Stress-Induced Reinstatement of Nicotine Preference Requires Dynorphin/Kappa Opioid Activity in the Basolateral Amygdala

Abstract

The dynorphin (DYN)/kappa-opioid receptor (KOR) system plays a conserved role in stress-induced reinstatement of drug seeking for prototypical substances of abuse. Due to nicotine's high propensity for stress-induced relapse, we hypothesized that stress would induce reinstatement of nicotine seeking-like behavior in a KOR-dependent manner. Using a conditioned place preference (CPP) reinstatement procedure in mice, we show that both foot-shock stress and the pharmacological stressor yohimbine (2 mg/kg, i.p.) induce reinstatement of nicotine CPP in a norbinaltorphimine (norBNI, a KOR antagonist)-sensitive manner, indicating that KOR activity is necessary for stress-induced nicotine CPP reinstatement. After reinstatement testing, we visualized robust c-fos expression in the basolateral amygdala (BLA), which was reduced in mice pretreated with norBNI. We then used several distinct but complementary approaches of locally disrupting BLA KOR activity to assess the role of KORs and KOR-coupled intracellular signaling cascades on reinstatement of nicotine CPP. norBNI injected locally into the BLA prevented yohimbine-induced nicotine CPP reinstatement without affecting CPP acquisition. Similarly, selective deletion of BLA KORs in KOR conditional knock-out mice prevented foot-shock-induced CPP reinstatement. Together, these findings strongly implicate BLA KORs in stress-induced nicotine seeking-like behavior. In addition, we found that chemogenetic activation of Gαi signaling within CaMKIIα BLA neurons was sufficient to induce nicotine CPP reinstatement, identifying an anatomically specific intracellular mechanism by which stress leads to reinstatement. Considered together, our findings suggest that activation of the DYN/KOR system and Gαi signaling within the BLA is both necessary and sufficient to produce reinstatement of nicotine preference.

Significance statement: Considering the major impact of nicotine use on human health, understanding the mechanisms by which stress triggers reinstatement of drug-seeking behaviors is particularly pertinent to nicotine. The dynorphin (DYN)/kappa-opioid receptor (KOR) system has been implicated in stress-induced reinstatement of drug seeking for other commonly abused drugs. However, the specific role, brain region, and mechanisms that this system plays in reinstatement of nicotine seeking has not been characterized. Here, we report region-specific engagement of the DYN/KOR system and subsequent activation of inhibitory (Gi-linked) intracellular signaling pathways within the basolateral amygdala during stress-induced reinstatement of nicotine preference. We show that the DYN/KOR system is necessary to produce this behavioral state. This work may provide novel insight for the development of therapeutic approaches to prevent stress-related nicotine relapse.

Keywords: basolateral amygdala; conditioned place preference; kappa opioid receptors; nicotine; reinstatement; stress.

Figure 1.

KOR activation is necessary for foot-shock stress-induced nicotine reinstatement. A, Timeline of behavioral testing schedule and protocol. B, Representative traces of locomotor behavior (distance traveled) during each stage of testing (pre, post, extinction, foot shock reinstatement, prime). C, Foot-shock stress before testing (day 8) reinstates nicotine CPP in a norBNI (10 mg/kg, i.p.)-sensitive manner. D, Locomotor responses are decreased during nicotine treatment (days 2 and 4, P.M. session) compared with saline-treated controls. E, Neither foot-shock stress nor norBNI pretreatment changes locomotor behavior during reinstatement testing. n = 12–16 per group, *p < 0.05.

Figure 2.

KOR activation is necessary for yohimbine-induced nicotine reinstatement. A, Experimental timeline for behavioral testing and protocols. B, Yohimbine (2 mg/kg, i.p.) 5 min before testing reinstates nicotine CPP in a norBNI (10 mg/kg, i.p.)-sensitive manner. C, Nicotine decreases locomotor behavior compared with saline controls. D, Locomotor responses are unchanged during CPP testing by pretreatment with norBNI. E, KOR^−/− and DYN^−/− show similar CPP scores as WT controls during the posttest and nicotine prime reinstatement test, but do not reinstate after yohimbine treatment. F, KOR^−/− and DYN^−/− mice show similar decrease in locomotion after nicotine treatment as seen in WT controls. G, KOR^−/− and DYN^−/− mice do not differ from WT controls in distance traveled during CPP or reinstatement tests. For B–D, n = 8–10 per group; for E–G, n = 5–9 per group; *p < 0.05.

Figure 3.

KOR activation is necessary for increased BLA c-fos expression after yohimbine-induced reinstatement. A, Timeline of behavioral events/treatment before perfusions after yohimbine-induced reinstatement to visualize c-fos expression within the BLA. B, Percentage of neurons with both c-fos+ and Nissl stain is increased compared with saline controls or mice pretreated with norBNI (n = 6 mice per group; *p < 0.05), of c-fos+ cells shown in C. C, Representative confocal images (20×) of BLA c-fos expression from each group quantified in B. Nissl (blue) and c-fos (red) staining. White arrow points to c-fos+- and Nissl+-labeled cell. Scale bars, 50 μm.

Figure 4.

KOR activation in the BLA is required for yohimbine-induced nicotine reinstatement. A, Experimental timeline. norBNI was injected into the BLA and mice were tested for yohimbine reinstatement 17 d later. B, Diagram of bilateral norBNI injection into the BLA (coordinates: ML ± 2.9, AP −1.8, DV −5). C, Representative heat maps showing activity during yohimbine reinstatement of mice bilaterally injected with vehicle (top) and norBNI (bottom). D, Local norBNI in the BLA before behavioral testing prevents yohimbine-induced nicotine CPP reinstatement without affecting nicotine place preference acquisition or prime reinstatement. E, Nicotine decreases locomotor responses to nicotine to a greater extent in mice injected with vehicle than mice injected with norBNI locally into the BLA. F, No changes in locomotor activity were seen during post, yohimbine reinstatement, or prime testing between groups. n = 8–12 per group, *p < 0.05.

Figure 5.

KORs in the BLA are necessary for foot-shock stress-induced nicotine CPP reinstatement. A, Diagram of cre-induced conditional knock-out of KORs in KOR floxed mice (KOR cKO) and generation of KOR cKO^tdTomato mouse line from the cross betweem KOR cKO × Ai9-tdTomato. Cre-recombinase packaged into an adeno-associated virus with a GFP tag, when infects cells with floxed gene, conditionally cuts out exon 4 from the KOR gene, preventing it from being transcribed and simultaneously labels the cell with GFP. B, Diagram of viral injections into BLA of KOR cKO mice. C, Confocal images (20×) of AAV5-cre-GFP (left, green) expressed in the BLA of a KOR cKO^tdTomato mouse, cre-induced tdTomato expression (middle, red), and a merged image of the two showing that GFP + cells colabel with tdTomato+ cells in the BLA, confirming the recognition of cre in floxed cells. Scale bars, 50 μm. D, Timeline of BLA viral injections and behavioral testing. E, Confocal (10×) of AAV5-cre-GFP (left) and AAV5-GFP (right) expression in the BLA 6 weeks after injection. Staining: GFP (green) and Nissl (blue). Scale bars, 50 μm. F, Diagram with marks indicating viral spread in mice included in behavioral dataset. G, Selective deletion of KORs from the BLA in KOR cKO mice attenuates foot-shock stress-induced nicotine CPP reinstatement without changing CPP acquisition or prime-induced reinstatement. H, No differences were seen in locomotor activity locomotor response to nicotine during conditioning. I, No changes in activity were seen during any testing stage. n = 6–9 per group, *p < 0.05.

Figure 6.

Chemogenetic activation of Gαi signaling in the BLA is sufficient to induce reinstatement of nicotine CPP. A, Diagram of hM4D(G_i) DREADD viral injections into BLA and subsequent behavioral experiments. B, Diagram of viral injection into the BLA (left) and IHC 20× confocal images of hM4D(G_i) DREADD (middle) and control (mCitrine packaged under CaMKIIα promoter with no receptor, right) viral expression in the BLA. mCitrine (cyan), c-fos (red), and Nissl (blue). Scale bars, 50 μm. C, Diagram with marks that indicate viral spread in mice included in behavioral dataset. D, Activation of hM4D(G_i) DREADDs in the BLA by CNO is sufficient to induce nicotine CPP reinstatement. E, No differences in locomotor activity before or after DREADD activation between Gαi and controls. n = 9–10 per group, *p < 0.01.