Kappa opioid receptor agonist U50,488 inhibits dopamine more in caudal than rostral nucleus accumbens core

Abstract

The nucleus accumbens (NAc) core is involved in regulating stress and shaping reward seeking behaviours. Multiple neuromodulators, including dynorphin/kappa opioid receptor (KOR) and dopamine systems, converge in this area to influence behavioural outcomes. KOR activation acutely inhibits dopamine release and chronically depresses overall dopamine transmission. Recently, studies in the NAc shell have revealed that the impact of KOR activation on behaviour is regionally specific, and these rostro-caudal differences are likely driven by greater control of KORs over dopamine inhibition in the caudal compared with rostral subregion. Given the importance of NAc core, particularly the interaction between KORs and dopamine in regulating reward seeking behaviours, we examined the impact of KOR activation on dopamine release and uptake along the rostro-caudal axis in the NAc core of male and female mice. Using ex vivo fast scan cyclic voltammetry, we observed that KOR mediated inhibition of dopamine release was significantly greater in caudal compared with rostral NAc core with no significant sex differences observed. These data suggest that KORs regulate dopamine release differentially along the rostro-caudal axis, providing a new axis on which to examine the process by which the KOR/dopamine system controls reward encoding.

1 |. INTRODUCTION

The nucleus accumbens (NAc) core heavily contributes to the regulation of responses to negative and positive stimuli, thereby affecting homeostatic and affective states. It is a heterogeneous region, which receives glutamatergic inputs from the basolateral amygdala, prefrontal cortex, ventral hippocampus and the paraventricular thalamus,^1–3 serotonergic inputs from the dorsal raphe⁴ and dopaminergic inputs from the ventral tegmental area (VTA). The VTA dopaminergic projection to the NAc core shapes reinforcement learning and motivated behaviours,^5–8 with both reinforcing and aversive stimuli facilitating dopamine (DA) release in the NAc.⁹ The NAc core also contains dynorphin producing cell bodies and axon terminals.¹⁰ Dynorphin is a neuropeptide released in response to stress, drugs and drug withdrawal^11–13; it is also the endogenous ligand of the inhibitory G-protein coupled kappa opioid receptor (KOR).^14,15 In the NAc core, KORs are located on pre-synaptic DA terminals^16–18 and exhibit a profound inhibitory control over DA release.^19–24 KORs are colocalized with DA transporters (DATs) and modulate DAT function, augmenting the reuptake of DA and further reducing DA release.^18,25,26 Previous studies have shown that KORs and DATs can exist together in protein complexes, with activation of KORs promoting the formation of these complexes and ultimately augmenting DAT activity in an ERK 1/2 dependent manner.²⁵ Overall, KOR activation inhibits DA transmission, leading to a hypodopaminergic state, which may contribute to anhedonia. It is important to note that KORs are also located post-synaptically on striatal medium spiny neurons and exert additional downstream effects on neurotransmission and therefore behavioural outcomes.^3,27

The mesolimbic DA system and the dynorphin/KOR system both regulate affective states and addictive behaviours.^23,28,29 Predominantly, studies have shown that chronic KOR activation produces aversion and promotes drug seeking and taking behaviours.^{20,21,24,28,30} Animal models of repeated or prolonged stress exposure promote excessive drug seeking and taking, which can be reversed by the inhibition of KORs. For example, augmented cocaine seeking after repeated forced swim stress exposure was attenuated following the administration of the KOR antagonist nor-binaltorphimine in rats.³¹ Similarly, adolescent social isolation²³ and chronic intermittent ethanol induced increases in ethanol consumption were reduced by KOR antagonists.²⁴ Interestingly, however, these KOR-blockade mediated reductions in drug responding and consumption were only observed in animals previously exposed to drugs or stress, suggesting that these exposures result in changes to KOR function, which were crucial in the drug seeking and taking behaviours.

In contrast to the relatively consistent findings of beneficial effects of KOR antagonists on drug- and alcohol-related behaviours, KOR agonist effects are highly variable. The KOR agonist U50,488 has been shown to both increase and decrease drug self-administration, depending on the dose and the timing of the exposure.^28,29,32 Acute KOR activation is known to rapidly produce anhedonia, which can influence the reinforcing properties of drugs of abuse, including cocaine. Thus, when a misused drug is experienced during acute anhedonia, close to the time of agonist administration, there may be either enhanced or reduced propensity to take the drug, depending on the behavioural assay.^28,32 At later time points, typically one or more hours after agonist exposure,²⁸ findings are more consistent and tend to mimic chronic stress-associated KOR activation, such as increased reinforcing effects of the drug and self-administration behaviours, although variables such sex,^33,34 and drug dose^24,35 affect the results. The temporal effects of KOR activation may be a result of differential temporal dynamics of KOR interaction with various proteins that regulate DA transmission, potentially affecting perceived reward value. The combined effects of chronic stress or drug exposure on DA and KOR systems likely alter the interaction between these systems, further affecting reward value perception and thus influencing behavioural outcomes.

Recent studies have reported opposing behavioural effects of dynorphin/KOR system activation in different subregions of the NAc shell.^36,37 Optical stimulation of dynorphinergic neurons in dorsal NAc shell was positively reinforcing and induced conditioned place preference; in contrast, optical stimulation of these neurons in the ventral NAc shell elicited robust conditioned place aversion.³⁷ Similarly, activation of KORs in the rostral NAc shell stimulated hedonic orofacial responses to sucrose and produced conditioned place preference whereas activation of KORs in the caudal NAc suppressed these hedonic responses and produced conditioned place aversion.³⁶ The opposing behaviour observed following selective KOR activation of the subregions in the NAc shell may be linked to greater modulation of DA by KORs in caudal versus rostral NAc shell.³⁸ Despite the growing body of literature on rostro-caudal differences in the NAc shell, the adjacent region of the core remains to be examined for similar subregional effects.

Modulation of KORs alters drug-induced elevations in DA release in both shell and core NAc.^22,23,28,29 Given the opposing influence of KORs in the subregions of the shell, it is possible that KORs differentially regulate DA release along the rostro-caudal axis in the core as well. Thus, in this study, we have examined the interaction between the KOR and DA systems in the rostral and caudal NAc core in both male and female mice. Based on previous literature, we anticipate that KOR-induced inhibition of DA release is greater in caudal compared with rostral NAc core.

2 |. MATERIAL AND METHODS

2.1 |. Subjects

Adult male and female C57Bl/6J mice (N = 12; six males and six females bred at Wake Forest School of Medicine) were pair housed in AAALAC-accredited facilities on a 12-h light/dark cycle (lights on at 6 AM). Mice received ad libitum chow (Laboratory Rodent Diet 5001, Lab Diet, St. Louis, MO, USA) and water. Experiments were approved by the Institutional Animal Care and Use Committee at Wake Forest School of Medicine and followed the NIH Guide for the Care and Use of Laboratory Animals (protocol number: A19–104). The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies.³⁹

2.2 |. Ex vivo fast scan cyclic voltammetry (FSCV)

To determine the effects of KOR stimulation on DA dynamics in the rostral compared with caudal NAc core, ex vivo FSCV was used to measure DA release under baseline conditions and in the presence of a selective KOR agonist, (−)-U50,488 hydrochloride (Tocris, Minneapolis, MN, USA).^40,41 Male and female mice (n = 6/group) were sacrificed 3 h into the light phase, and their brains were rapidly removed and prepared as previously described.⁴² A vibrating tissue slicer was used to prepare 300 μm thick coronal brain sections containing NAc core—One rostral slice (+1.34 to +1.10 from Bregma)⁴³ and one caudal slice (+0.86 to +0.62 from Bregma)⁴³ was taken from each animal, resulting in a within-subject design. These slices were immersed in oxygenated artificial cerebrospinal fluid (aCSF; 32°C) containing: 126 mM NaCl, 2.5 mM KCl, 1.2 mM NaH₂PO₄, 2.4 mM CaCl₂, 1.2 mM MgCl₂, 25 mM NaHCO₃, 11 mM glucose and 0.4 mM L-ascorbic acid, and the pH was adjusted to 7.4. Endogenous DA release was evoked by single electrical pulse stimulation (monophasic+, 4 ms, 350 μA) applied to the tissue every 5 min, and DA was detected using a triangular scanning waveform (−0.4 to +1.2 and back to 0.4 V, Ag vs AgCl) at the rate of 400 V/s. Electrode placements for recordings were distributed throughout the NAc core (lateral, medial and ventral) over the experiments. After stable DA responses were obtained, cumulative concentrations of U50,488 (0.01–1.0 μM)^23,24,38,44 were bath applied to rostral and caudal NAc core slices acquired from the same animal. Subsequently, increasing concentrations of U50,488 were bath applied once DA release to the previous concentration stabilized. Recording electrodes were calibrated upon termination of each experiment with known concentrations of DA (3 μM). All ex vivo FSCV data were analysed for stimulated DA release, and the uptake portion of the curve was fitted to an exponential decay model and expressed as tau or time to 33% of the peak height (unit in seconds).^45,46 These analyses were carried out using the peak and decay function of the Demon Voltammetry and Analysis software.⁴⁵

2.3 |. Statistical analysis

Statistical analysis of the FSCV data was conducted using GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA). Baseline DA release and uptake data for male and female mice were analysed together using repeated measures (RM) two-way analysis of variance (ANOVA), with sex and region as the independent variables and DA release or uptake (tau, exponential decay constant) as the dependent variables. The U50,488 concentration response curves were analysed using an RM two-way ANOVA. The dependent variables were μM DA release or tau, and the independent variables were rostral/caudal subregions of NAc core and U50,488 concentration. First, the analysis for U50,488 effects on DA release and tau were conducted separately in male and female mice. Regional differences in efficacy and potency were analysed using paired two-tailed Student’s t-test. To analyse sex differences in KOR modulation of DA transmission, a mixed model two-way ANOVA was used (sex, between-subject; subregion, within-subject). Significant effects from ANOVAs were followed up by Sidak’s pairwise post-hoc analysis. All data are reported as mean ± standard error of the mean. Significance was set at p < 0.05. Changes in release and tau from baseline to a single U50,488 dose were compared using paired two-tailed Student’s t-test. The paired two-tailed Student’s t-test was used to evaluate the percent baseline of tau from 10 nM to 30 nM of U50,488. FSCV data analysis was performed under blinded conditions. Sample size was selected based on previous studies examining the KOR system function in the NAc core.^22–24

3 |. RESULTS

3.1 |. Stimulated DA release and tau in rostral and caudal NAc core

To determine whether stimulated DA release was comparable or different along the rostro-caudal axis in the NAc core, a single pulse electrical stimulation was applied in rostral or caudal NAc core from female and male mice (n = 6/group), and DA release and rate of uptake (using exponential decay constant, tau) were measured using ex vivo FSCV. Representative DA signals are shown in Figure 1A (rostral, top; caudal, bottom; male, blue; female, pink). Shaded areas within each section indicate general electrode placements. A two-way ANOVA comparing DA release in rostral and caudal NAc core (RM) in female and male mice revealed no significant differences in region [F (1,10) = 0.5564, p = 0.4729], or of sex [F (1, 10) = 0.1866, p = 0.6749], and no interaction between the variables [F (1, 10) = 0.03705, p = 0.8512] (Figure 1B). Comparison of DA uptake rate (tau) between the two subregions (RM) in the two sexes also did not reveal significant differences (region: [F (1, 10) = 2.187, p = 0.1700]; sex: [F (1, 10) = 2.119, p = 0.1762] and interaction: [F (1, 10) = 1.305, p = 0.2800]; Figure 1C).

FIGURE 1.

Evoked dopamine release and uptake in rostral and caudal nucleus accumbens (NAc) core. (A) Representative traces of transient dopamine signals in response to single pulse electrical stimulation in rostral (top, dashed line) and caudal (bottom, solid line) NAc core of male (middle, blue) and female (right, pink) mice with the target area highlighted on the coronal slices diagram (left). (B) Stimulated dopamine release. There was no significant difference in baseline dopamine release in rostral compared with caudal NAc core in female (white/pink—rostral; solid pink—caudal) or male (white/ blue—rostral; solid blue—caudal) mice. There were also no significant sex differences in evoked dopamine release. (C) Dopamine uptake as measured by tau was also not different between the rostral and caudal NAc core in female (white/pink—rostral; solid pink—caudal) or male (white/ blue—rostral; solid blue—caudal) mice. No sex differences were observed in uptake. C, caudal; KOR, kappa opioid receptor; R, rostral.

3.2 |. KOR mediated inhibition of DA is enhanced in the caudal NAc core subregion

To determine the magnitude of KOR mediated inhibition of DA release along the rostro-caudal axis in NAc core, cumulative concentrations of U50,488 were bath applied to slices containing rostral or caudal NAc core from female and male mice. In males, an RM two-way ANOVA comparison of rostral and caudal subregions of the NAc core (Figure 2A) revealed main effects of U50,488 concentration [F (4, 20) = 53.41, p < 0.0001] and subregion [F (1, 5) = 14.07, p = 0.0133], but there are no significant interaction between U50,488 concentration and subregion [F (4, 20) = 2.167, p = 0.1099]. Post-hoc Sidak’s pairwise comparisons revealed that U50,488 significantly inhibited DA release to a greater extent in the caudal compared with rostral NAc shell at the 0.03 (p = 0.0003), 0.1 (p < 0.0001), 0.3 (p = 0.0058) and 1.0 μM (p = 0.0238) concentrations. A paired t-test comparison of the EC50 between rostral and caudal regions also showed a significant difference [t = 3.992, df = 5, p = 0.0104] (Figure 2B). The maximal effect of U50,488-induced inhibition of DA release was trending towards being significantly greater in the caudal compared with rostral subregion of the NAc core [t = 2.343, df = 5, p = 0.0661] (Figure 2C). An RM two-way ANOVA comparison of the rostral and caudal subregions of the NAc core of females (Figure 2D) revealed main effects of U50,488 concentration [F (4, 20) = 170.6, p < 0.0001] and subregion [F (1, 5) = 11.46, p = 0.0196], but no significant interaction between U50,488 concentration and subregion [F (4, 20) = 0.6904, p = 0.6072]. Post-hoc Sidak’s pairwise comparisons revealed that U50,488 significantly inhibited DA release to a greater extent in the caudal compared with rostral NAc core at 0.01 (p = 0.0006), 0.03 (p = 0.0002), 0.1 (p < 0.0001) 0.3 (p < 0.0001) and 1.0 μM (p < 0.0001) concentrations. A paired t-test comparison of the EC50 between rostral and caudal subregions also showed a significant difference [t = 3.024, df = 5, p = 0.0293] (Figure 2E). Moreover, the maximal effect of U50,488-induced inhibition of DA release was significantly greater in the caudal compared with rostral subregion of the NAc core [t = 2.640, df = 5, p = 0.0460] (Figure 2F).

FIGURE 2.

Impact of kappa opioid receptor (KOR) activation on dopamine release in rostral and caudal nucleus accumbens (NAc) core. (A) KOR activation mediated inhibition of dopamine release was greater in caudal (closed circles and solid line) compared with rostral (open circles and dotted line) NAc core in male mice. (B) Potency of KORs (EC50) was greater in the caudal (closed circles), indicated by lower EC50, compared with rostral (open circles) NAc core of male mice. (C) Maximal KOR-mediated reduction in dopamine release, efficacy, was statistically trending to be greater in the caudal (closed circles) compared with rostral (open circles) NAc core of male mice. (D) In female mice, KOR activation mediated inhibition of dopamine release was greater in caudal (closed circles and solid line) compared with rostral (open circles and dotted line) NAc core. This augmented function was driven by greater potency (E) and efficacy (F) of KORs in the caudal (closed circles) compared with rostral (open circles) NAc core. U50,488, KOR agonist; C, caudal; EC50, concentration of drug that results in the half-maximal response in the tissue; KOR, kappa opioid receptor; NAc, nucleus accumbens; R, rostral. **** p < 0.0001, main effect of U50,488 concentration; # p < 0.0001, δδδ p < 0.001, δδ p < 0.01, δ p < 0.05, post hoc pairwise comparison.

Direct comparison of KOR activation-mediated inhibition of DA release between males and females in the rostral NAc core using a two-way ANOVA revealed a main effect of U50,488 (RM) [F (4, 40) = 65.04, p < 0.0001], but not sex [F (1, 10) = 0.2113, p = 0.6556], and there are no interactions between the two variables [F (4, 40) = 0.3528, p = 0.8406] (Figure 3A). Similar analysis of the caudal NAc core showed a main effect of U50,488 (RM) [F (4, 40) = 189.3, p < 0.0001], but no effect of sex [F (1, 10) = 1.298, p = 0.2811] or interaction effect [F (4, 40) = 1.432, p = 0.2411] (Figure 3B).

FIGURE 3.

Impact of kappa opioid receptor (KOR) activation on dopamine release in males vs females in the rostral and caudal nucleus accumbens (NAc) core. (A) KOR activation mediated inhibition of dopamine release was not significantly different in the rostral NAc core of female (pink) and male (blue) mice. (B) Likewise, KOR activation mediated inhibition of dopamine release was not significantly different in the caudal NAc core of female (pink) and male (blue) mice. U50, 488, KOR agonist. **** p < 0.0001.

3.3 |. KOR did not alter DA uptake in any of the NAc core subregions

To determine the impact of KOR activation on DAT function, we quantified percent of baseline tau as a measure of uptake at the 10 and 30 nM doses of U50,488, as tau cannot be reliably quantified when DA release is very low, which occurs at higher concentrations of U50,488. The percent change in tau from the 10 to 30 nM doses were examined using a paired t-test and revealed that tau was not significantly changing across the two doses of U50,488 (male rostral [t = 0.06185, df = 5, p = 0.9531]; male caudal [t = 0.3770, df = 5, p = 0.7217]; female rostral [t = 0.7163, df = 5, p = 0.5059]; female caudal [t = 1.755, df = 5, p = 0.1396]) (Figure 4).

FIGURE 4.

Impact of kappa opioid receptor (KOR) activation in the rostral and caudal nucleus accumbens (NAc) core on uptake of dopamine. The percent change in tau at both the 10 and 30 nM doses of U50,488 did not show consistent changes in any of the groups analysed. F, female; M, male; R, rostral; C, caudal.

4 |. DISCUSSION

Here, we have documented a rostral-to-caudal gradient of KOR activity, with less DA inhibition via KOR in the rostral compared with the caudal area of the NAc core. This sub-regional based dichotomy appears to be a function of anatomical location, that is, independent of magnitude of DA innervation, as we observed comparable DA release in rostral and caudal NAc core in males and females, suggesting that the dopaminergic projection from the VTA is equivalent across the rostro-caudal axis and between sexes. Indeed, a recent tract tracing study showed that the mesolimbic projection is equally dense across the subregions, although the projection neurons themselves may be distinct.⁴⁷ A previous study using rats has shown sex differences in magnitude of DA release between males and females, with evoked accumbal DA release being greater in females compared with males because of greater DA synthesis in females.^33,48,49 However, these studies were all done in rats, whereas our work here and others carried out in mice have found no sex differences.⁵⁰ The lack of differences in DA uptake across the rostro-caudal axis and across sexes suggests that there is no difference in DAT expression or function. The greater variability observed in our female data could be, in part, because of the oestrous cycling effects or dispersed electrode placements within the NAc core.

Although no differences were observed in baseline DA release and uptake, KOR activation was shown to inhibit DA release to a greater extent in the caudal subregion of the NAc core in female and male mice. KORs inhibit DA release because of terminal hyperpolarization via increased potassium channel conductance.⁵¹ The increased inhibition of DA in the caudal subregion could be driven by differential expression levels of KORs on DA terminals in the rostral and caudal accumbens core. To date, there have been no studies that have examined these differences. The differential magnitude of KOR-activation induced inhibition of DA release appears to be driven by a difference in potency, demonstrated by a reduction in EC50, as well as in efficacy, shown by greater maximal effect in inhibition of DA release. Although the mechanisms underlying the differential KOR agonist effects are unknown, ligand affinity can be influenced by many factors that influence G-protein coupling, including properties of the lipid bilayer and intracellular milieu.^52,53 Thus, the intracellular cascade triggered by U50,488 binding may be different along the rostro-caudal axis of the NAc core. For example, individual Gα-subunits differentially affect conformation and ligand affinity of the KORs.⁵³ It is possible that these various Gα-subunits are expressed in distinct proportions in DA terminals in rostral and caudal NAc core, leading to the unequal efficacy and potency in the two subregions.

The regional differences in KOR control over DA release are comparable in both sexes. Previous studies have shown sex differences in KOR-mediated DA inhibition and associated behaviours, with females exhibiting less DA inhibition and behavioural responses following KOR activation, but no differences in KOR mRNA.^33,34,54 It is important to note that these are in vivo studies, and KOR agonist was administered intraperitoneally. Thus, it is possible that the observed sex differences are driven by differential KOR expression and/or function in brain areas other than the NAc core. Another study examining the effect of KOR activation on aversion, analgesia and DA transmission showed that KOR-mediated inhibition of DA release in the NAc is blunted in female mice.⁵⁵ This blunting of KOR inhibitory control over DA release occurs via oestradiol mediated phosphorylation of G-protein receptor protein kinase 2, ultimately reducing G-protein signalling and is thus dependent on the oestrous cycle.⁵⁵ In the work presented herein, we did not track the female’s cycles, as we aimed to examine the effects of acute KOR activation and avoid any repeated stress. It is possible that it is the reason that we do not see sex differences, as our sample size could be spread across different stages of the oestrous cycle. However, we do not anticipate that the intracellular signalling pathway by which oestradiol mediated G-protein signalling to be any different across the rostro-caudal axis.

In addition to DA release, we also evaluated the exponential decay of the uptake portion of the curve by the measure tau.^45,46 At very low DA release concentrations, such as those caused by U50,488, the uptake is obscured as return to baseline appears to be slower than at the higher release concentrations. To limit this effect from affecting our assessment of uptake, we evaluated tau at only the two lowest doses of U50,488. We did not observe a significant directional change from the 10 to the 30 nM dose of U50,488, suggesting that the uptake was not consistently being affected in either direction. Previous studies showed KOR activation rapidly increased DAT activity; however, they were conducted using an in vitro analysis using rat tissue and a single exposure of the agonist.²⁵ The data presented herein used an ex vivo method in mice following a cumulative concentration response curve; thus, the assay preparation, agonist dose, species differences and KOR activation time could potentially explain the discrepancy.

In this study, we have shown that although DA release and uptake rate are not different across the rostro-caudal axis within NAc core, KOR control over these measures is significantly enhanced in the caudal versus rostral subregion. Activation of KORs in rostral NAc shell has been reported to promote conditioned place preference, whereas KOR activation in the caudal shell subregion produces conditioned place avoidance.³⁶ Given the involvement of the DA system in shaping hedonic and anhedonic states, it is possible that these behavioural differences are produced because of, at least in part, differential impact of KORs over DA transmission across the rostro-caudal axis. Particularly, KOR activation inhibits DA release significantly more in the caudal compared with the rostral NAc core. The differences in KOR regulation of DA release along the rostro-caudal axis observed in the current study likely gate behavioural outcomes as reported in previous studies.^36,38 KORs are ubiquitously present on afferents into the NAc and may bias activity towards direct or indirect pathways in the NAc, further influencing behavioural outcomes.⁵⁶ More research is necessary to pinpoint exact mechanisms that may be involved in the differential interaction between KORs and DATs across the rostro-caudal axis in NAc core and its impact on motivated and affective behaviours.