Blockade of Cannabinoid CB1 Receptors in the Dorsal Periaqueductal Gray Unmasks the Antinociceptive Effect of Local Injections of Anandamide in Mice

Abstract

Divergent results in pain management account for the growing number of studies aiming at elucidating the pharmacology of the endocannabinoid/endovanilloid anandamide (AEA) within several pain-related brain structures. For instance, the stimulation of both Transient Receptor Potential Vanilloid type 1 (TRPV1) and Cannabinoid type 1 (CB1) receptors led to paradoxical effects on nociception. Here, we attempted to propose a clear and reproducible methodology to achieve the antinociceptive effect of exogenous AEA within the dorsal periaqueductal gray (dPAG) of mice exposed to the tail-flick test. Accordingly, male Swiss mice received intra-dPAG injection of AEA (CB1/TRPV1 agonist), capsaicin (TRPV1 agonist), WIN (CB1 agonist), AM251 (CB1 antagonist), and 6-iodonordihydrocapsaicin (6-IODO) (TRPV1 selective antagonist) and their nociceptive response was assessed with the tail-flick test. In order to assess AEA effects on nociception specifically at vanilloid or cannabinoid (CB) substrates into the dPAG, mice underwent an intrinsically inactive dose of AM251 or 6-IODO followed by local AEA injections and were subjected to the same test. While intra-dPAG AEA did not change acute pain, local injections of capsaicin or WIN induced a marked TRPV1- and CB1-dependent antinociceptive effect, respectively. Regarding the role of AEA specifically at CB/vanilloid substrates, while the blockade of TRPV1 did not change the lack of effects of intra-dPAG AEA on nociception, local pre-treatment of AM251, a CB1 antagonist, led to a clear AEA-induced antinociception. It seems that the exogenous AEA-induced antinociception is unmasked when it selectively binds to vanilloid substrates, which might be useful to address acute pain in basic and perhaps clinical trials.

Keywords: anandamide; antinociception; cannabinoid substrates; periaqueductal gray; vanilloid substrates.

FIGURE 1

Timeline of the tail-flick test showing basal TFLs (dark gray boxes) and test TFLs (light gray boxes) recordings as well as injection procedures performed at the Exps. 1–3.

FIGURE 2

(A) Schematic representation (left) and photomicrograph (right) of the mouse brain. Both frames (left and right) correspond to –4.16 mm from bregma. (B) Schematic representation of microinjections sites within dPAG. Number of dots are representative and fewer than the actual number of animals due to overlapping. Histology revealed that all positive injection sites were between –4.16 and –4.60 mm from bregma (based on Paxinos and Franklin, 2004).

FIGURE 3

Lack of effects of AEA (A) and AM251 (D) and effects of capsaicin (B), WIN (C), and 6-IODO (E) injected into the dPAG on the TFL of mice exposed to the tail-flick test. Microinjection was performed at “zero” time. N = 4–7. Dots in the line chart represent mean ± SEM. Two-way ANOVA (repeated measures) followed by Duncan post hoc test. ^∗P < 0.05 compared to vehicle-treated animals. ^#P < 0.05 compared to vehicle-treated animals until 20 min post-treatment.

FIGURE 4

Antinociceptive effects of intra-dPAG AM251 microinjection followed by local AEA on the TFL of mice exposed to the tail-flick test. Microinjections were performed at “zero” time. N = 4–7. Dots in the line chart represent mean ± SEM. ^∗P < 0.05 compared to vehicle-treated animals. Two-way ANOVA followed by Duncan post hoc test.

FIGURE 5

Lack of effects of intra-dPAG 6-IODO microinjection followed by local AEA on the TFL of mice exposed to the tail-flick test. Microinjections were performed at “zero” time. N = 4–6. Dots in the line chart represent mean ± SEM. Two-way ANOVA followed by Duncan post hoc test.