Role of ventral medial prefrontal cortex in incubation of cocaine craving

Abstract

Cue-induced drug-seeking in rodents progressively increases after withdrawal from cocaine, suggesting that cue-induced cocaine craving incubates over time. Here, we explored the role of the medial prefrontal cortex (mPFC, a brain area previously implicated in cue-induced cocaine seeking) in this incubation. We trained rats to self-administer cocaine for 10days (6h/day, infusions were paired with a tone-light cue), and then assessed after 1 or 30 withdrawal days the effect of exposure to cocaine cues on lever presses in extinction tests. We found that cue-induced cocaine-seeking in the extinction tests was higher after 30 withdrawal days than after 1day. The time-dependent increases in extinction responding were associated with large (ventral mPFC) or modest (dorsal mPFC) increases in ERK phosphorylation (a measure of ERK activity and an index of neuronal activation). After 30 withdrawal days, ventral but not dorsal injections of muscimol+baclofen (GABAa+GABAb receptor agonists that inhibit neuronal activity) decreased extinction responding. After 1 withdrawal day, ventral but not dorsal mPFC injections of bicuculline+saclofen (GABAa+GABAb receptor antagonists that increase neuronal activity) strongly increased extinction responding. Finally, muscimol+baclofen had minimal effect on extinction responding after 1day, and in cocaine-experienced rats, ventral mPFC injections of muscimol+baclofen or bicuculline+saclofen had no effect on lever presses for an oral sucrose solution. The present results indicate that ventral mPFC neuronal activity plays an important role in the incubation of cocaine craving.

Figure 1. Effect of cue-induced cocaine seeking in extinction tests on p-ERK levels

(A) Outline of experimental timeline. Four groups of rats self-administered cocaine for 6 h/d for 10 d. On withdrawal days 1 or 30, the rats were either tested or not tested for cocaine seeking in extinction tests, and their brains were removed for p-ERK analysis. (B) Test for cue-induced cocaine seeking: data are mean±sem number of lever-presses on the previously active lever and on the inactive lever during the tests for cocaine seeking performed under extinction conditions after 1 day or 30 days of withdrawal. During the test sessions, cocaine was not available and lever presses resulted in the delivery of the tone-light cue previously paired with cocaine. All rats were chronically housed in the self-administration chambers during training; the rats in the 30 days withdrawal groups were housed in their home cage in the animal facility after the self-administration training. Non-reinforced lever responding after 30 withdrawal days was higher than after 1 withdrawal day. * Different from Day 1, p<0.01, n = 9–11 per group). (C-D) p-ERK levels in dorsal or ventral mPFC on day 1 or 30 of withdrawal in the test or no test conditions; data are depicted as percent of mean Day 1 No Test condition. p-ERK positive cells are indicated with white arrows. Rats in the Extinction Test condition were trained to self-administer cocaine and were exposed to the cocaine cues in a 30 min extinction test after 1 or 30 withdrawal days. Rats in the No Test condition were trained to self-administer cocaine and were not exposed to the cocaine cues after 1 or 30 withdrawal days. Exposure to cocaine cues in the extinction tests increased the number of p-ERK positive cells (white arrows) in both dorsal and ventral mPFC after 30 days but not 1 day of withdrawal; this effect was more pronounced in ventral mPFC than in dorsal mPFC. Abbreviations: ACg, Anterior cingulate cortex; PL, Prelimbic cortex; IL, infralimbic cortex. # Different from the other 3 groups, p<0.05.

Figure 2. Effect of dorsal and ventral mPFC muscimol+baclofen injections on day 30 extinction responding

(A–B) Test for cocaine seeking after dorsal or ventral mPFC injections of vehicle or a mixture of muscimol+baclofen (0.03+0.3 nmol/side). Data are mean±SEM number of non-reinforced presses on the previously active lever and on the inactive lever during the extinction tests. (C–D) p-ERK levels in the dorsal and ventral mPFC after dorsal or ventral mPFC injections of vehicle or muscimol+baclofen. Data are depicted as a percent of the mean±sem of the vehicle condition. Ventral but not dorsal mPFC injections of muscimol-baclofen significantly decreased extinction responding; both ventral and dorsal mPFC injections of muscimol+baclofen significantly decreased local p-ERK levels. (E) Approximate anatomical location of the injectors for vehicle (open circles) and muscimol+baclofen (closed circles). Numbers indicate the approximate anterior-posterior distance in mm from Bregma. * Different from vehicle, p<0.05 (n=8–12 per group).

Figure 3. Effect of dorsal and ventral mPFC bicuculline+saclofen injections on day 1 extinction responding

(A–B) Test for cocaine seeking after dorsal or ventral mPFC injections of vehicle or a mixture of bicuculline+saclofen (50+50 ng/side). Data are mean±SEM number of non-reinforced presses on the previously active lever and on the inactive lever during the extinction tests. (C–D) pERK levels in the dorsal and ventral mPFC after dorsal or ventral mPFC injections of vehicle or bicuculline+saclofen. Data are depicted as a percent of the mean±SEM of the vehicle condition. Ventral but not dorsal mPFC injections of bicuculline+saclofen increased extinction responding; both ventral and dorsal mPFC injections of bicuculline+saclofen increased local p-ERK levels. (E) Approximate anatomical location of the injectors for vehicle (open circles) and (closed circles) bicuculline+saclofen. Numbers indicate approximate anterior-posterior distance in mm from Bregma. * Different from vehicle, p<0.05 (n= n=5–6 and 12–13 for dorsal and ventral mPFC groups, respectively).

Figure 4. Effect of ventral mPFC muscimol+baclofen or bicuculline+saclofen injections on sucrose self-administration

Data are mean±SEM number of responses on the active and inactive levers after bilateral injections of vehicle, muscimol+baclofen (0.03+0.3 nmol/side), and bicuculline+saclofen (50+50 ng/side) (n=5). No significant effects were observed.