Kappa opioid receptors on dopaminergic neurons are necessary for kappa-mediated place aversion

Abstract

Kappa-opioid receptor (KOR) agonists have dysphoric properties in humans and are aversive in rodents. This has been attributed to the activation of KORs within the mesolimbic dopamine (DA) system. However, the role of DA in KOR-mediated aversion and stress remains divisive as recent studies have suggested that activation of KORs on serotonergic neurons may be sufficient to mediate aversive behaviors. To address this question, we used conditional knock-out (KO) mice with KORs deleted on DA neurons (DAT(Cre/wt)/KOR(loxp/loxp), or DATCre-KOR KO). In agreement with previous findings, control mice (DAT(Cre/wt)/KOR(wt/wt) or WT) showed conditioned place aversion (CPA) to the systemically administered KOR agonist U69,593. In contrast, DATCre-KOR KO mice did not exhibit CPA with this same agonist. In addition, in vivo microdialysis showed that systemic U69,593 decreased overflow of DA in the nucleus accumbens (NAc) in WT mice, but had no effect in DATCre-KOR KO mice. Intra- ventral tegmental area (VTA) delivery of KORs using an adeno-associated viral gene construct, resulted in phenotypic rescue of the KOR-mediated NAc DA response and aversive behavior in DATCre-KOR KO animals. These results provide evidence that KORs on VTA DA neurons are necessary to mediate KOR-mediated aversive behavior. Therefore, our data, along with recent findings, suggest that the neuronal mechanisms of KOR-mediated aversive behavior may include both dopaminergic and serotonergic components.

Figure 1

Cre-mediated recombination of the KOR gene is specific to dopamine transporter mRNA-expressing neurons in the VTA region. (a) PCR screen for Cre-mediated KOR exon 2 deletion. Genomic DNAs were prepared from the indicated tissues from a DATCre-KORflox mouse. PCR analyses show KOR deletion is specific to the olfactory bulb and SN/VTA regions. KOR deletion was not detected in other brain areas or tissues analyzed. (b and c) Representative images of dual fluorescence in-situ hybridization of DAT mRNA (green) and KOR mRNA (red) in the VTA of a DATCre-KOR KO (c) and control (b) mouse. Blue signal represents DAPI nucleic acid stain. Arrows indicate cells expressing DAT mRNA. DAT-positive neurons co-express KOR mRNA in WT animals (b). As predicted, DAT-positive neurons do not express KOR mRNA in DAT-KOR KO mice (c). In addition, KOR mRNA was present in DAT-negative neurons in both WT and KO animals (arrowheads in b and c).

Figure 2

Conditional deletion of KORs on DA neurons abolished NAc DA response to systemically administered KOR agonist U69,593 and development of KOR-mediated CPA. (a and b) Representative images of dual fluorescence in-situ hybridization of DAT mRNA (green) and KOR mRNA (red) in the VTA of a WT (DAT^Cre/wt KOR^wt/wt; A) and KO (DAT^Cre/wt KOR^loxp/loxp; B) mouse. Blue signal represents DAPI nucleic acid stain. Arrows indicate cells expressing DAT mRNA. DAT-positive neurons co-express KOR mRNA in control animals (a). As predicted, DAT-positive neurons do not express KOR mRNA in DATCre-KOR KO mice (b). In addition, KOR mRNA was present in DAT-negative neurons in both genotypes (arrowheads in a and b). (c) Time course of basal and U69,593-induced NAc DA overflow in WT (filled circles) and KO (open circles) mice. The data are expressed as mean % of baseline±SEM. (d) AUC values of DA levels after U69,593 challenge in WT (filled bars) and KO (open bars) animals. **Significant difference between WT and KO animals. (e) Time spent in vehicle- (open bars) or U-69,593- (filled bars) paired compartments during post-test. The data are expressed as mean of the time spent±SEM. *A significant difference between time spent in vehicle- and U69,593-paired compartments. (f) Conditioning score defined as time spent in vehicle- or drug-paired place during post-test minus that spent in the same place during post-test. The data are expressed as mean of the time spent±SEM. * and ** reflect significant difference between vehicle- and U69,593-paired compartments and between WT and KO animals, respectively.

Figure 3

Viral rescue of KOR function in DATCre-KOR KO mice VTA restored NAc DA response to KOR agonist and KOR-mediated CPA. (a and b) Representative images of the VTA region in DATCre-KOR KO mice injected with AAV2-CAG-GFP (a) or AAV2-CAG-KOR (b) viral vectors. (a) Arrows show DAT immuno-stained cells (red) co-expressing GFP. GFP was also expressed in DAT-negative neurons (arrowheads). (b) Arrows show KOR mRNA signal (red probe) co-localized with DAT mRNA signal (green probe). Blue signal represents DAPI nucleic acid stain. (c) Time course of basal and U69,593-induced NAc DA overflow in AAV2-CAG-GFP- (open squares) and AAV2-CAG-KOR-injected (filled squares) mice. The data are expressed as mean % of baseline±SEM. (d) AUC values of DA levels after U69,593 challenge in AAV2-CAG-GFP- (filled bars) and AAV2-CAG-KOR-injected (open bars) animals. **Significant difference between AAV2-CAG-GFP and AAV2-CAG-KOR groups. (e) Time spent in vehicle- (open bars) or U-69,593- (filled bars) paired compartments during post-test. The data are expressed as mean of the time spent±SEM. *A significant difference between time spent in vehicle- and U69,593-paired compartments. (f) Conditioning score defined as time spent in vehicle- or drug-paired place during post-test minus that spent in the same place during post-test. The data are expressed as mean of the time spent±SEM. * and ** reflect a significant difference between vehicle- and U69,593-paired compartments and between AAV2-CAG-GFP- and AAV2-CAG-KOR-injected animals, respectively.