Ventral tegmental afferents in stress-induced reinstatement: the role of cAMP response element-binding protein

Abstract

The transcription factor cAMP response element-binding protein (CREB) is required for stress- but not drug-induced reinstatement of cocaine conditioned place preference. To reveal the neural circuitry associated with this CREB dependence, we injected a retrograde tracer into the ventral tegmental area (VTA) and identified afferents that were activated after stress or cocaine exposure in both naive and cocaine-conditioned mice. Neuronal activation, as assessed by Fos expression, was greatly reduced in the dorsal and ventral bed nucleus of the stria terminalis (BNST), lateral septum, and nucleus accumbens shell in mice lacking CREB (CREBαΔ mice) after a 6 min swim stress but not after cocaine exposure (20 mg/kg). Additionally, activation of VTA afferent neurons in the ventral BNST and the infralimbic cortex in CREBαΔ mice was blunted in response to stress. This pattern of neuronal activation persisted in mice that were conditioned to a cocaine place preference procedure before stress exposure. Furthermore, lidocaine inactivation (0.4 μl, 4%) studies demonstrated the necessity of BNST activation for swim-stress-induced reinstatement of cocaine-conditioned reward. Together, the present studies demonstrate that CREB is required for the activation of a unique circuit that converges on the dopamine reward pathway to elicit reinstatement of drug reward and points to the BNST as a key intersection between stress and reward circuits.

Figure 1.

CREB mutant mice exhibit decreased Fos protein expression in response to stress but not to cocaine. Although wild-type mice exhibit an increase in Fos protein in the ventral (a) and dorsal (b) BNST, lateral septum (c), and the nucleus accumbens shell (d) in response to forced swim stress, CREBαΔ mutant mice do not. This decreased stress response is not seen in the nucleus accumbens core (e), lateral hypothalamus (f), prelimbic cortex (g), or infralimbic cortex (h). No differences were seen between the genotypes in response to an acute cocaine injection (a–h). *p < 0.05, **p < 0.01, ***p < 0.001 compared with control; ^#p < 0.05 pairwise comparison for wild-type versus mutant forced swim test (FST).

Figure 2.

Fos immunoreactivity after swim stress and acute cocaine administration. Example photomicrographs demonstrating that wild-type mice exhibit an increase in Fos protein expression within the vBNST (b, d) and IL (g, i) after forced swim stress or acute cocaine administration. CREBαΔ mutant mice do not show stress-induced increases in Fos protein expression within the vBNST (c), whereas expression within the IL remains intact (h). No differences are seen in the response to acute cocaine in CREBαΔ mutant mice (e, j) FST, Forced swim test.

Figure 3.

Naive CREB mutant mice exhibit a decrease in immobility during swim stress compared with wild-type littermates; however, conditioned place preference-experienced animals do not. Throughout the forced swim test in experiment I (a), mutant mice exhibited lower levels of immobility than their wild-type littermates. However, in experiment II (b), no differences were seen between the genotypes. *p < 0.05, **p < 0.01, ***p < 0.001 compared with wild type.

Figure 4.

Animals with Fluorogold injections that filled the VTA unilaterally were included in double-labeled analysis. Schematic representation of injection sites from wild-type (a–c) and mutant (d–f) mice that were exposed to the forced swim test (FST). The injection sites and immediate spread of FG for individual animals are indicated by black (WT) or gray (mutant) circles and corresponding animal numbers. Cocaine and control injection sites were comparable.

Figure 5.

CREB mutant mice exhibit a decrease in stress-activated cells that project to the VTA. Although wild-type mice exhibit an increase in Fos-activated cells that project to the ventral tegmental area (doubled-labeled Fos/FG cells) in the ventral BNST (a) and the infralimbic cortex (b), CREBαΔ mutant mice do not. Although no differences were seen between the genotypes in response to acute cocaine, there was an increase in double-labeled cells in two regions that did not show stress-induced alterations, the dorsal BNST (c) and the lateral hypothalamus (d). **p < 0.01, ***p < 0.001 compared with control; ^#p < 0.05 pairwise comparison for wild-type versus mutant forced swim test (FST).

Figure 6.

Double labeling of Fos and Fluorogold immunoreactivity after forced swim stress. Example photomicrographs demonstrating the increase in Fos protein expression within VTA afferents in the vBNST (b) and IL (e) of wild-type mice after forced swim stress (FST). These specific increases in Fos immunoreactivity within cells that project to the VTA was not seen within CREBαΔ mutant mice (c, f). AC, Anterior commissure.

Figure 7.

Cocaine-induced conditioned place preference behavior in both mutant and wild-type mice and extinction training eliminated this preference. Both wild-type and mutant mice paired with cocaine showed a significant place preference to the cocaine-paired side on test day (drug, F_(1,72) = 43.12, p < 0.0001). After extinction training, this preference was no longer present. ***p < 0.001 compared to PreTest.

Figure 8.

CREB mutant mice exhibit blunted stress-induced Fos protein expression independent of previous drug conditioning. Although wild-type mice exhibit an increase in Fos protein in the ventral (a) and dorsal (b) BNST, lateral septum (c), and the nucleus accumbens shell (d) in response to forced swim stress, CREBαΔ mutant mice do not. Although no interactions were present between genotype and drug pretreatment, a decrease in stress-induced Fos expression in the ventral and dorsal BNST was present in both genotypes after cocaine conditioning. Comparable stress-induced Fos expression was seen in both genotypes in the nucleus accumbens core (e), lateral hypothalamus (f), prelimbic cortex (g), or infralimbic cortex (h). *p < 0.05, **p < 0.01 compared with No Stress condition, #p < 0.05, ##p < 0.01 genotype comparison of wild type versus mutant within the FST groups.

Figure 9.

CREB mutant mice exhibit a decrease in stress-activated cells that project to the VTA independent of previous drug conditioning. Although wild-type mice exhibit an increase in Fos-activated cells that project to the ventral tegmental area (doubled-labeled Fos/FG cells) in the ventral (a) and dorsal (b) BNST, lateral hypothalamus (c), and the infralimbic cortex (d), CREBαΔ mutant mice do not. No differences in Fos-activated cells that project to the VTA were seen as a result of drug treatment during CPP. *p < 0.05, ***p < 0.001 compared with No Stress condition; ##p < 0.01, ###p < 0.001 genotype comparison of wild type versus mutant within the FST groups.

Figure 10.

Stress-induced reinstatement requires BNST activation. a, Vehicle- and lidocaine-treated mice exhibit similar levels of preference for the cocaine-paired side on the posttest day compared with their preferences before conditioning and similar levels of extinction. However, on reinstatement test day, only vehicle-treated animals exhibit a significant preference for the drug-paired side. **p < 0.01, extinction versus reinstatement; ***p < 0.001, posttest versus pretest. b, Microinjector placements within the BNST for all cocaine-conditioned animals.

Figure 11.

Inactivation of the BNST increases swimming in response to a forced swim stress. When lidocaine was infused into the BNST, mice exhibited a decrease in the time spent immobile during the last 4 min of a 6 min swim stress. No changes were seen when lidocaine was infused into the lateral ventricle. *p < 0.05, lidocaine versus vehicle.

Figure 12.

Inactivation of the BNST blunts activation of stress-induced reinstatement circuitry. Lidocaine-treated mice exhibit a blunted response to stress-induced reinstatement as indicated by a decrease in the number of Fos-positive cells within the VTA (a), CeA (b), and NAc core (c). No differences were seen between the groups within the NAc shell (d), prelimbic (e) or infralimbic (f) cortices, or the lateral hypothalamus (g). *p < 0.05, vehicle versus lidocaine.