A Critical Evaluation of Terpenoid Signaling at Cannabinoid CB1 Receptors in a Neuronal Model

Abstract

In addition to phytocannabinoids, cannabis contains terpenoids that are claimed to have a myriad of effects on the body. We tested a panel of five common cannabis terpenoids, myrcene, linalool, limonene, α-pinene and nerolidol, in two neuronal models, autaptic hippocampal neurons and dorsal root ganglion (DRG) neurons. Autaptic neurons express a form of cannabinoid CB1 receptor-dependent retrograde plasticity while DRGs express a variety of transient receptor potential (TRP) channels. Most terpenoids had little or no effect on neuronal cannabinoid signaling. The exception was nerolidol, which inhibited endocannabinoid signaling. Notably, this is not via inhibition of CB1 receptors but by inhibiting some aspect of 2-arachidonoylglycerol (2-AG) production/delivery; the mechanism does not involve reducing the activity of the 2-AG-synthesizing diacylglycerol lipases (DAGLs). Nerolidol was also the only terpenoid that activated a sustained calcium response in a small (7%) subpopulation of DRGs. In summary, we found that only one of five terpenoids tested had notable effects on cannabinoid signaling in two neuronal models. Our results suggest that a few terpenoids may indeed interact with some components of the cannabinoid signaling system and may therefore offer interesting insights upon further study.

Keywords: CB1; G protein-coupled receptor; cannabinoid; cannabis; terpene; terpenoid.

Figure 1

Structures of selected terpenoids.

Figure 2

Myrcene, linalool, limonene andα-pinene do not alter cannabinoid signaling in autaptic hippocampal neurons. (A) Myrcene (1 µM) did not alter EPSCs. (B) Myrcene also did not have an effect on DSE responses. (C) Linalool (1 µM) slightly increased EPSCs. (D) However, linalool had no effect on DSE responses. (E) Limonene (1 µM) did not alter EPSC sizes (F) and did not significantly inhibit DSE responses. (G) α-pinene (1 µM) did not alter EPSCs. (H) Similarly α-pinene did not inhibit DSE responses. * p < 0.05 one-sample t-test vs. 1.0 or vs. baseline at 3 s of depolarization. Mean ± SEM shown.

Figure 3

Nerolidol has complex interaction with cannabinoid signaling. (A) Nerolidol (1 μM) does not alter EPSC sizes. (B) Nerolidol substantially inhibits DSE responses at 1 μM but not at 100 nM. (C) 2-AG signaling is not diminished by treatment with nerolidol. *** p < 0.001, **** p < 0.0001 at 3 and 10 s depolarizations via 2-way ANOVA with Bonferroni post hoc test.

Figure 4

Nerolidol does not alter the basal activity of DAGLα. (A) Sample Enzchek lipase activity time course in the presence of vehicle (black) or nerolidol (3 μM, red) in DAGLα-transfected CHO cells. (B) Summarized results show lipase activity in DAGLα-expressing CHO cells in response to vehicle or nerolidol (n = 3).

Figure 5

Nerolidol does not compete for binding at the active site of DAGLβ. (A) DAGL-binding probe HT-01 is competed by DAGLβ blocker KT109 (100 nM) but not by nerolidol. (B) Summary of results from 2 experiments. NS by one tailed t-test vs. 1.0 (1.0 = no effect).

Figure 6

Nerolidol, linalool and myrcene do not reliably activate calcium responses in DRGs. (A) Sample population response using a calcium sensor in DRGs treated with nerolidol (1 μM) followed by capsaicin (1 μM). (B) Infrequently (7% of neurons), nerolidol elicited a sustained calcium response as shown in these sample time courses. (C) Time course showing responses to linalool (1 μM) as in A. (D) Rarely (0.86%) linalool elicited a response in DRGs. (E) Time course showing responses to myrcene (1 μM) as in A. (F) Myrcene rarely (1.3%) elicited a calcium response in DRGs.

Figure 7

Limonene and pinene did not activate calcium responses in DRGs. (A) Sample population response using a calcium sensor in DRGs treated with limonene (1 μM) followed by capsaicin (1 μM). (B) Population response to pinene (1 μM) treatment as in A.