Canniboids and Pain Relief

Could The CB1-TRPV1 Crosstalk be a Target
Our pain marker-TRPV1-was recently referenced in an interesting study. Here researchers suggest that spatial distribution of the CB1 receptor and TRPV1 contributes to the complexity of their functional interaction.

They conclude: "Therefore, in order to utilise the analgesic potential of endogenous anandamide fully in PSN, ways to interfere with the mechanisms, through which the CB1 receptor promotes anandamide-responsiveness and sensitisation of TRPV1, must be identified. Due to the co-expression of TRPV1 and the CB1 receptor in various areas of the brain, the CB1 receptor – TRPV1 crosstalk we characterised in the present study could also have relevance in brain signal processing."


Chen J, Varga A, Selvarajah S, Jenes A, Dienes B, Sousa-Valente J, Kulik A, Veress G, Brain SD, Baker D, Urban L, Mackie K, Nagy I. Spatial Distribution of the Cannabinoid Type 1 and Capsaicin Receptors May Contribute to the Complexity of Their Crosstalk. Sci Rep. 2016 Sep 22;6:33307. doi: 10.1038/srep33307.

The SDS-FRL method reveals two major types of CB1 receptor and TRPV1 spatial distribution in DRG neurons. (A,B) Double immunogold labelling for TRPV1 (12 nm particles; arrows) and the CB1 receptor (5 nm; arrowheads) reveals that the immunoreactivity for both proteins is moderate (A) to strong (B) on the protoplasmic face (P-face) but not on the exoplasmic face (E-face) of plasma membrane of PSN. In some patches of putative somatic membrane of PSN, the great majority of immunoparticles for TRPV1 and CB1 are isolated from each other (A), whereas on other patches of the membrane immunoparticles for TRPV1 and CB1 form co-clusters (arrow + arrowhead in B). Note the occasional co-clustering of particles labelling TRPV1 (double arrows in B). Scale bars = 0.2 μm

Methods: Sodium dodecyl sulfate (SDS)-digested freeze-fracture replica immunolabeling Immunohisto-chemical labeling for electron microscopy was performed. Briefly, adult (150–200 g) male Sprague-Dawley rats (n = 4) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and perfused transcardially with 25 mM PBS, followed by 2% paraformaldehyde and 15% saturated picric acid in 0.1 M phosphate buffer (PB). Sections from DRGs were cut at a thickness of 110 μm. The slices were cryoprotected in a solution containing 30% glycerol in 0.1 M PB and then frozen by a high-pressure freezing machine (HPM 100; Leica, Austria). Frozen samples were inserted into a double replica table and fractured into two pieces at −130 °C and replicated by carbon deposition (5 nm thick), platinum (2 nm), and carbon (15 nm) in a freeze-fracture replica machine (BAF 060; BAL-TEC, Lichtenstein). They were digested in a solution containing 2.5% SDS and 20% sucrose in 15 mM Tris-HCl at 80 °C for 18 h then at 60 °C for 18 h. The replicas were washed in 25 mM PBS containing 0.05% bovine serum albumin (BSA; Roth, Germany) and then incubated in a blocking solution containing 5% BSA for 1 h. Subsequently, replicas were incubated in a mixture of primary antibodies (TRPV1 raised in guinea pig against the 22 amino acids of the C terminus (YTGSLKPEDAEVFKDSMVPGEK; Neuromics, USA), the dilution was 1:1000 and CB1 receptor (Prof. K. Mackie, raised in goat; Veress et al. 2014), dilution was 1:4000) in 50 mM Tris-buffered saline (TBS) containing 1% BSA. After several washes, replicas were reacted with a mixture of gold-coupled donkey anti-guinea pig (for TRPV1; 12 nm) and rabbit anti-goat (for CB1, 5 nm) secondary antibodies (1:30; BioCell Research Laboratories, Cardiff, UK) in 25 mM TBS containing 5% BSA. The anti-TRPV1 antibody from Neuromics provided better staining in the replicas than then the anti-TRPV1 antibody from Chemicon, which we tried in a pilot study. They were then washed and picked up on 100-mesh grids. The specificity of the antibodies was tested on tissues dissected from TRPV1 and CB1 knock out mice.

We will keep you posted as we discover new research on targeting CB1 for pain. That said, could this crosstalk be central to medical marijuana and its ability to ameliorate certain forms of pain?

Medical Marijuana Research

What we are learning.

I have reported here Medical Marijuana research and its therapeutic value for treating pain. The specifics posts feature publications that reference use of our Pain and Inflammation Research Markers. These include: J. Desroches, J.-F. Bouchard, L. Gendron, P. Beaulieu. Involvement of cannabinoid receptors in peripheral and spinal morphine analgesia ☆ Neuroscience, Volume 261, 7 March 2014, Pages 23–42.http://dx.doi.org/10.1016/j.neuroscience.2013.12.030.
Iryna A. Khasabova,Sergey Khasabov, Justin Paz, Catherine Harding-Rose, Donald A. Simone, and Virginia S. Seybold. Cannabinoid Type-1 Receptor Reduces Pain and Neurotoxicity Produced by Chemotherapy. The Journal of Neuroscience, 16 May 2012, 32(20): 7091-7101; doi: 10.1523/​JNEUROSCI.0403-12.2012

Here researchers study the impact of Marijuana on memory impairment using our one of our 5-HT Serotonin Antibodies: Xavier Viñals, Estefanía Moreno, Laurence Lanfumey, Arnau Cordomí, Antoni Pastor, Rafael de La Torre, Paola Gasperini, Gemma Navarro, Lesley A. Howell, Leonardo Pardo, Carmen Lluís, Enric I. Canela, Peter J. McCormick, Rafael Maldonado, Patricia Robledo. Cognitive Impairment Induced by Delta9-tetrahydrocannabinol Occurs through Heteromers between Cannabinoid CB₁ and Serotonin 5-HT_2A Receptors. Published: July 9, 2015DOI: 10.1371/journal.pbio.1002194.
Impact on Memory: Activation of cannabinoid CB1 receptors (CB1R) by delta9-tetrahydrocannabinol (THC) produces a variety of negative effects with major consequences in cannabis users that constitute important drawbacks for the use of cannabinoids as therapeutic agents. For this reason, there is a tremendous medical interest in harnessing the beneficial effects of THC. Behavioral studies carried out in mice lacking 5-HT2A receptors (5-HT2AR) revealed a remarkable 5-HT2AR-dependent dissociation in the beneficial antinociceptive effects of THC and its detrimental amnesic properties We found that specific effects of THC such as memory deficits, anxiolytic-like effects, and social interaction are under the control of 5-HT2AR, but its acute hypolocomotor, hypothermic, anxiogenic, and antinociceptive effects are not. In biochemical studies, we show that CB1R and 5-HT2AR form heteromers that are expressed and functionally active in specific brain regions involved in memory impairment. Remarkably, our functional data shows that costimulation of both receptors by agonists reduces cell signaling, antagonist binding to one receptor blocks signaling of the interacting receptor, and heteromer formation leads to a switch in G-protein coupling for 5-HT2AR from Gq to Gi proteins. Synthetic peptides with the sequence of transmembrane helices 5 and 6 of CB1R, fused to a cell-penetrating peptide, were able to disrupt receptor heteromerization in vivo, leading to a selective abrogation of memory impairments caused by exposure to THC. These data reveal a novel molecular mechanism for the functional interaction between CB1R and 5-HT2AR mediating cognitive impairment. CB1R-5-HT2AR heteromers are thus good targets to dissociate the cognitive deficits induced by THC from its beneficial antinociceptive properties. 

Figures: 5-HT2AR and CB1R form heteromers in transfected cells. In (A), BRET saturation experiments were performed in HEK-293T cells transfected with 0.025 μg of 5-HT2AR-Rluc cDNA and increasing amounts of CB1R-YFP cDNA (0.05 μg to 1.5 μg, black curve), with 0.5 μg of dopamine D1R-Rluc cDNA and increasing amounts of CB1R-YFP cDNA (0.5 μg to 6 μg, yellow line), or with 0.025 μg of 5-HT2AR-Rluc cDNA and increasing amounts of adenosine A1R-YFP cDNA (0.05 μg to 1.5 μg, red line). The relative amount of BRET is given as a function of 100 x the ratio between the fluorescence of the acceptor (YFP) and the luciferase activity of the donor (Rluc). BRET is expressed as milli BRET units (mBU) and is given as the mean ± standard deviation (SD) of 3–6 experiments grouped as a function of the amount of BRET acceptor. In (B), a schematic representation of fluorescence complementation experiments is depicted in the left panel showing that fluorescence only appears after the YFP Venus hemiprotein complementation due to the proximity of two receptors fused to hemi-YFP Venus proteins (cYFP or nYFP). In the right panel, fluorescence at 530 nm was detected in HEK-293T cells transfected with different amounts of cDNA corresponding to both 5-HT2AR-cYFP and CB1R-nYFP (equal amount for each construct), but not in negative controls in which cells were transfected with cDNA corresponding to 5-HT2AR-cYFP and the noninteracting adenosine A1 receptor-nYFP or CB1R-nYFP and the noninteracting dopamine D1 receptor-cYFP. One-way ANOVA followed by a Dunnett’s multiple comparison test showed a significant fluorescence over basal values in HEK-293T cells (** p< 0.01, *** p< 0.001). In (C), PLAs were performed in HEK-293T cells expressing CB1R and 5-HT2AR. Confocal microscopy images (superimposed sections) are shown in which heteromers appear as green spots. In all cases, cell nuclei were stained with DAPI (blue). Scale bars = 20 μm. In (D), PLAs were performed in nontransfected HEK-293T cells, cells transiently transfected with 0.5 μg of CB1R or 5-HT2AR cDNA (negative controls, white columns), or with increasing amounts of CB1R and 5-HT2AR cDNA (black columns). In each case, the ratio between the number of green spots and the number of cells showing spots (ratio r) was calculated. One-way ANOVA followed by a Dunnett’s multiple comparison test showed a significant PLA staining over nontranfected cells (*** p <; 0.001). doi:10.1371/journal.pbio.1002194.g004

Yes, memory impairment must be considered as a side effect of Medical Marijuana, I am confident there are human subjects that would volunteer for cognition studies to get a clearer picture of the short and long term impact on users.