This post will bounce back and forth between a THC study with a CB1 knock out mouse  and a terpene study from Michael Streicher’s laboratory  The post will quickly examine some membrane biophysics studies that back up some of the perplexing terpene results and offer new clues of CB1 receptor dynamics.This post will be tricky and may be considered an endocannabinoid tetrad too. The crazy thing is that we do not know if exercise induced tetrads have anything to do with endocannabinoids.
- Analgesia, exercise for neuropathic pain Analgesic aspects of wheel running was covered on the endocannabinoid page.
- Hypothermia with exercise? Surely not! Perhaps running wheel anandamide release would shut down thermo generation in brown fat. Exercise and brown fat termogenesis as been addressed in the literature but will not be a topic of this post.
- Hypolocomotion with exercise??? Perhaps after a workout. How does one distinguish between an endocannabinoid effect or simply being tired? The running wheel mice in the Fuss (2015) study in the endocannabinoid page were more likely to voluntarily venture into and explore a box.
- Catalepsy is a condition characterized by waxy rigidity of the limbs, which may be placed in various positions that are maintained for a time, lack of response to stimuli, mutism, and inactivity” It is highly likely that both studies featured in this post considered catalepsy a central nervous system phenomenon: fine tuning of motions gone to extremes. CB1 is present in skeletal muscle. Skeletal muscle CB1 signalling has been shown to increase muscle fatigue via modulation of calcium release and reuptake into the sacroplasmic reticulum. 
The first thing to notice is that there are no obvious structural commonalities. Anandamide and THC have a penta-alkyl group and some cis double bonds. We are about to learn that the terpene commonality is the membrane component cholesterol. Note that both terpenes and cholesterol ar products of geranyl pyrophosphate synthesis pathways.
In the CB1 knockout study body temperatures of the mice were recorded with a rectal probe 50 minutes after injection of the test compound. 
Note that both THC and the synthetic agonist HL210 decreased the body temperature by 6-8oC. In the terpene study mouse body temperatures were also recorded with a rectal thermometer 50 minutes after injection with the test compound. 
Figure 3 from LaVigne 2021 has been adapted slightly to compare trends of the four featured terpenes. The black symbols are before and the colored after. Mice were injected with 200mg /kg terpenes. Temperatures were taken 30 minutes after injection.
- 1st set of columns, All of the terpenes except pinene reduced the body temperature to at least that of the CB1 agonist WIN alone.
- 2nd set of columns Adding the CB1 agonist tended to increase the temperature reduction.
- 3rd set of columns The CB1 antagonist Rimonabant blocked some but not all of the temperature reduction.
- 4th set of columns.. On a very qualitative level… linalool and geraniol are closer in structure to limonene when one allows for conformational flexibility. We find ourselves in the realm folk wisdom that drinking lemonade with A2A agonist limonene to cool down and coffee with A2A antagonist caffeine to warm up. How much of this due to the temperatures of the drinks themselves?
In very simple terms mice were video recorded moving about a white chamber (40 × 40 × 40 cm) 1 h after the injection of THC or vehicle. The distance they traveled in 15 minutes was recorded. This site reviewed the use of the open field test to measure anxiolytic effects of the running wheel released endo cannabinoid anandamide. Is a mouse not moving because it doesn’t care or because it is anxious? The mouse could also just have a bad cause of skeletal muscle fatigue. 
LaVigne et al (2021) also used an open field test. In this study the field was a 30×28 cm box with a black floor and white walls. Mice were recorded, injected, and then recorded again 10-15 minutes latter.
In these graphs the black dotted lines are the mobile time with the solvent used to dissolve the terpene, alone. The red dotted lines are 5.6 mg/kg WIN55,212-2 (CB1 agonist) alone. Note that with the exception of geraniol, The tepenes synergize with the CB1 agonist to reduce mobile time. Again, the CB1 antagonist Rimonabant reverses the terpene reduction of motion. Oddly the hypolocomotion effect of terpenes least like limonene, β-pinene and α-humulene, are more inhibited by the A2A antagonist Istradefyllene
In the CB1 knock out study the ring test was performed on a vertical tube (5.5 cm diameter) 60 minutes after injection with THC. Immobility was the percentage of the 4 minutes session in which the mouse was motionless.  If the mouse jumped off the tube, it was palced back on.  The the previous figure panel 4C. A drug effect of 1400% means that at the highest dose of THC the wild type mice spent 14x as much time motionless on the tube.
In the terpene study the ring test was performed using a method that entails gently placing a mouse on a wire ring suspended from the surface. During a 5 minute interval the number of times the in which the mouse remained motionless was recorded.Motion in this referenced method was defined as all voluntary movements except those associated with breathing. 
The two terpenes that least resemble limonene are inhibited by the andenosine A2A antagonist.
Mice were placed on a hot plate heated to heated to 52°C.  The time before they showed first signs of discomfort (licking or flinching of the hind paws, jumping). The cutoff time was 60 sec. In the formalin test consisted of injecting 20 μl of a 5% solution under the dorsal surface of the right hind paw and monitored for signs of pain. They were timed until they showed signs of discomfort: jumping, paw licking or flinching. Mice were not kept on the hot plate longer than one minute.
Note that little difference was observed between wild type and CB1 knock out mice treated with THC in the tail flip assay.  The tail flip response mght be mediated by another THC receptor.
The terpene study only subjected the distal portion of the mouse tail to a water bath set to 52 °C. The time for the mouse to flick its tail out of the water was recorded.  Mice were then injected with compounds intraperitoneally (i.p.). The tail flick assay was repeated over a time course of 2 hours.
The terpenes clearly elicit a tail flip response. Win alone and the terpene alone tend to be about the same. The terpene plus Win is additive. Rimonabant inhibits the increase in latency.
Points and discussion from LaVigne 
The LaVigne saga, published in the prestigious Nature journal spinoff Scientific Reports continued with demonstrating low affinity of these terpenes for CB1 and limited down stream signalling.  Some points they made in their discussion shift the paradigm of thinking regarding the entourage effect of terpenes and THC.
(1) direct modulation of membrane dynamics, shifting CB1 activation equilibrium to favor the activated receptor;
All of this makes complete and total sense. Cholesterol and terpenes share geranyl pyrophosphate as an intermediate. Linalool and Geraniol have certain structural features in common with membrane stabilizing cholesterol.
(2) terpene modulation of endocannabinoid synthesis and/or degradation, which would then result in CB1 activation by these endocannabinoids. “It has been suggested that membrane composition and dynamics heavily influence the cannabinoid receptor“
Terpenes and fungal membranes
The cell walls of Candida albicans fungal cells were removed enzymatically leaving only the “prptp[;asts.” The electron paramagnetic resonance ( EPR) probes 5- and 16-doxylstearic acid were used to label the protoplasts.  Doxyl is a lipophillic free radical with an unpaired electron that yield information on membrane fluidity 5- and 16 carbons into the interior of the bilayer. Linaool was found to have a fluidizing effect in superficial regions of and a rigidifying effect in deeper layers. Phase transitioning temperature was also a factor as well as leakage of cellular components. 
Another study examined the influence of humulene and mercene on lipid monolayers containing the fungal version of cholesterol, egosterol.  Brewster angle microscopy was used to measure changes in the packing pressures of the monolayers in response to humulene.  Model fungal lipid bilayers were prepared from 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and ergosterol.  Changes in membrane fluidity was assessed with the fluorescence anisotropy probe DPH. When membrane fluidity is high, DPH tumbles fast such that it may rotate into a different plane before it emits a photon at a lower energy (longer wavelength) than the polorized photon with which it was excited. If the membrane containing the DPH probe is rigid, most emitted light will be in the same plane as the light with which it was excited. The lower the anisotropy, the more the probe is tumbling. In this case, the lower r, the more fluid the membrane.
These data showing that the fungal version of cholesterol, ergosterol, can interact with humulene has interesting implications of a review on cholesterol targeting anandamide to CB1. 
- Cholesterol may be a non protein targeting agent in lipid rafts that also include .sphingolipids . 
- Anandamide has been localized between TMH6 and TMH7 of CB1, the latter of which displays two potential cholesterol binding domains, one in each leaflet of the membrane 
- This review discusses serum albumin as a carrier of anandamide, anandamide aggregates, and different conformations of this neruotransmitter. 
Terpene targets thoughts
Much of this website seems to currently be devoted to “this terpene binds to some allosteric site on GABAA.” The remarkable thing about hte Lavigne (2021) study is that it was opened the door to the possibility that terpenes might also target any membrane molecule that is regulated by cholesterol. Not reviewed in this post, cholesterol modulates G protein coupled receptor dimerization. Does this include CB1/adenosine A2A heterodimers? How does the cholesterol and other lipid content in the vacinity of a presynaptic neuron differ from that of CB1 on brown fat cells or skeletal muscle? Can a terpene dissolve well in one and not the other?
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