Endocannabinoid regulation of acute and protracted nicotine withdrawal: effect of FAAH inhibition

Abstract

Evidence shows that the endocannabinoid system modulates the addictive properties of nicotine. In the present study, we hypothesized that spontaneous withdrawal resulting from removal of chronically implanted transdermal nicotine patches is regulated by the endocannabinoid system. A 7-day nicotine dependence procedure (5.2 mg/rat/day) elicited occurrence of reliable nicotine abstinence symptoms in Wistar rats. Somatic and affective withdrawal signs were observed at 16 and 34 hours following removal of nicotine patches, respectively. Further behavioral manifestations including decrease in locomotor activity and increased weight gain also occurred during withdrawal. Expression of spontaneous nicotine withdrawal was accompanied by fluctuation in levels of the endocannabinoid anandamide (AEA) in several brain structures including the amygdala, the hippocampus, the hypothalamus and the prefrontal cortex. Conversely, levels of 2-arachidonoyl-sn-glycerol were not significantly altered. Pharmacological inhibition of fatty acid amide hydrolase (FAAH), the enzyme responsible for the intracellular degradation of AEA, by URB597 (0.1 and 0.3 mg/kg, i.p.), reduced withdrawal-induced anxiety as assessed by the elevated plus maze test and the shock-probe defensive burying paradigm, but did not prevent the occurrence of somatic signs. Together, the results indicate that pharmacological strategies aimed at enhancing endocannabinoid signaling may offer therapeutic advantages to treat the negative affective state produced by nicotine withdrawal, which is critical for the maintenance of tobacco use.

Figure 1. Spontaneous nicotine withdrawal measured after 7 days of nicotine exposure (5.2 mg/rat/day).

Rats previously exposed to nicotine showed (A) increased occurrence of overall withdrawal signs compared to non-exposed controls. Overall withdrawal signs were obtained for each animal by accumulating the number of events belonging to different categories of somatic withdrawal manifestations (wet dog shakes/tremors, gasp/writhes, chattering/chews) scored at 16 hours from nicotine discontinuation. Nicotine exposed rats also showed: (B) decreased distance (cm) traveled in the open field at the 16-hour time point; (C) reduced percentage (%) in open arms time measured at 34 hours from nicotine patch removal, and (D) rapid body weight gain (g). Values represent the mean (±SEM) of N = 6 subjects per group. In A, B and C, *p<0.05, **p<0.01 difference vs. control. In D, **p<0.01, ***p<0.001 difference vs. nicotine group at T = 0 h.

Figure 2. Spontaneous nicotine withdrawal measured 16 hours after nicotine patches (5.2 mg/rat/day) removal was not prevented by administration of URB597 (0.0, 0.1 and 0.3 mg/kg, i.p.).

(A) overall withdrawal symptoms, (B) wet dog shakes/tremors, (C) chattering/chews, (D) gasp/writhes, (E) yawns. Values represent the mean (±SEM) of N = 8 subjects per group. #p<0.05, ##p<0.01, difference vs. “C+0.0” group.

Figure 3. URB597 blocked nicotine withdrawal-induced anxiety-like behavior, as assessed in the elevated plus-maze and in the shock-probe defensive burying tests 34 hours after nicotine discontinuation.

(A) Animals previously exposed to nicotine (N+0.0) showed significant reduction in percent (%) open arm time compared to controls (C+0.0). URB597 returned % open arms time to control levels. Values represent the mean (±SEM) of N = 10–11 subjects per group. (B) Animals previously exposed to nicotine (N+0.0) showed significant increase in burying time (seconds) compared to controls (C+0.0). URB597 dose-dependently returned burying time to control levels. Values represent the mean (±SEM) of N = 7–8 subjects per group; *p<0.05, **p<0.01 compared to nicotine exposed receiving vehicle; #p<0.05, difference from non-nicotine exposed controls.