) and by funds from the Joan L. and Julius H. Jacobson Biomedical Professorship. We thank Dr. Robert Steven, Dr. Tomer Avidor-Reiss, and Dr. Vera Hapiak for reviewing and editing the manuscript and the C. elegans Genetics Center and the National Bioresources Center for null strains.
The authors declare no competing financial interests.
- Correspondence should be addressed to Richard Komuniecki, Department of Biological Sciences, University of Toledo, 2801 W. Bancroft Street, Toledo, OH 43606. rkomuni{at}uoft02.utoledo.edu
