“A Critical Evaluation of Terpenoid Signaling at Cannabinoid CB1 Receptors in a Neuronal Model”
Wesley Corey, Indiana University, Department of Psychological and Brain Sciences
Abstract
Δ9-THC, the main psychoactive constituent of cannabis, binds to CB1 receptors in the endogenous cannabinoid signaling system. This system also includes receptors CB2, GPR18, and others. Aside from Δ9-THC, hundreds of related phytocannabinoids and terpenoids are found in cannabis. Terpenoids are cannabinoid-associated scented terpenes, though these can be found in many other plants, such as fruits. Fueled by legal changes and strong consumer interest, these compounds have been promoted as having beneficial effects in relation to pain, mood, and inflammation.
We have tested the action of several terpenoids on CB1 signaling in a well-characterized, neuronal model. These neurons express CB1 receptors, and also synthesize and metabolize the endocannabinoid 2-AG. We tested five terpenoids: myrcene, limonene, linalool, α-pinene, and nerolidol. Most compounds had little or no effect on cannabinoid signaling. However, nerolidol was found to have dual, opposing actions. Nerolidol reduced cannabinoid signaling, likely by inhibiting endocannabinoid synthesis, and was observed to enhance maximal CB1 signaling. This suggests that nerolidol has both presynaptic and post-synaptic effects on cannabinoid signaling. Besides nerolidol, most terpenoids tested in a neuronal model had little interaction with CB1-based signaling at concentrations that are likely to be encountered by consumers of cannabis-related products.
Introduction
Cannabis has a long history of use that stretches back thousands of years. However, recent legalization in Uruguay, Canada, and several American states, has increased cannabis popularity. The majority of attention has been given to the two phytocannabinoids that are the most prevalent in cannabis: tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). Δ9-THC exerts its psychoactive effects primarily through cannabinoid CB1 receptors (Matusuda et al., 1990) that are widely distributed in the brain (Herkenham et al., 1990). Cannabinoid CB1 receptors regulate important physiological systems such as pain, mood, movement, and memory (Piomelli, 2003).
Lately there has been a strong, growing interest in other phytocannabinoids and terpenoids, many of which are marketed for supposed health benefits. Terpenoids give cannabis its characteristic odor and are variants of terpenes, a larger class of organic compounds. Several cannabis-derived terpenes such as myrcene, α-pinene, humulene, limonene, linalool, nerolidol, and caryophyllene have attracted the interest of companies and consumers. These have been described as having various alleged effects, and are often proposed to be synergistic with phytocannabinoids (reviewed in (Russo, 2011)). These effects include altered Δ9-THC psychoactivity (myrcene), mood (limonene), inflammation (caryophyllene), sedation (nerolidol), and memory (α-pinene). Terpenoids are structurally unrelated to phytocannabinoids and are generally recognized as safe by the FDA. As a result, terpenoids may see a more rapid embrace by an industry interested in cannabis.
There have been few, if any systematic studies of terpenoid interaction with the cannabinoid signaling system. Therefore, we tested several of the widely promoted terpenoids for their interaction with endogenous cannabinoid signaling in a well-characterized, CB1-based, neuronal model, as described in approximately 25 publications (Straiker and Mackie, 2005). In this model, autaptic hippocampal neurons express CB1 receptors, the cellular machinery to synthesize and metabolize the endocannabinoid 2-AG, and several other forms of CB1-mediated neuronal plasticity (Kellogg et al., 2009; Straiker et al., 2009; Straiker and Mackie, 2007). This system allows us to test direct agonism/antagonism of CB1 receptors and the interaction with other components of cannabinoid signaling, including potential synergism with endocannabinoids.
Methods
Hippocampal culture preparation: All procedures in this study were approved by the Animal Care Committee of Indiana University, and conform to the Guidelines of the National Institutes of Health on the Care and Use of Animals. Mouse hippocampal neurons, isolated from the CA1-CA3 region, were cultured on micro-islands (Bekkers and Stevens, 1991; Furshpan et al., 1076). Neurons were obtained from animals (aged postnatal day 0-2) and plated on a feeder layer of hippocampal astrocytes previously laid down (Levison and McCarthy, 1991). Cultures were grown in highglucose (20mM) DMEM containing 10% horse serum, without mitotic inhibitors. Neurons were used for recordings after eight days in culture and no more than three hours after removal from culture medium.
Electrophysiology: When a single neuron is grown on a small island of permissive substrate, it forms synapses — or “autapses”— onto itself. Experiments were preformed on isolated autaptic neurons. Whole-cell, voltage-clamp recordings were performed at room temperature using an Axopatch 200A amplifier. Extracellular solution (ECS) contained 119mM NaCl, 5mM KCl, 2.5mM CaCl2, 1.5mM MgCl2, 30mM glucose, and 20mM HEPES. Continuous flow of solution through the bath chamber (~2 mL/min) ensures rapid drug application and clearance. Drugs were prepared as stocks, and diluted into ECS to their final concentrations.
Recording pipettes of 1.8-3MΩ resistance were filled with 121.5mM KGluconate, 17.5mM KCl, 9mM NaCl, 1mM MgCl2, 10mM HEPES, 0.2mM EGTA, 2mM MgATP, and 0.5mM LiGTP. Access resistance and holding current were monitored. Only cells with stable access resistance and holding current were included in data analysis. Cellular membrane potential was held at -70mV and excitatory postsynaptic currents (EPSCs) were induced every 20 seconds by triggering an unclamped action current with a 1.0msec depolarization. The resulting waveform consists of a downward spike, representing inward sodium currents, followed by slower EPSCs. The size of recorded EPSCs was calculated by integrating the induced currents. Calculating charge value in this way yields indirect measures of neurotransmitter release, and minimizes effects of cable distortion on currents far from the recording electrode.
After establishing a 10-20 second, 0.5 Hz baseline, depolarization-induced suppression of excitation (DSE) was achieved by depolarizing to 0mV for 50msec, 100msec, 300msec, 500msec, 1sec, 3sec, and 10sec. Depolarizations were followed by resumptions of a 0.5 Hz stimulus for 20-80+ seconds, allowing EPSCs to recover to baseline. This determines sensitivity of synapses to DSE induction. Before DSE stimulus, baseline values were normalized to one to allow comparison. DSE inhibition values are presented as fractions of 1, i.e. 50% inhibition form the baseline response is 0.50± standard error of the mean. The xaxis of DSE-depolarization-response curves is log-seconds of the depolarization duration used to elicit DSE. These curves were obtained to determine pharmacological properties of endogenous 2-AG signaling. The data are fitted with a nonlinear regression, allowing calculation of an ED50, the effective dose or duration of depolarization at which 50% inhibition is achieved. Significance was calculated using two-way ANOVA with a Bonferroni test. Terpenoids were obtained from Sigma-Aldrich and 2-AG was purchased from Cayman Chemical.
Results
Myrcene and linalool do not alter CB1 signaling
Myrcene is known for its proposed ability to enhance the cannabis high. A compound like myrcene could directly activate cannabinoid receptors, enhance signaling through positive allosteric modulation, enhance synthesis of endocannabinoids, or inhibit endocannabinoid metabolism.
We found that myrcene had no significant effect on cannabinoid signaling at 1μM. We tested myrcene’s impact on CB1 signaling in autaptic hippocampal neurons using whole-cell, patch-clamp recording. At 1μM, myrcene did not alter EPSCs (F(6,60)=0.6523, P=0.6881), indicating myrcene does not directly alter neurotransmission in this model.
Additionally, myrcene did not alter depolarization-induced suppression of excitation (DSE), a form of endogenous 2-AG and CB1-mediated retrograde signaling present in autaptic hippocampal neurons. Longer depolarizations result in greater inhibition of EPSCs, yielding a “depolarized dose-response” curve. A compound that enhances cannabinoid signaling is expected to shift this curve to the left, as seen with CB1 positive allosteric modulators as seen in this model (Mitjavila et al., 2018). Conversely, an inhibitor should shift the curve rightward, as seen with CB1 negative allosteric modulators (Straiker er al., 2015). We found that myrcene does not affect the DSE response curve at 1μM.
Linalool is a monoterpene proposed to act as an anxiolytic (Linck et al., 2010). Linalool is commonly associated with lavender, but has recently been proposed to interact with the cannabinoid signaling system. At 1μM, we find no effect on neurotransmission (data not shown) or significant alteration in DSE responses (F(6,60)=1.113, P=0.3656).
Limonene exerts a modest antagonistic effect on CB1 signaling
Limonene, a cyclic monoterpene associated with citrus, is described by cannabis vendors as being anxiolytic, anti-depressant, and beneficial for memory (Carvalho-Freitas and Costa, 2002; Komiya et al., 2006). While limonene did not alter neurotransmission directly, it modestly inhibited cannabinoid signaling (F(6,48)=2.256, P=0.0058).
Nerolidol has dual action on CB1 signaling
Nerolidol is a sesquiterpene described as having sedative properties (Russo 2011). Nerolidol did not directly alter neurotransmission, but did effect cannabinoid signaling. Nerolidol concentration-dependently inhibited DSE, shifting the DSE response curve to the right. This was found at 1μM but not 100nM ((F(6,72)=9.418, P<0.0001);(F6,96)=0.8496, P=0.5350)). In DSE, 2-AG is produced post-synaptically and acts on presynaptic CB1 receptors, a retrograde inhibition of synaptic transmission. If nerolidol inhibits CB1 presynaptically, then we would see similar inhibition of bath-applied 2-AG. However, nerolidol did not inhibit 2-AG signaling (F(4,44)=0.4721, P=0.7559). This suggests that nerolidol reduces 2-AG production post-synaptically. Intriguingly this experiment unmasked a separate effect – an enhanced maximal CB1 activation. This suggests that nerolidol has two separate, opposing effects on cannabinoid signaling.
α-Pinene antagonizes CB1 signaling
α-Pinene is a monoterpene found in conifers and believed to enhance memory (Perry et al., 2000). α-Pinene did alter neurotransmission, but like limonene, it did reduced CB1 signaling (F(6,84)=3.198, P=0.0071).
Discussion
Terpenoids are cannabinoid-associated terpenes that are continuing to find their way into consumer interests due to their often-claimed therapeutic benefits, though there is little understanding of their actual effects on cannabinoid signaling. We tested several of the more widely promoted terpenes: myrcene, linalool, limonene, α-pinene, and nerolidol in a well-characterized neuronal model that utilizes endogenous cannabinoid signaling. Our chief findings are that most terpenoids either had no effect, or a modest inhibitory effect on cannabinoid signaling at 1μM. The exception was nerolidol, which had dual, opposing effects. Nerolidol reduced cannabinoid signaling, likely by inhibiting endocannabinoid synthesis, but also enhanced maximal CB1 signaling. This suggests that nerolidol acts both pre-synaptically and post-synaptically in neurons.
Partly because they are marketed in the context of cannabis, terpenoids are promoted as having effects via the cannabinoid signaling system. Our results suggest that this is unlikely, at least through cannabinoid CB1 receptors, though they do not rule out effects through other cannabinoid-associated receptors such as CB2 or GPR18. We only tested a select panel of often-cited compounds, but there are likely other terpenoids that could be of interest, especially given that one terpenoid, as noted above, exhibited an interesting signaling profile.
An important question has to do with the likely physiological concentration of a given terpenoid. 1μM was chosen as a starting concentration because it represents a ceiling physiological concentration likely to be encountered by consumers. In one study, goats (55kg) were fed one gram of limonene and α- pinene, yielding peak blood-plasma concentrations of 1μM for limonene, and 600nM for α-pinene (Poulopoulou et al., 2012). In an additional study, humans were massaged with 1.5g of lavender essential oil (containing 24.7% linalool) and peak blood-plasma concentrations were found to be 600nM at 19min after application, with a half-life of 13.8min (Jäger et al., 1992). However, it is unlikely that these same concentrations would be seen in cannabis users. Terpenoid levels are low, with <1% for a given terpenoids in most cannabis strains (Potter, 2009). Terpenoid yields from cannabis products (cigarettes, brownies, etc) are therefore likely to be relatively low.
Cannabis-associated terpenoids are promoted as having healthy and beneficial effects, and it is often proposed that these are achieved through the cannabinoid signaling system. Our tests of a panel of five often-cited terpenoids show that all but one of these compounds has no positive effect on cannabinoid signaling. The exception proved to be pharmacologically interesting, with complex and opposing effects on CB1 cannabinoid signaling. Though more research is called for, our work suggests that most terpenoids are unlikely to interact with CB1-based cannabinoid signaling.
Impact Statement
Cannabis has long history of use that stretches back thousands of years and recent legalization in Uruguay, Canada, and several US states has increased cannabis use and positive mentality relating to all things cannabis. Terpenoids are cannabis related compounds that have attracted the interest of companies, and as a result they have been marketing these compounds into loosely regulated consumer products, such as creams and food items. Terpenoids are often proposed to act in a synergistic way with the cannabinoid signaling system (Russo, 2011), but their interaction with this system has not been investigated systematically, which is needed to arm the public with information. Using a well-characterized neuronal model we tested a panel of 5 terpenoids (myrcene, α-pinene, limonene, linalool, and nerolidol) that have generated the most interest, and we found that all but one had either no effect or inhibited cannabinoid signaling, which suggests that most terpenoids do not activate cannabinoid signaling.
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