Cannabinoids Activate Monoaminergic Signaling to Modulate Key C. elegans Behaviors

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 α_2A-adrenergic-like octopamine (OA) receptor, OCTR-1, and a 5-HT_1A-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 STATEMENTCannabis 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.

Keywords: C. elegans; cannabinoid; monoamine; neuromodulation; nociception.

Figure 1.

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.

Figure 2.

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.

Figure 3.

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). I_HK represents the current induced upon a switch from low-K⁺ to high-K⁺ Ringer's solution. I_Ligand 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).

Figure 4.

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.

Figure 5.

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).

Figure 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 α_2A-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.