Locus coeruleus kappa-opioid receptors modulate reinstatement of cocaine place preference through a noradrenergic mechanism

Abstract

Activation of kappa-opioid receptors (KORs) in monoamine circuits results in dysphoria-like behaviors and stress-induced reinstatement of drug seeking in both conditioned place preference (CPP) and self-administration models. Noradrenergic (NA) receptor systems have also been implicated in similar behaviors. Dynorphinergic projections terminate within the locus coeruleus (LC), a primary source of norepinephrine in the forebrain, suggesting a possible link between the NA and dynorphin/kappa opioid systems, yet the implications of these putative interactions have not been investigated. We isolated the necessity of KORs in the LC in kappa opioid agonist (U50,488)-induced reinstatement of cocaine CPP by blocking KORs in the LC with NorBNI (KOR antagonist). KOR-induced reinstatement was significantly attenuated in mice injected with NorBNI in the LC. To determine the sufficiency of KORs in the LC on U50,488-induced reinstatement of cocaine CPP, we virally re-expressed KORs in the LC of KOR knockout mice. We found that KORs expression in the LC alone was sufficient to partially rescue KOR-induced reinstatement. Next we assessed the role of NA signaling in KOR-induced reinstatement of cocaine CPP in the presence and absence of a α2-agonist (clonidine), β-adrenergic receptor antagonist (propranolol), and β(1)- and β(2)-antagonist (betaxolol and ICI-118,551 HCl). Both the blockade of postsynaptic β(1)-adrenergic receptors and the activation of presynaptic inhibitory adrenergic autoreceptors selectively potentiated the magnitude of KOR-induced reinstatement of cocaine CPP but not cocaine-primed CPP reinstatement. Finally, viral restoration of KORs in the LC together with β-adrenergic receptor blockade did not potentiate KOR-induced reinstatement to cocaine CPP, suggesting that adrenergic receptor interactions occur at KOR-expressing regions external to the LC. These results identify a previously unknown interaction between KORs and NA systems and suggest a NA regulation of KOR-dependent reinstatement of cocaine CPP.

Figure 1

Selective KOR antagonism in the LC attenuates KOR-induced reinstatement of cocaine place preference. (a, left) Image of a coronal section (plane −5.40 mm from bregma) highlighting the LC; (right) representative photomicrograph of tyrosine hydroxylase (TH, green) and phosphorylated-ERK (pERK, purple) in the LC of WT mice injected with either NorBNI (2.5 μg/side) or saline. White arrows indicate colocalization of pERK and TH. All scale bars are 100 μm. (B) NorBNI treatment decreases co-immunoreactivity of pERK and TH compared with Saline treatment (n=3 slices per brain from three brains for each group; ***p=0.0001; LC Saline vs LC NorBNI; Student's t-test). (c) Timeline of cocaine place preference/reinstatement experiment, indicating previous injection of into the LC. (d) Cocaine preference scores, calculated as post-test minus pre-test on the cocaine-paired side and U50,488 (5 mg/kg i.p.)-induced reinstatement scores of extinguished cocaine place preference (For all results, mice were classified as extinguished if the extinction post-test was within 15% of the pre-test values). Data show a significant reduction in KOR-induced reinstatement of cocaine place preference following local norBNI infusion into the LC. Data represent the mean preference (s)±SEM, n=8–13 (*p<0.05, LC Saline/U50 vs LC NorBNI/U50 and no surgery control vs LC NorBNI/U50; one-way ANOVA followed by Bonferroni's post-hoc test). (e) No significant difference in locomotor activity was observed, measured as distance travelled (cm) during the 3-day conditioning period in the drug-paired side. Data represent the mean distance (cm)±SEM, n=8–13.

Figure 2

Viral restoration and activation of KORs exclusively in the LC is sufficient for KOR-induced reinstatement of CPP. (a) Schematic of the KOR lentiviral (Lenti-KOR) construct. LTR, long terminal repeats; RRE, Rev response element; FLAP, HIV-1 flap sequence; PGK, phosphoglycerate kinase promoter; WPRE, Woodchuck hepatitis virus post-transcriptional regulatory element; PPT, polypurine tract; U3, Unique 3′ region. (b) Representative photomicrograph demonstrating LC targeting (TH, red) via expression of the Lenti-GFP reporter virus (green). Dashed lines indicate the border of the fourth ventricle. Arrows indicate representative colocalization of GFP and TH. All scale bars are 100 μm. (c) Representative photomicrographs of KOR rescue in KOR KO. The LC is anatomically identified with a fluorescent Nissl stain (blue) and the presence of the fourth ventricle (dashed lines). The rescue of KORs in the LC following injection of Lenti-KOR is identified using a chicken anti-KOR antibody (red). All scale bars are 100 μm. (d) KOR immunoreactivity, measured as average intensity in the LC, is rescued in Lenti-KOR-injected mice (n=8 slices from eight brains for each group; ****p<0.0001; Lenti-GFP vs Lenti-KOR; Student's t-test). (e) Timeline of the lentiviral injections, cocaine place preference, and KOR-induced reinstatement of cocaine place preference. (f) Cocaine preference scores, calculated as post-test minus pre-test on the cocaine-paired side and U50,488-induced (5 mg/kg i.p.) reinstatement scores of extinguished cocaine place preference in LC-injected mice. Data show that viral restoration of KORs within the LC partially recovers KOR-mediated reinstatement of cocaine place preference. Data represent mean time±SEM, n=6 (*p<0.05; Lenti-KOR/U50 vs Lenti-GFP/U50; Student's t-test). (g) No significant difference in locomotor activity was observed, measured as distance (cm)±SEM, n=6 in Lenti-KOR/U50 vs Lenti-GFP/U50 groups during the 3-day conditioning period.

Figure 3

The magnitude of KOR-induced reinstatement to cocaine preference requires noradrenergic signalling. (a) Timeline of the KOR-induced reinstatement of cocaine preference experiment, Sal, Saline, Prop, Propranolol, Clon, Clonidine. (b) Representative Ethovision behavioral traces of mouse activity post cocaine conditioning, extinction and propranolol/U50-induced reinstatement. (c) Mice injected with clonidine (α₂-adrenergic receptor agonist, 0.03 mg/kg, i.p.) 30 min before treatment with U50,488 (5 mg/kg i.p.). Clonidine alone did not induce reinstatement. Data represent the mean preference (s)±SEM, n= 10–12 (*p<0.05, Saline/Saline vs Saline/U50; **p<0.001, Saline/U50 vs Clonidine/Saline; ****p<0.0001, Saline/U50 vs Clonidine/U50, Saline/Saline vs Clonidine/U50; one-way ANOVA followed by Bonferroni's post-hoc test). (d) Mice were injected with propranolol (β-adrenergic receptor antagonist, 10 mg/kg i.p.) 30 min before treatment with U50,488 (5 mg/kg, i.p). Propanolol alone did not induce reinstatement. Data show that blockade of noradrenergic signaling via α₂-adrenergic receptor agonist or β-adrenergic receptor antagonist causes a potentiation in KOR-mediated reinstatement of cocaine place preference behavior. Data represent the mean preference (s)±SEM, n=7–13 (*p<0.05, Saline/U50 vs Propranolol/U50; ****p<0.0001, Saline/Propranolol vs Propranolol/U50; one-way ANOVA followed by Bonferroni's post-hoc test). (e) Locomotor activity measured in 5-min bins during reinstatement phase. Data represent mean distance travelled (cm)±SEM, n=7–13. To identify which β-adrenergic receptor subtype may be modulating the potentiation of KOR-induced reinstatement, betaxolol (Betax; selective β₁-adrenergic receptor antagonist, 10 mg/kg i.p.) or ICI-11855 (ICI; selective β₂-adrenergic receptor antagonist, 5 mg/kg i.p.) was systemically injected before U50,488-induced reinstatement. (f) Betaxolol, but not ICI-118551, significantly potentiated U50,488-induced reinstatement. Data represent the mean preference (s)±SEM, n=6–12 (*p<0.05, Saline/U50 vs Betax/U50; **p<0.01, Saline/U50 vs Propranolol/U50; one-way ANOVA, Bonferroni's post-hoc test). (g) Locomotor activity, measured as average distance travelled, in 5-min bins during reinstatement phase. Data represent mean distance travelled (cm)±SEM, n= 6–12.

Figure 4

Propanolol does not potentiate cocaine prime-induced reinstatement or effect KOR-mediated place aversion. (a) Timeline of cocaine place preference, including propranolol pretreatment during the 3-day conditioning period. (b) Propanolol does not alter conditioning to cocaine. Data represent the mean preference (s)±SEM, n=6–12; one-way ANOVA, Bonferroni's post-hoc test ***p<0.001, Saline vs Propranolol/Cocaine; ***p<0.01, Saline vs Cocaine). (c) Treatment with propranolol before cocaine conditioning did not alter locomotor activity recorded during the 30-min conditioning session (n=6–12; ****p<0.0001, Day 3 Saline vs Cocaine, Day 4 Saline vs Cocaine; **p<0.01, Day 3 Saline vs Propranolol/Cocaine; *p<0.05 Day 4 Saline vs Prop/Coc; two-way ANOVA, Bonferroni's post-hoc test). (d) Timeline of cocaine place preference, extinction and cocaine-primed reinstatement. (e) Propanolol does not potentiate cocaine-induced reinstatement to drug seeking. Data represent the mean preference (s)±SEM, n=6–12. (f) Timeline of U50,488 conditioned place aversion. (g) The potentiating effect of propranolol is not due to enhancement of KOR-mediated aversion behavior. No significant difference was observed between the groups (n=8–16).

Figure 5

Rescue of KORs in the LC and block of β-adrenergic receptors does not block potentiation of KOR-induced reinstatement. (a) Timeline of KOR-induced reinstatement experiment, including injection of lentivirus. (b) Cocaine preference scores, calculated as post-test minus pre-test on the cocaine-paired side and propranolol (10 mg/kg i.p.)/U50,488-induced (5 mg/kg i.p.) reinstatement scores in LC-injected mice. Data represent means±SEM, n=8 (*p<0.05; Lenti-KOR, Propranolol/U50 vs Lenti-GFP, Propranolol/U50; Student's t-test). Propanolol-induced potentiation of U50,488-induced reinstatement to cocaine CPP is absent in KOR KO mice in which KORs have been virally re-expressed in the LC. (c) No significant difference in locomotor activity was observed, measured as distance (cm)±SEM, n= 8 in Lenti-KOR vs Lenti-GFP groups during the 3-day conditioning period. (d) Hypothetical model representing the interaction sites between dynorphin/KOR and noradrenergic neural systems in the modulation of KOR-mediated reinstatement of cocaine place preference. Proposed model shows that KOR activity in the LC is likely to act upstream of previously identified KOR circuits involved in KOR-mediated behavioral responses. This is confirmed in the current study where local norBNI infusion into the LC does not completely block KOR-induced reinstatement to cocaine CPP. In addition, noradrenergic circuits negativity regulate the magnitude of these responses (as identified by antagonism studies in this report). Output of KOR-induced inhibition of LC activity and modulation of noradrenergic outflow are likely mechanisms for the potentiating effect of noradrenergic blockade isolated in this report. The bed nucleus of the stria terminalis (BNST) and the basolateral amygdala (BLA) are likely output structures given their enriched noradrenergic terminal projections and expression of β₁-adrenergic receptors. DA, dopamine; 5-HT, serotonin; k, kappa opioid receptor, NE, norepinephrine.