Spatial Distribution of the Cannabinoid Type 1 and Capsaicin Receptors May Contribute to the Complexity of Their Crosstalk

Abstract

The cannabinoid type 1 (CB1) receptor and the capsaicin receptor (TRPV1) exhibit co-expression and complex, but largely unknown, functional interactions in a sub-population of primary sensory neurons (PSN). We report that PSN co-expressing CB1 receptor and TRPV1 form two distinct sub-populations based on their pharmacological properties, which could be due to the distribution pattern of the two receptors. Pharmacologically, neurons respond either only to capsaicin (COR neurons) or to both capsaicin and the endogenous TRPV1 and CB1 receptor ligand anandamide (ACR neurons). Blocking or deleting the CB1 receptor only reduces both anandamide- and capsaicin-evoked responses in ACR neurons. Deleting the CB1 receptor also reduces the proportion of ACR neurons without any effect on the overall number of capsaicin-responding cells. Regarding the distribution pattern of the two receptors, neurons express CB1 and TRPV1 receptors either isolated in low densities or in close proximity with medium/high densities. We suggest that spatial distribution of the CB1 receptor and TRPV1 contributes to the complexity of their functional interaction.

Figure 1. Anandamide- and capsaicin-evoked responses are partially segregated in cultured primary sensory neurons.

(A) A typical response to anandamide (10 μM) recorded from cultured rat PSN by conventional whole-cell voltage-clamp. A sub-population of cells responds to anandamide. The response is washed off within a few tens of seconds after stopping anandamide application, which is indicated by the bar above the current trace. (B) Concentration-response relationship of anandamide in cultured rat PSN (data were fitted with Hill’s equation: y = Vmax*x^n/(k^n + x^n)). The calculated EC₅₀ is 2.2 μM; the maximal effect was reached at 30 μM. (C,D) A typical whole-cell current from a cultured rat PSN during anandamide (bar, 30 μM) application. Capsaicin-evoked response from the same neuron (D). Bar indicates capsaicin (500 nM) application. A group of cultured PSN, which are regarded as “anandamide- and capsaicin-responsive” (ACR) neurons, exhibits this response pattern. (E,F) About a third of the rat PSNs are capsaicin-only-responsive neurons (COR). Typical whole-cell voltage-clamp recording from a COR neuron, which fails to respond to anandamide (E; bar, 30 μM) but exhibits a response to 500 nM capsaicin (F). Bar indicates capsaicin application. (G) Changes in the intracellular Ca²⁺ concentrations were measured by a conventional ratiometric approach in cultured PSN isolated from wild type (WT) or TRPV1 knock out (TRPV1^−/−) mice following the application of 30 μM anandamide (ANA), 500 nM capsaicin (CAP), 30 μM mustard oil (MO), 50 mM KCl and 5 μM ionomycin (ION). A typical recording of Ca²⁺ transients from a PSN which is isolated from a WT mouse and responds to both anandamide and capsaicin. (H) A typical recording of Ca²⁺ transients from a neuron which is isolated from a WT mouse and responds to capsaicin but not to anandamide. (I) Cells isolated from TRPV1^−/− mice do not respond to either anandamide or capsaicin, however a sub-population of these neurons responds to mustard oil.

Figure 2. Rimonabant reduces the amplitude of anandamide- and capsaicin-evoked currents only in ACR neurons without affecting the ratio of ACR and COR cells.

(A) Whole-cell currents evoked by 30 μM anandamide and subsequent 500 nM capsaicin applications were also recorded when 200 nM rimonabant (Rimo), a highly selective and specific antagonist/inverse agonist of the CB1 receptor was continuously applied to the cells through the bath solution in order to find the effect of the CB1 receptor on anandamide- and capsaicin-evoked responses in ACR and COR neurons. The presence of rimonabant in the bath solution does not change the ratio of ACR and COR neurons (p = 1, Fisher’s exact test). (B) Average maximum amplitudes of anandamide-evoked currents produced by cultured rat PSN in the control medium and in the presence of 200 nM rimonabant (Rimo). Rimonabant significantly reduces the amplitude of anandamide-evoked currents (p = 0.006, Student’s t-test). (C) Average maximum amplitudes of capsaicin-evoked currents produced by ACR and COR type cultured rat PSN in control medium and in the presence of 200 nM rimonabant (Rimo). The average maximum amplitude of the capsaicin-evoked currents in COR type neurons is significantly smaller (^$p = 0.002, Student’s t-test) than that in ACR type neurons in the control bath solution. While the presence of rimonabant significantly reduces the average maximum amplitude of capsaicin-evoked currents in ACR neurons (*p = 0.001, Student’s t-test), it has no significant effect on the average maximum amplitude of capsaicin-evoked currents in COR neurons (p = 0.34, Student’s t-test).

Figure 3. Deletion of the CB1 receptor reduces both anandamide- and capsaicin-evoked responses in ACR neurons and changes the ACR – COR ratio.

(A) Conventional ratiometric approach was used to find the effect of deleting the CB1 receptor in Biozzi ABH mice on anandamide- and capsaicin-evoked calcium transients in ACR and COR type cultured PSN. The ratio of ACR and COR neurons in cultures prepared from wild type (WT-CB1) and CB1 receptor knock out (CB1^−/−) mice. Deletion of the CB1 receptor significantly reduces the number of ACR neurons (p < 0.0001, Fisher’s exact test), whereas it significantly increases the number of COR neurons (p < 0.0001, Fisher’s exact test). The overall number of capsaicin-sensitive cells (ACR + COR) however is not changed significantly (p = 0.56, Fischer’s exact test, see Results). (B) Average amplitudes of anandamide-evoked calcium transients in ACR neurons collected from wild type (WT-CB1) and CB1 receptor knock out (CB1^−/−) mice. Deletion of the CB1 receptor results in a significant reduction in anandamide-evoked calcium transients (p = 0.02, Student’s t-test). (C) Average amplitudes of capsaicin-evoked calcium transients in ACR and COR neurons collected from wild type (WT-CB1) and CB1 receptor knock out (CB1^−/−) mice. Deletion of the CB1 receptor results in significantly reduced amplitude of capsaicin-evoked calcium transients in ACR (p = 0.01, Student’s t-test) but not in COR neurons (p = 0.42, Student’s t-test).

Figure 4. Capsaicin-evoked Ca²⁺ transients are significantly reduced by rimonabant in ACR but not in COR cultured rat PSN.

(A) Conventional ratiometric approach was used to find the effect of the CB1 receptor antagonist/inverse agonist rimonabant (200 nM; RIMO) on capsaicin-evoked calcium transients in cultured rat PSN. (A) 100 nM capsaicin (CAP) was applied in every 2 minutes followed by 30 μM anandamide (A) and 50 mM KCl. The recording shows calcium transients evoked by consecutive capsaicin application in an ACR type neuron in a control experiment when no rimonabant was applied. Capsaicin induces the typical desensitisation. (B) During the recording from another ACR type neuron, rimonabant (RIMO) was applied immediately after the first capsaicin application until the end of the second capsaicin application. Rimonabant reduces the amplitude of the Ca²⁺ transient evoked by the second capsaicin application (arrow). In this neuron, the capsaicin-evoked response exhibits partial recovery following the removal of rimonabant. (C) Average amplitudes of calcium transients evoked by consecutive application of 100 nM capsaicin in every 2 minutes in ACR type neurons either without (C; black bars) or with the application of 200 nM rimonabant (R; red bars). In control experiments capsaicin induces significant desensitisation (*p < 0.0001, Student’s t-test both for the second and third responses). Rimonabant produces a significant reduction in the amplitude of the calcium transients evoked by the second (^$p = 0.0001, Student’s t-test) and third capsaicin (^$p < 0.0001, Student’s t-test) applications. (D) Calcium transients evoked by consecutive capsaicin application in a COR type neuron (note the lack of response to anandamide). (E) Rimonabant (RIMO) has no effect on the amplitude of the Ca²⁺ transient evoked by the second capsaicin application in a COR neuron. (F) Average amplitudes of calcium transients evoked by consecutive application of 100 nM capsaicin in every 2 minutes in COR type neurons either without (C; black bars) or with the application of 200 nM rimonabant (R; red bars). In control experiments capsaicin induced desensitisation (*p < 0.0001, Student’s t-test). Rimonabant does not produce any significant change in the amplitude of the calcium transients evoked by the capsaicin applications.

Figure 5. The CB1 receptor is expressed by all ACR type and the majority of COR type cultured rat cultured PSN.

(A) After determining the types of cultured rat PSN by whole-cell voltage-clamp recordings by anandamide (30 μM) and capsaicin (500 nM) application, 76 cells were collected for single-cell PCR. 10 COR type, 11 ACR type and 8 “anandamide- and capsaicin non-responding” neurons (NR) were successfully processed to find out whether the neuron expressed the CB1 receptor. The sizes of the amplicons generated to specific primers for CB1 receptor (127 bp) or the house-keeping molecule GAPDH (380 bp) were indistinguishable from the predicted sizes as it is shown by these typical gel images. L indicates the size marker. Note the presence of the GAPDH and the lack of CB1 amplicon in a COR neuron. (B) All the ACR cells and 8 of 10 COR neurons express the CB1 receptor in this sample of neurons. In addition 50% of the non-responding cells also express the CB1 receptor.

Figure 6. 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.

Figure 7. Morphometric analysis of electron microscopic images reveals three types of neurons.

(A) Based on results of the multivariate statistical tool principal component analysis (PCA; see Supplementary Figure 11) we built a partial least square discriminant analysis (PLS-DA) model to find classify the groups found by PCA. PLS-DA identified three principal components which together accounted for ~80% of the variability of data. (B) The PLS-DA score plot reveals the presence of well-separated three groups of neurons. (C) The loading plot shows the contribution of the variables to the separation of the three groups. (D) The variable importance on projection plot shows that all variables contributed significantly to the separation of the groups. The contribution of TRPV1-CB1 average distance (TR-CB dist), TRPV1 density (TR dens) and CB1 receptor density (CB dens) is highly significant. TR-CB crit: number of TRPV1-CB receptor critical distances; TR-TR crit: number of TRPV1-TRPV1 critical distances; TR-TR dist: TRPV1-TRPV1 average distance; CB-TR dist: CB1 receptor-TRPV1 average distance. (E,F) Bar charts showing average values of morphometric data of neurons belonging to the three groups shown in (B). Statistical analysis (ANOVA followed by Fischer’s post-hoc test) shows that neurons is Group 1 (yellow) are different from neurons both in Group 2 (red) and 3 (purple) in CB1 receptor density (CB dens), TRPV1 – CB1 receptor average distances (TR-CB dist) and the number of TRPV1 – CB1 receptor critical distances (TR-CB crit). Neurons in Group 2 (red) are different from neurons in Group 3 (purple) mainly in the distribution of TRPV1 (TRPV1 density (TR dens); TRPV1-TRPV1 average distance (TR-TR dist) and the number of TRPV-TRPV1 critical distance (TR-TR crit)). CB1 receptor-TRPV1 average distances (CB-TR dist) are also different between neurons in Group 2 and 3. distribution. The number of CB1 receptor TRPV1 critical distances (CB-TR crit) is not different between the 3 groups.

Figure 8. TRPV1 and the CB1 receptor are engaged in PSN.

Immunoblots showing TRPV1/CB1 receptor heteromers in the rat DRG. Whole cell lysates were prepared from DRG and immunoprecipitation assays using anti-CB1 (A) and anti-TRPV1 (B) antibodies were carried out as detailed in Materials and Methods. The resulting immune complexes were analysed by immunoblotting using anti-TRPV1 (A) and anti-CB1 (B) antibodies. Immunoprecipitating antibodies and immunoblotting antibodies were omitted from positive and negative controls, respectively. TRPV1 was detected as a ~100 kDa band, while the CB1 receptor was detected as a doublet with molecular weight ~55 and ~63 kDa in the immunoprecipitated samples.