Nicotine-induced antinociception, rewarding effects, and physical dependence are decreased in mice lacking the preproenkephalin gene

Abstract

It has been shown previously that the endogenous opioid system may be involved in the behavioral effects of nicotine. In the present study, the participation of endogenous enkephalins on nicotine responses has been investigated by using preproenkephalin knock-out mice. Acute nicotine-induced hypolocomotion remained unaffected in these mice. In contrast, antinociception elicited in the tail-immersion and hot-plate tests by acute nicotine administration was reduced in mutant animals. The rewarding properties of nicotine were then investigated using the place-conditioning paradigm. Nicotine induced a conditioned place preference in wild-type animals, but this effect was absent in knock-out mice. Accordingly, in vivo microdialysis studies revealed that the enhancement in dopamine extracellular levels in the nucleus accumbens induced by nicotine was also reduced in preproenkephalin-deficient mice. Finally, the somatic expression of the nicotine withdrawal syndrome precipitated in nicotine-dependent mice by mecamylamine was significantly attenuated in mutant animals. In summary, the present results indicate that endogenous opioid peptides derived from preproenkephalin are involved in the antinociceptive and rewarding properties of nicotine and participate in the expression of physical nicotine dependence.

Figure 1.

Effects of acute nicotine on locomotion in preproenkephalin-deficient (black bars) and wild-type (white bars) mice. Horizontal (A) and vertical (B) locomotion were measured 5 min after nicotine administration (0, 1, 3, and 6 mg/kg, s.c.). Data are expressed as mean ± SEM of photocell counts during a 10 min period in wild-type (saline, n = 34; nicotine, 1 mg/kg, n = 21; nicotine, 3 mg/kg, n = 20; nicotine, 6 mg/kg, n = 15) and knock-out (saline, n = 33; nicotine, 1 mg/kg, n = 17; nicotine, 3 mg/kg, n = 18; nicotine, 6 mg/kg, n = 15) mice. ^★★p < 0.01, when compared with the saline group of the same genotype (Dunnett's test).

Figure 2.

Antinociceptive effects of acute nicotine in preproenkephalin-deficient (black bars) and wild-type (white bars) mice. Antinociceptive responses in the hot-plate (A) and tail-immersion (B) tests were measured at 15 and 16 min, respectively, after nicotine administration (0, 1, 3, and 6 mg/kg, s.c.). Data are expressed as mean ± SEM of percentage of maximum possible effect (MPE) in wild-type (saline, n = 34; nicotine, 1 mg/kg, n = 21; nicotine, 3 mg/kg, n = 19; nicotine, 6 mg/kg, n = 14) and knock-out (saline, n = 33; nicotine, 1 mg/kg, n = 17; nicotine, 3 mg/kg, n = 17; nicotine, 6 mg/kg, n = 15) mice. ^★★p < 0.01, when compared with the saline group of the same genotype. ^☆p < 0.05, comparison between genotypes (Dunnett's test).

Figure 3.

Rewarding effects of nicotine in preproenkephalin-deficient and wild-type mice on the place-conditioning paradigm. Place conditioning was induced by the administration of saline (Sal) (wild-type mice, n = 39; knock-out mice, n = 37) and 0.25 mg/kg (Nic 0.25) (wild-type mice, n = 15; knock-out mice, n = 14), 0.5 mg/kg (Nic 0.5) (wild-type mice, n = 24; knock-out mice, n = 19), and 1 mg/kg (Nic 1) (wild-type mice, n = 16; knock-out mice, n = 15) of nicotine (s.c.). The figure shows the time spent in the drug-paired compartment during preconditioning (white bars) and test (black bars) phases. All values are expressed as mean ± SEM. ^★★p < 0.01, paired two-tailed Student's t test.

Figure 4.

Effects of mecamylamine (1 mg/kg, s.c.) pretreatment on nicotine-induced conditioned place preference (0.5 mg/kg, s.c.) in wild-type animals. The figure shows the time spent in the drug-paired compartment during preconditioning (white bars) and test (black bars) phases. All values are expressed as mean ± SEM. ^★★p < 0.01, paired two-tailed Student's t test; n = 13-15 per group.

Figure 5.

A, Effects of nicotine (0.5 and 1 mg/kg, s.c.) and saline on DA concentrations in dialysates obtained by in vivo microdialysis from the NAc of preproenkephalin-deficient and wild-type mice. The arrow indicates nicotine or saline administration at time 0. Dialysate samples were taken every 15 min during 1.5 h before and 3 h after injection. B, Bar graphs of AUC values for the DA concentration in dialysates after nicotine or saline administration in knock-out (black bars) and wild-type (white bars) mice. All values are expressed as mean ± SEM. ^★★p < 0.01, when compared with the saline group of the same genotype; ^☆p < 0.05 comparison between genotypes (Dunnett's test).

Figure 6.

Representative coronal section (20 μm) of the mouse brain stained with cresyl violet illustrating the placement of the probe in the nucleus accumbens (bregma, +0.98) (A). In B, a diagram showing the representative probe placements between bregma +1.70 and +0.86 mm.

Figure 7.

Mecamylamine-precipitated nicotine withdrawal in preproenkephalin-deficient and wild-type mice. Abstinence was precipitated by acute mecamylamine administration (1 mg/kg, s.c.) after 6 d of nicotine perfusion (25 mg/kg/d) by using subcutaneous minipumps. A global withdrawal score was calculated for each animal by giving each individual sign a relative weight (A). A decrease in the number of scratches was the main change observed during nicotine withdrawal in mutant mice (B). Data are expressed as mean ± SEM in wild-type (white bars) and knock-out (black bars) mice (n = 12-15 mice for each group). ^★p < 0.05; ^★★p < 0.01, when compared with the saline group of the same genotype; ^☆p < 0.05; ^☆☆p < 0.01, comparison between genotypes (one-way ANOVA).

Figure 8.

Diagram showing the possible role of the endogenous enkephalinergic system in the modulation of nicotine positive-rewarding effects. Nicotine exposure could induce the release of endogenous enkephalins in the VTA by a direct mechanism or indirectly through glutamate transmission. The subsequent activation by these opioid peptides of μ-opioid receptors located in GABAergic neurons would remove the inhibitory effect that these GABAergic cells exert on VTA DA neurons, thus contributing to a net increase of DA release in the NAc. Ach, Acetylcholine; ENK, enkephalins; GLUT, glutamate; PFC, prefrontal cortex; PFC, prefrontal cortex; (-) indicates inhibition; (+) indicates stimulation. The arrows indicate the main possible sites of action of nicotine to explain its modulatory effects on opioid transmission.