Involvement of cannabinoid type 1 receptor in fasting-induced analgesia

Abstract

The endocannabinoid system (ECS) is known to modulate not only food intake but also pain, especially via the cannabinoid type 1 receptor (CB1R) expressed throughout the central nervous system and the peripheral tissues. Our previous study demonstrated that fasting produces an analgesic effect in adult male mice, which is reversed by intraperitoneal (i.p.) administration of CB1R antagonist (SR 141716). In the present study, we further examined the effect of CB1R expressed in the peripheral tissues. In the formalin-induced inflammatory pain model, i.p. administration of peripherally restricted CB1R antagonist (AM 6545) reversed fasting-induced analgesia. However, intraplantar administration of SR 141716 did not affect fasting-induced analgesia. Furthermore, mRNA expression of CB1R did not change in the formalin model by fasting in the dorsal root ganglia. The formalin-induced c-Fos expression at the spinal cord level was not affected by fasting, and in vivo recording from the superficial dorsal horn of the lumbar spinal cord revealed that fasting did not affect formalin-induced neural activity, which indicates minimal involvement of the spinal cord in fasting-induced analgesia. Finally, when we performed subdiaphragmatic vagotomy to block the hunger signal from the gastrointestinal (GI) system, AM 6545 did not affect fasting-induced analgesia, but SR 141716 still reversed fasting-induced analgesia. Taken together, our results suggest that both peripheral and central CB1Rs contribute to fasting-induced analgesic effects and the CB1Rs in the GI system which transmit fasting signals to the brain, rather than those in the peripheral sensory neurons, may contribute to fasting-induced analgesic effects.

Keywords: Fasting-induced analgesia; cannabinoid receptor 1; endocannabinoid system; vagotomy.

Figure 1.

The effect of peripherally restricted CB1R antagonist on fasting-induced analgesia in formalin-induced acute inflammatory pain model. (A) Experimental design and schedule for formalin test. (B) Time course of spontaneous pain behavior following intraplantar (i.pl) injection of formalin in free-fed and 24 h fasted mice who received either AM 6545 or SR 141716, respectively. We adopted the result of SR 141716 from our recent publication (“The analgesic effect of refeeding on acute and chronic inflammatory pain” by Jeong-Yun Lee and Grace J. Lee et al. is licensed under CC BY 4.0). (C) Formalin-induced pain behavior was divided into two phases and analyzed. As compared with free fed-vehicle groups, formalin-induced pain behavior decreased in the 24 h fasted-vehicle group only at 2 phase. As compared with 24 h fasted-vehicle group, AM 6545 and SR 141716 reversed fasting-induced analgesia. ***p<0.001, ****p<0.0001 ((C) one-way ANOVA followed by Tukey test).

Figure 2.

The effect of fasting on nociceptive signaling in the peripheral sensory neurons in formalin-induced acute inflammatory pain model. (A) The effect of intraplantar (i.pl) injection of CB1R antagonist on fasting-induced analgesia. Experimental design and schedule for formalin test (a). Time course of spontaneous pain behavior following injection of formalin (b, c). Formalin-induced pain behavior was divided into two phases, and the second phase was analyzed (d). SR 141716 (i.pl., 10 µg) did not affect the analgesic effect of fasting in the formalin-induced acute inflammatory pain model. (B) The mRNA expression of CB1R (cnr1) (a) and activating transcription factor 3 (ATF3) (b) in DRG. 24 h fasting did not affect the mRNA expression of cnr1 and ATF3 in formalin-induced acute inflammatory pain model ((A), (B) unpaired t-test).

Figure 3.

The effect of fasting on nociceptive signaling in the spinal cord in the formalin-induced acute inflammatory pain model. (A) Formalin-induced c-Fos protein expression in the spinal cord of lumbar (L4-L5) segments. Representative expression of c-Fos protein in L4-L5 (a). The quantification of c-Fos protein expression (b). The total number of c-Fos positive neuron from lamina I to VI and the number of c-Fos positive neuron from lamina I to II (superficial dorsal horn) were counted. (B) Formalin-induced c-Fos mRNA expression in the spinal cord of lumbar segments. As compared with the free-fed group, formalin-induced c-Fos expression was not different in the 24 h fasted group. (C) An example of continuous chart recording showing spinal neuronal unit firings in response to cutaneous formalin injection in free-fed mice (a). The lowest trace in the right are shown in an expanded timescale. Arrowheads indicate unit neuronal firings during the action (in the 2^nd phase) of formalin. The time-course of the average number of unit firing shown in (Ca) showing a formalin-induced biphasic response (b). Insets show ten superimposed single unit firings indicated by arrowheads in a. 24 fasted mice also showed a similar formalin-induced biphasic response in the spinal dorsal horn (see Results). The number of unit firings in the 2^nd phase between free-fed and 24 fasted groups was not different (c) ((A), (B), (C) unpaired t-test).

Figure 4.

The effect of subdiaphragmatic vagotomy on fasting-induced analgesia in formalin-induced acute inflammatory pain model. (A) Time course of spontaneous pain behavior following intraplantar injection of formalin. (B) Formalin-induced pain behavior was divided into two phases, and the second phase was analyzed. (C) As compared to the sham free-fed group, subdiaphragmatic vagotomy significantly reduced formalin-induced pain behavior. Following subdiaphragmatic vagotomy, AM 6545 did not affect fasting-induced analgesia. SR 141716 reversed the effect of fasting/vagotomy-induced analgesia ***p<0.001, ****p<0.0001 ((B) one-way ANOVA followed by Tukey test).