Role of Central Amygdala Neuronal Ensembles in Incubation of Nicotine Craving

Abstract

The craving response to smoking-associated cues in humans or to intravenous nicotine-associated cues in adult rats progressively increases or incubates after withdrawal. Here, we further characterized incubation of nicotine craving in the rat model by determining whether this incubation is observed after adolescent-onset nicotine self-administration. We also used the neuronal activity marker Fos and the Daun02 chemogenetic inactivation procedure to identify cue-activated neuronal ensembles that mediate incubation of nicotine craving. We trained adolescent and adult male rats to self-administer nicotine (2 h/d for 12 d) and assessed cue-induced nicotine seeking in extinction tests (1 h) after 1, 7, 14, or 28 withdrawal days. In both adult and adolescent rats, nicotine seeking in the relapse tests followed an inverted U-shaped curve, with maximal responding on withdrawal day 14. Independent of the withdrawal day, nicotine seeking in the relapse tests was higher in adult than in adolescent rats. Analysis of Fos expression in different brain areas of adolescent and adult rats on withdrawal days 1 and 14 showed time-dependent increases in the number of Fos-positive neurons in central and basolateral amygdala, orbitofrontal cortex, ventral and dorsal medial prefrontal cortex, and nucleus accumbens core and shell. In adult Fos-lacZ transgenic rats, selective inactivation of nicotine-cue-activated Fos neurons in central amygdala, but not orbitofrontal cortex, decreased "incubated" nicotine seeking on withdrawal day 14. Our results demonstrate that incubation of nicotine craving occurs after adolescent-onset nicotine self-administration and that neuronal ensembles in central amygdala play a critical role in this incubation.

Significance statement: The craving response to smoking-associated cues in humans or to intravenous nicotine-associated cues in adult rats progressively increases or incubates after withdrawal. It is currently unknown whether incubation of craving also occurs after adolescent-onset nicotine self-administration. The brain areas that mediate such incubation are also unknown. Here, we used a rat model of incubation of drug craving, the neuronal activity marker Fos, and the Daun02 chemogenetic inactivation method to demonstrate that incubation of nicotine craving is also observed after adolescent-onset nicotine self-administration and that neuronal ensembles in the central nucleus of the amygdala play a critical role in this incubation in adult rats.

Keywords: Daun02; Fos; central amygdala; incubation of craving; nicotine; orbitofrontal cortex.

Figure 1.

Incubation of nicotine craving in adolescent and adult rats. A, Timeline of the experimental procedure. B, Nicotine self-administration training in adolescents (n = 43) and adults (n = 43) rats. Data are mean ± SEM numbers of infusions and active and inactive lever presses during the 12 training days. C, Nicotine seeking in the extinction tests at different withdrawal days. Data are mean ± SEM numbers of nonreinforced presses on the previously active lever and on the inactive lever. *Different from day 1, p < 0.05 within each age group, p < 0.05. n = 7–13 rats per group. SA, Self-administation.

Figure 2.

Incubation of nicotine craving in adolescent and adult rats is associated with increased neuronal activity in mPFC, OFC, NAc, and amygdala. Data are mean ± SEM numbers of Fos-IR neurons in dorsal and ventral mPFC (A, B), OFC (C), NAc core and shell (D, E), and central and basolateral amygdala (F, G) of adolescent and adult rats exposed to the extinction tests on withdrawal days 1 or 14. Left, Mean ± SEM Fos-labeled neurons. Center, Representative photomicrographs of Fos expression from the different groups (scale bar, 100 μm). Right, Sampling regions. *Different from day 1 within each age group, p < 0.05. n = 10–13 per group. SA, Self-administation.

Figure 3.

Double-labeling of Fos with β-gal and NeuN in the OFC and CeA. A, percentage of Fos + β-gal and Fos + NeuN double-labeled neurons in OFC and CeA in adolescent (left) and adult rats (right) perfused after extinction tests on withdrawal day 14. Pictures of double-labeling of adult Fos + β-gal and Fos + NeuN in the OFC (B, C) and CeA (D, E) of adult Fos-lacZ rats that underwent an extinction test on withdrawal day 14. Red, β-gal or NeuN (top); green, Fos (middle); yellow, Fos + β-gal and Fos + NeuN double-labeled neurons (bottom). Scale bar, 50 μm. F, Brain images indicating sampling regions for the OFC (top) and CeA (bottom).

Figure 4.

Daun02 injections into the OFC of adult Fos-lacZ rats had no effect on incubated nicotine seeking on withdrawal day 14. A, Timeline of the experimental procedure. B, Nicotine self-administration. Data are mean ± SEM of nicotine infusions and active and inactive lever presses of all rats (total n = 29) during the 12 training days. C, Extinction responding. Data are mean ± SEM of presses on the previously active lever and on the inactive lever during the extinction test after 14 withdrawal days. D, Cannula placements. Numbers indicate millimeters from bregma. E, β-gal expression. Left, Mean± SEM β-gal-positive neurons in the OFC in the four groups. Right, Representative photomicrographs of β-gal expression in the OFC of rats in the different treatment groups. We injected the rats with Daun02 or vehicle 75 min after a short 15 min extinction session in the nicotine self-administration context or exposure to a novel context on withdrawal day 11. n = 6–8 per group.

Figure 5.

Daun02 injections into the CeA of adult Fos-lacZ rats decreased incubated nicotine seeking on withdrawal day 14. A, Timeline of the experimental procedure. B, Nicotine self-administration. Data are mean ± SEM of nicotine infusions and active and inactive lever presses of all rats (total n = 42) during the 12 training days. C, Extinction responding. Data are mean ± SEM of presses on the previously active lever and on the inactive lever during the extinction test after 14 withdrawal days. D, Cannula placements. Numbers indicate mm from bregma. E, β-gal expression. Left, Mean ± SEM β-gal-positive neurons in the CeA in the four groups. Right, Representative photomicrographs of β-gal expression in the CeA of rats in the different treatment groups. We injected the rats with Daun02 or vehicle 75 min after a short 15 min extinction session or exposure to a novel context on withdrawal day 11. n = 9–12 per group.