Abstract
The dynorphin/κ-opioid receptor (KOR) system has been previously implicated in the regulation of cognition, but the neural circuitry and molecular mechanisms underlying KOR-mediated cognitive disruption are unknown. Here, we used an operational test of cognition involving timing and behavioral inhibition and found that systemic KOR activation impairs performance of male and female C57BL/6 mice in the differential reinforcement of low response rate (DRL) task. Systemic KOR antagonism also blocked stress-induced disruptions of DRL performance. KOR activation increased ‘bursts’ of incorrect responses in the DRL task and increased marble burying, suggesting that the observed disruptions in DRL performance may be attributed to KOR-induced increases in compulsive behavior. Local inactivation of KOR by injection of the long-acting antagonist nor-BNI in the ventral tegmental area (VTA), but not the infralimbic prefrontal cortex (PFC) or dorsal raphe nucleus (DRN), prevented disruption of DRL performance caused by systemic KOR activation. Cre-dependent genetic excision of KOR from dopaminergic, but not serotonergic neurons, also blocked KOR-mediated disruption of DRL performance. At the molecular level, we found that these disruptive effects did not require arrestin-dependent signaling, because neither global deletion of G-protein receptor kinase 3 (GRK3) nor cell-specific deletion of GRK3/arrestin-dependent p38α MAPK from dopamine neurons blocked KOR-mediated DRL disruptions. We then showed that nalfurafine, a clinically available G-biased KOR agonist, could also produce DRL disruptions. Together, these studies demonstrate that KOR activation in VTA dopamine neurons disrupts behavioral inhibition in a GRK3/arrestin-independent manner and suggests that KOR antagonists could be beneficial for decreasing stress-induced compulsive behaviors.
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Introduction
Dynorphin, an endogenous opioid peptide released following stress, activates the κ-opioid receptor (KOR) to produce depression-like behaviors (Mague et al, 2003; Knoll and Carlezon, 2010) and aversion (Shippenberg and Herz, 1986). In humans, acute KOR activation with selective and highly efficacious KOR agonists or the natural products from the Salvia divinorum plant (Salvinorin A; a selective KOR agonist) produces potent psychotomimetic and hallucinogenic effects (Johnson et al, 2011), suggesting that KOR activation may also contribute to cognitive disruptions following a behavioral stress experience.
Although it is challenging to measure psychotomimetic drug effects using animal models, KOR activation has been shown to alter multiple domains of cognition in rodents, including attention, memory, and impulsivity (Nemeth et al, 2010; Cole et al, 2013). KOR activation has no effect on impulse control (Paine et al, 2007) or impulsive choice, but does disrupt behavioral performance in a response inhibition task (Walker and Kissler, 2013). Degradation of response inhibition may be indicative of a loss of inhibitory control, a cognitive feature that is disrupted in psychiatric illnesses such as substance use disorder (Jentsch and Taylor, 1999), affective disorders (Murphy et al, 1999), and compulsive disorders (Chamberlain et al, 2005). KOR activation following stress may exacerbate behavioral symptoms or contribute to the etiology of these diseases. Although KOR antagonists are in development for the therapeutic goal of decreasing stress-induced mood disorders and relapse of substance abuse (Carroll and Carlezon, 2013), it is unknown whether KOR antagonists may also be useful for decreasing stress-induced cognitive disruptions or compulsive behaviors.
Behavioral and cognitive disruptions in patients with obsessive–compulsive disorder (OCD) can worsen following stressful events (Fornaro et al, 2009). Many of the symptoms observed in OCD may be a consequence of loss of inhibitory control (Chamberlain et al, 2005) leading to obsessive or compulsive thoughts and behaviors. One method to investigate disruption of inhibitory control in rodents is through the use of the differential reinforcement of low response rate (DRL) task. The DRL task requires an animal to withhold responding for a set wait period before making a reinforced response (Sidman, 1955). Responses occurring before the end of the wait period (nonreinforced responses) reset the wait period. This task has previously been used to measure temporal discrimination (Sidman, 1956), antidepressant efficacy (O’Donnell et al, 2005), and impulsive action (Selleck et al, 2015). Nonspecific opioid receptor activation in the prefrontal cortex disrupts performance in the DRL task (Selleck et al, 2015), demonstrating that opioid receptor activation decreases inhibitory control. Stress-induced release of dynorphin would be expected to disrupt cognitive performance, but the specific nature of these disruptions, the sites of action in brain, and the cellular mechanisms underlying these disruptions are not yet known. Here, we find that KOR activation in dopamine neurons of the ventral tegmental area (VTA) disrupts inhibitory control to increase compulsive responses. We also determine that KOR-mediated DRL disruptions are likely to use G-protein receptor kinase 3 (GRK3)/arrestin-independent intracellular signaling pathways, indicating that some G-biased KOR agonists may produce compulsive responses.
Materials and methods
Subjects
Male and female C57BL/6 mice (n=174) ranging from 3 to 10 months of age were used in these experiments.
Drugs
The (±)U50488H (U50488), nor-binaltorphimine (nor-BNI), and nalfurafine were provided by the National Institute of Drug Abuse Drug Supply Program (Bethesda, MD). U50488 (5 and 10 mg/kg), nor-BNI (10 mg/kg), or nalfurafine (50 μg/kg) was dissolved in saline and administered intraperitoneally (i.p.) in a volume of 10 ml/kg. The nor-BNI (2.5 μg/μl) dissolved in sterile artificial cerebrospinal fluid (ACSF) was intracranially microinjected.
Procedures
Differential reinforcement of low rates of responding (DRL)
Following training to discriminate between active and inactive nose poke holes, mice were trained in a 60-min DRL procedure (Horwood et al, 2001). During a DRL session, a single nose poke led to food reward delivery. Following the first reinforced nose poke, subjects were required to withhold responding for a specified wait period. Nose pokes that occurred before the end of the wait period reset the wait period and were nonreinforced. Once trained for stable performance on the DRL task (below 40% error over at least 4 days; 15–25 training sessions), animals underwent sessions where they received saline treatment on a baseline DRL day, and a U50488 treatment the following day immediately before a DRL session. For microinjection studies, male mice were used that had previously been tested for the systemic effect of KOR activation in the DRL task. Male mice were trained in the DRL task, and then received a single microinjection of nor-BNI (2.5 μg/μl; long-acting κ-antagonism by JNK activation; Bruchas et al, 2007; Land et al, 2009) into the infralimbic prefrontal cortex (PFC; n=4), dorsal raphe nucleus (DRN; n=4), or VTA (n=10). Controls received an injection of ACSF into the VTA (n=8). Mice recovered from surgery for 3 days before retraining for stable performance in the DRL task (3–5 days). Following retraining, mice received a saline baseline test day and U50488 the following day. Conditional and global knockout mice and their littermate controls received two DRL sessions with 5 mg/kg U50488 and one DRL session with 10 mg/kg U50488. Each test was separated by at least 5–10 daily sessions of DRL training between the drug test days to ensure that there were no persistent effects of drug administration on DRL performance. Each drug test day was preceded by a saline baseline DRL test day.
Forced-swim stress
To induce stress, C57BL/6 male mice (n=8) were exposed to a modified forced-swim test as previously described (McLaughlin et al, 2003). Briefly, the modified-Porsolt forced-swim paradigm used was a 2-day procedure in which mice swam in 30 °C water for 15 min the first day following a baseline DRL session, and then 6 min during each of four trials on the second day without the opportunity to escape. Mice were tested for DRL performance within 10 min following the second day of stress (stress 1). After 1 week, mice received the 2-day FSS procedure (stress 2) with nor-BNI (10 mg/kg; i.p.) or saline 24 h pretreatment (n=4 per group) and DRL performance was tested the following day.
Marble burying
C57BL/6 male mice were placed in a novel rectangular context (50.8 × 25.4 × 25.4 cm; corn cob bedding packed to 5 cm depth) housed in a sound- and light-attenuating cabinet for a 30 min habituation period. Mice then received an injection of U50488 (n=10) or saline (n=9) and were immediately returned to the context for another 30 min habituation period. Mice were removed from the context and 18 marbles (16 mm diameter) were placed on the bedding. Mice were immediately returned for a 30 min marble burying test. Total number of marbles buried (at least 2/3 covered) at the end of the 30 min session were counted by an experimenter blinded to drug treatment (Deacon, 2006).
Data Analysis
Error percentage was calculated by the number of responses that led to a reset of the wait time (nonreinforced responses) divided by total number of responses (reinforced+nonreinforced responses). Stable performance in the DRL task was defined as at least 4 days with <40% error (Sinden et al, 1986). After mice showed stable performance, they received baseline (saline) and U50488 test days. Mice that did not show stable performance also received baseline and test days to match drug treatment between cage-mates. Thus, mice that showed >40% error during the DRL baseline day were removed from analysis. Experimenters were blind to genotype (Figures 4e–h and 5) and group assignment (Figures 4a–d). Interresponse time (IRT) was the time between each nose poke on the active port. MATLAB software (version R2016a) was used to extract burst response data. Data were analyzed with t-tests or two-way ANOVAs as required by experimental designs for DRL, fixed ratio 1 (FR1), fixed ratio 5 (FR5), and marble burying experiments. For experiments in Figure 4, data from two baseline days and two U50488 days were averaged for analysis. The post hoc comparisons were conducted with Sidak’s test. For all statistical tests, the α was set to 0.05.
Results
Systemic KOR Activation Disrupts DRL Performance in Male Mice
Following training for stable reinforcement under a DRL 15 s schedule, we tested the effect of systemic KOR activation on DRL performance in male mice. The experimental paradigm is diagrammed (Figure 1a). Responses during a DRL session could be either reinforced or nonreinforced, and either response resets the 15 s wait period. A subset of nonreinforced responses occurred with RT intervals of <1 s. These ‘burst responses’ have been described as a consequence of reward omission and a disruption in positive feedback for reinforced responses (Kramer and Rilling, 1970). Mice (n=13) were administered saline before a DRL session on a baseline day, and 1 day later received a KOR agonist (U50488) immediately before a DRL session. A representative raster plot of responses in the DRL task is shown from a male mouse following saline and then U50488 pretreatment (Figure 1b).
Administration of U50488 (5 mg/kg) significantly increased the number of nonreinforced responses (t12=2.78, p=0.016) and significantly decreased the number of reinforced responses (t12=2.54, p=0.026) during the DRL test session compared with the saline test session, but there was no significant difference between saline and U50488 treatment in total response number (Figure 1c). Treatment with U50488 significantly increased percent error during the DRL session (t12=6.59, p<0.0001) compared with the baseline saline day (Figure 1d). To investigate the temporal patterns of responding in the DRL task following KOR activation, we compared IRTs during U50488 and saline treatment days (Figure 1e). There was a significant main effect on IRT (F(10, 120)=39.86, p<0.0001), a main effect of Treatment (F(1, 12)=16.67, p=0.002), and an interaction between IRT and Treatment (F(10, 120)=24.4, p<0.0001). There was a significant difference between saline and U50488 treatment in the 0–3 s IRT bin (p<0.0001), indicating a loss of inhibitory control of behavior (Selleck et al, 2015). The loss of inhibitory control caused by KOR activation was evident as a significant increase in burst responding (t12=2.85, p=0.015) (Figure 1f).
Stress-induced release of dynorphin generated similar disruptions to those observed with systemic U50488 treatment in a separate cohort of mice. Following a repeated forced-swim stress procedure (stress 1), mice (n=8) showed a significant increase in percent error (t8=2.93, p=0.022). These mice were then retrained and retested following a second repeated forced-swim stress (stress 2) with saline (n=4) or nor-BNI (n=4) pretreatment. Mice that received saline before stress 2 showed a significant increase in percent error (t4=3.59, p=0.037), but mice that received nor-BNI before stress 2 did not show a significant increase in percent error (Figure 1g). Together, these results demonstrate that pharmacological or stress-induced KOR activation impaired performance in the DRL task by disrupting behavioral inhibition and indicate that KOR activation increased compulsive responses to unexpected reward omissions.
Systemic KOR Activation Disrupts DRL Performance in Female Mice
KOR activation has sex-dependent effects on depressive behaviors (Russell et al, 2014; Chartoff and Mavrikaki, 2015), but it is unknown whether there are sex differences in KOR-mediated cognitive disruption. Female mice (n=10) showed lower error rates during initial training in the 15 s DRL task compared with male mice (data not shown). Female mice were instead trained with a 25 s DRL schedule to ensure that DRL performance was learned, rather than reflecting a baseline rate of performance. Figure 2a shows a raster plot of responses in the DRL task from a representative female mouse with saline then U50488 pretreatment. Following training for stable performance in the 25 s DRL task, KOR activation (U50488; 5 mg/kg) significantly increased nonreinforced responses (t9=4.38, p=0.002) and total responses (t9=2.70, p=0.024). There was no significant effect of U50488 on reinforced responses compared with saline treatment (Figure 2b). However, there was a significant increase in percent error (t9=11.48, p<0.0001) during the DRL task in female mice (Figure 2c). A two-way ANOVA (IRT and Treatment as factors; Figure 2d) showed a significant main effect on IRT (F(15, 135)=27.9, p<0.0001), a main effect of Treatment (F(1, 9)=20.9, p=0.001), and an interaction between IRT and Treatment (F(15, 135)=16.21, p<0.0001). There was a significant difference between saline and U50488 treatment in the 0–3 s (p<0.0001) and 3–6 s time bins (p=0.031). KOR activation also significantly increased (t9=4.19, p=0.002) burst responding (Figure 2e). These results show that despite procedural differences in the DRL paradigm (ie, wait times), male and female mice display qualitatively similar disruptions in DRL performance efficiency following KOR activation and KOR-mediated disruptions of cognition may not be sex dependent.
KOR Activation Does Not Affect FR Responding and Increases Marble Burying
In the DRL task, burst responses have been hypothesized to occur when the delivery of reward is ambiguous or omitted (Sidman, 1956). To determine whether the burst responding caused by KOR activation could be attributed to a nonspecific change in operant performance or compulsive increases in responding, we tested the effect of KOR activation in two fixed ratio tasks. Male mice (n=7) were trained in the FR1 procedure, where one nose poke led to the delivery of one food pellet. Following stable performance in the FR1 task, mice were given saline on one day and U50488 (5 mg/kg) the following day. There was no significant difference in the total number of reinforced responses following U50488 treatment (Figure 3a), and unlike mice trained in the DRL task, all responses were separated by at least 1 s, showing that FR1-trained animals did not produce burst responses with saline or U50488 treatment. We trained a separate cohort of male mice in an FR5 task (n=6), where five nose pokes led to the delivery of one food pellet. KOR activation did not significantly alter the number of total responses during an FR5 session (Figure 3b) and produced no burst responses following a reinforced response. These experiments demonstrated that when reward delivery was predictable, KOR activation did not promote compulsive burst responses.
However, when tested in a task producing mild anxiety, such as in the marble burying test (Deacon, 2006), KOR activation with U50488 significantly (t17=2.71, p=0.015) increased the number of marbles buried (Figure 3c). These results suggest that KOR-mediated increases in burst responses may be specific to anxiogenic or ambiguous reward contexts.
KORs in the VTA and in Dopamine Neurons Are Required for KOR-Mediated DRL Disruptions
The infralimbic PFC, DRN, and VTA contain KORs (Mansour et al, 1987) and have been implicated in response inhibition (Dalley et al, 2011). To identify the brain regions involved in KOR-mediated DRL disruptions, male mice were trained in the DRL task, and then received either ACSF into the VTA (Control; n=8) or a microinjection of nor-BNI (2.5 μg/μl) bilaterally into the PFC (n=4) or VTA (n=10) or unilaterally into the DRN (n=4) (Figure 4a). For nonreinforced responses, a two-way ANOVA with Treatment (Saline; U50488) and Brain Region (PFC; DRN; VTA; Control) as factors showed that there was a significant effect of Treatment (F(1, 22)=36.71, p<0.0001) and a significant interaction between Treatment and Brain Region (F(3, 22)=10.16, p=0.0002). Nonreinforced responses were different between saline and U50488 treatment days in Control (p=0.0003) and DRN (p=0.0001) groups, but not in VTA or PFC (Figure 4b). There was a significant effect of U50488 treatment (F(1, 22)=28.02, p<0.0001) and Brain Region (F(3, 22)=4.22, p=0.017) on percent error, and a significant interaction between Treatment and Brain Region (F(3, 22)=7.25, p=0.002). U50488 treatment significantly increased percent error in Control (p=0.003), DRN (p=0.024), and PFC (p=0.004) groups, but not in the VTA group (Figure 4c). This suggests that KOR activation in the VTA was required for disruptions in DRL performance. For burst responses (Figure 4d), there was a significant effect of Treatment (F (1, 23)=5.34, p=0.03). There was no significant effect of U50488 on reinforced responses, total responses, or IRTs in VTA injected mice (Supplementary Figure 1). Histological confirmation for VTA targeting is shown in Supplementary Figure 2.
These results suggested that KOR activation in the VTA was required for the U50488-mediated effects on DRL performance. Although the VTA primarily comprises dopaminergic neurons, it contains other cell types and is innervated by a broad variety of neurons (eg, serotonergic or GABAergic) that would be affected by KOR inactivation (Polter and Kauer, 2014). Dopaminergic and serotonergic neuron activity is important for impulsivity (Dalley and Roiser, 2012) and KOR-mediated aversion (Ehrich et al, 2015), but cognitive disruptions may occur through distinct cellular mechanisms. We tested whether KOR-mediated disruptions in DRL performance occurred in male mice having either global KOR knockout (KOR KO; n=13), or KOR conditionally removed from ePet1-Cre-expressing serotonergic neurons (KOR CKOPET; n=4), or KOR conditionally removed from DAT-Cre-expressing dopaminergic neurons (KOR CKODAT; n=11). Control mice (n=18) were KORlox/lox or KOXlox/+ littermates from CKO colonies (Figure 4e).
For nonreinforced responses, a two-way ANOVA with Treatment (Saline; U50488) and Genotype (Control; KOR CKOPET; KOR CKODAT; KOR KO) as factors showed a main effect of Treatment (F(1, 42)=16.63, p<0.001) and a nonsignificant trend toward an interaction between Treatment and Genotype (F(3, 42)=2.59, p=0.065; Figure 4f). For percent error, there was a significant effect of Treatment (F(1, 42)=36.4, p<0.001), Genotype (F(1, 42)=3.034, p=0.04), and an interaction between Treatment and Genotype (F(3, 42)=7.187, p<0.001). U50488 treatment significantly increased percent error in Control (p<0.0001) and KOR CKOPET (p=0.0001) groups, but not KOR CKODAT or KOR KO groups (Figure 4g). For burst responses, there was a significant effect of Treatment (F(1, 42)=26.1, p<0.0001) and a significant interaction between Treatment and Genotype (F(3, 42)=3.192, p=0.033). KOR activation significantly increased burst responses in Control (p=0.0007) and KOR CKOPET groups (p=0.0035), but not KOR CKODAT or KOR KO groups (Figure 4h). Analyses of reinforced responses, total responses, and IRTs for this experiment are shown in Supplementary Figure 3. Similar effects were observed when mice were given a 10 mg/kg dose of U50488 (Supplementary Figure 4). Similar to pharmacological blockade of VTA KORs, genetic excision of KORs from dopaminergic neurons prevents KOR-mediated increases in percent error.
KOR-Mediated DRL Disruptions Are GRK3/Arrestin Independent
The aversive effects of KOR activation have been attributed to GRK3/arrestin-dependent activation of the p38α mitogen-activated protein kinase (p38 MAPK) in dopamine neurons (Bruchas et al, 2007; Ehrich et al, 2015). We tested whether KOR-mediated cognitive disruptions had similar molecular requirements to KOR-mediated aversion by measuring KOR effects on DRL performance in mice with conditional knockout of p38 MAPK from dopaminergic neurons (p38αCKODAT). Male p38αCKODAT (n=4) and p38αlox/+ littermates (n=5) showed a main effect of Treatment in percent error (F(1, 7)=28.5, p=0.001) but no effect of Genotype and no interaction between Genotype and Treatment (Figure 5a). Before p38 activation, GRK3 phosphorylates KOR and promotes arrestin binding to KOR to initiate MAPK signaling (Bruchas et al, 2006). To test whether the KOR-mediated DRL disruptions were arrestin dependent, we used male GRK3 knockout mice (n=14) and wild-type littermates (n=9). There was a significant effect of Treatment (F(1, 21)=45.2, p<0.001), but no effect of Genotype and no interaction between Genotype and Treatment. We then tested whether a G-biased KOR agonist, nalfurafine (Schattauer et al, 2017), could produce deficits that were comparable to the unbiased KOR agonist U50488. Nalfurafine (50 μg/kg) pretreatment produced a significant increase in percent error (t12=9.125, p<0.0001). Analyses of nonreinforced responses and burst responses for these experiments are shown in Supplementary Figure 5. Together, these results demonstrate that KOR-mediated disruptions of DRL performance are GRK3/arrestin independent.
Discussion
The present study specifies the cellular and molecular pathways underlying KOR-mediated increases in compulsive responses. First, we found that systemic KOR activation disrupted inhibitory control and decreased response efficiency in the DRL task by increasing nonreinforced responses and burst responses in both male and female mice. Systemic KOR antagonism blocked disruptions of DRL performance caused by stress-induced release of dynorphin in male mice. The burst responses induced by pharmacological KOR activation could not be attributed to a simple disruption of operant responding, as KOR activation did not produce burst responding in an FR1 or FR5 task. Instead, U50488 administration promoted marble burying, suggesting that KOR activation increased compulsive behaviors in anxiogenic or uncertain environments. Second, we demonstrated that KOR-mediated disruptions in DRL performance were due to KOR activity in the VTA and KOR activation on dopamine neurons. Third, although arrestin-dependent signaling in dopaminergic neurons is required for KOR-mediated aversion (Ehrich et al, 2015), KOR-mediated cognitive disruptions can be generated via arrestin-independent intracellular signaling pathways. Together, these findings reveal a relationship between KOR activation and inhibitory control of behavior that may underlie interactions between stress and compulsivity.
Although KOR actions can have sex-dependent effects (Russell et al, 2014), we found that KOR agonism disrupted DRL performance in both male and female mice. Sex differences have been observed in stress circuitry (Goldstein et al, 2010) and impulsivity (Mitchell and Potenza, 2015), suggesting that there could be sex-specific effects of KOR activation on DRL performance. One issue in comparing male and female mouse behavior in the DRL task is that female rats acquire DRL more efficiently than male rats, and we observed the same sex difference in mice. This effect on DRL performance has been suggested to result from an effect of ovarian steroids (Beatty, 1973) or baseline differences in locomotor activity in males and females (van Hest et al, 1987). To account for baseline differences in DRL acquisition in the present study, female mice were trained on a DRL 25 s protocol, rather than a DRL 15 s protocol. Despite the differences in DRL procedure, KOR activation induced qualitatively similar deficits in males and females in the DRL task, demonstrating that the cognition disrupting effects of KOR activation are likely to be sex independent.
Performance in the DRL task can be disrupted by several different factors, including dysregulation of temporal discrimination, general locomotor alteration, and the loss of inhibitory control (Kramer and Rilling, 1970). Alterations in temporal discrimination would shift the peak of observed IRTs (Cho and Jeantet, 2010), but in these experiments, there was no broad shift in IRTs observed following KOR activation. Instead, there were increases in responses occurring within the 0–3 s IRT bin, indicating deficits in inhibitory control (Selleck et al, 2015). We assessed how inhibitory control may be affected in the DRL task by analyzing burst responses (IRTs <1 s) and found that burst responses increased following U50488 administration. If burst responses increased in operant tasks where reinforcement probability is consistently predictable (eg, an FR1 schedule of reinforcement), it would suggest that KOR activation produced a broad increase in compulsive responding. However, we observed no increased burst responses and no differences in total responses in an FR1 or FR5 task following KOR activation. Many studies have reported hypolocomotion induced by KOR activation (Paris et al, 2011), suggesting that the KOR-mediated increase in burst responses is unlikely to reflect a simple motoric effect. We found that compulsive responses could be observed following KOR activation in the marble burying task, and corroborated Rose et al (2016), who demonstrated that KOR antagonism decreased drug-induced marble burying. Marble burying may reflect a perseverative or compulsive response that is increased in anxiogenic settings, but is not consistently correlated with other anxiety behaviors (Thomas et al, 2009). Compulsive responses have been hypothesized to arise as a reaction to environmental uncertainty to cope with ambiguous or threatening stimuli (Holaway et al, 2006). Novel objects in the marble burying task may provoke compulsive behavior, or uncertainty in the DRL task about the relationship between responses and outcomes following a nonreinforced response could produce compulsive responding. In contrast, during the FR tasks, there is a stable relationship between responses and outcomes, leading to a lack of burst responses following KOR activation. In addition to evidence suggesting that KOR activation can enhance dopamine D2 receptor-mediated compulsive responses (Perreault et al, 2007), our data indicate that KOR activity may have potent effects on compulsive behaviors via dopaminergic circuits.
Dopamine neurons in the VTA encode discrepancies between expected and actual outcomes (Schultz et al, 1997), as well as convey information about whether outcomes are better or worse than expected (Hart et al, 2014). Alterations in reinforcement probability can modify dopamine neuron responsivity to aversive events (Matsumoto et al, 2016). KOR activation on dopamine neurons in the VTA can produce aversion (Ehrich et al, 2015), and potentiate cocaine reward (Ehrich et al, 2014). We found that KOR activation in the VTA but not the DRN was necessary for KOR-mediated DRL disruptions. Mice with KOR blockade in the PFC increased percent error as a result of nonsignificant decreases in reinforced responses and increases in nonreinforced responses, rather than increases in burst responses. Local KOR activation in the PFC or in the VTA can decrease dopamine release in the PFC (Margolis et al, 2006; Tejeda et al, 2013) and dopamine depletion in the PFC has been shown to disrupt DRL performance by increasing burst responding (Sokolowski and Salamone, 1994). KOR blockade on dopamine neurons in the PFC may block burst responses, but this may not be sufficient to overcome the percent error increasing effects of KOR activation in the VTA. It is also possible that KOR activation in the VTA could dysregulate the dopamine signals received in the corticostriatal network to disrupt normal feedback mechanisms that control behavioral inhibition (Jentsch and Taylor, 1999).
Cre-driven excision of KOR can simultaneously remove KOR activity from somatic and terminal regions of particular neuronal populations. We compared the effect of conditional knockout of KOR from dopaminergic or serotonergic neurons against littermate controls and mice with global deletion of KOR. KOR activation in dopaminergic, but not serotonergic neurons, was necessary for disruptions in DRL performance. KOR activation on serotonergic neurons is important for affective processing (Bruchas et al, 2011) and cocaine reward potentiation (Schindler et al, 2012), but may not contribute to this particular feature of cognition. Many serotonergic antidepressant drugs that are effective in decreasing compulsive behaviors (Kellner, 2010) are also effective in improving DRL performance (O’Donnell et al, 2005), and hence it is possible that chronic KOR activation could lead to serotonin-dependent disruptions in DRL performance. Conditional deletion of KOR from dopaminergic neurons prevented increases in burst responding, demonstrating that KOR activation on dopaminergic neurons may be important for generating responses to reward omissions. One challenge with a conditional deletion of KOR from dopamine neurons is that dopamine neurons in the substantia nigra (SN) also contain KORs (Tempel and Zukin, 1987). Although SN KORs do not contribute to aversion (Bals-Kubik et al, 1993), KORs in the SN could produce the observed cognitive disruptions or compulsive behaviors. However, in combination with the microinjection experiments targeting the VTA, we show that KOR activation on dopamine neurons of the VTA disrupts inhibitory control.
Ehrich et al (2015) demonstrated that KOR-mediated aversion requires GRK3/arrestin-dependent p38α MAPK activation in VTA dopamine neurons. However, we found no effect of global GRK3 deletion or p38α MAPK deletion from dopamine neurons on KOR-mediated DRL disruptions. These findings show that KOR effects on cognition are distinct from KOR-mediated effects on aversion and likely to be arrestin independent. We also found that nalfurafine, a G-biased KOR agonist (Schattauer et al, 2017), could produce the observed DRL disruptions. An important implication of this finding is that a highly G-biased KOR agonist used in the treatment of pain or itch without producing dysphoria might still be complicated by unwanted cognitive side effects at high doses (Chavkin, 2011).
In summary, our studies demonstrate that KOR activation in dopamine neurons in the VTA disrupts inhibitory control of behavior. Dopamine neurons have been shown to modulate compulsive behaviors in mice (Pascoli et al, 2015), and our results show that KOR activation in dopamine neurons may increase compulsive behaviors when reinforcement probability is ambiguous. In contrast, when reinforcement probability was well predicted, KOR activation had no effect or promoted inhibition of behavior. Together, these studies suggest that KOR activation disrupts behavioral inhibition in a reward context-dependent manner. Our results demonstrate that KOR antagonism during periods of chronic stress could decrease cognitive disruptions and may be beneficial for treating stress-mediated increases in compulsive responses. In agreement with our preclinical findings, there are some case studies reporting that opioid antagonists, such as naltrexone, can decrease compulsive behaviors (Kim, 1998) and buprenorphine, a μ-opioid receptor partial agonist and KOR antagonist, can decrease treatment resistant compulsive behaviors (Liddell et al, 2013). Future studies could identify the molecular mechanisms underlying KOR-mediated increases in compulsive behaviors to generate novel therapeutic interventions for stress-induced cognitive disruptions.
Funding and disclosure
The authors declare no conflict of interest.
References
Bals-Kubik R, Ableitner A, Herz A, Shippenberg TS (1993). Neuroanatomical sites mediating the motivational effects of opioids as mapped by the conditioned place preference paradigm in rats. J Pharmacol Exp Ther 264: 489–495.
Beatty WW (1973). Effects of gonadectomy on sex differences in DRL behavior. Physiol Behav 10: 177–178.
Bruchas MR, Land BB, Aita M, Xu M, Barot SK, Li S et al (2007). Stress-induced p38 mitogen-activated protein kinase activation mediates kappa-opioid-dependent dysphoria. J Neurosci 27: 11614–11623.
Bruchas MR, Macey TA, Lowe JD, Chavkin C (2006). Kappa opioid receptor activation of p38 MAPK is GRK3- and arrestin-dependent in neurons and astrocytes. J Biol Chem 281: 18081–18089.
Bruchas MR, Schindler AG, Shankar H, Messinger DI, Miyatake M, Land BB et al (2011). Selective p38α MAPK deletion in serotonergic neurons produces stress resilience in models of depression and addiction. Neuron 71: 498–511.
Bruchas MR, Yang T, Schreiber S, Defino M, Kwan SC, Li S et al (2007). Long-acting kappa opioid antagonists disrupt receptor signaling and produce noncompetitive effects by activating c-Jun N-terminal kinase. J Biol Chem 282: 29803–29811.
Carroll FI, Carlezon WA (2013). Development of κ opioid receptor antagonists. J Med Chem 56: 2178–2195.
Chamberlain SR, Blackwell AD, Fineberg NA, Robbins TW, Sahakian BJ (2005). The neuropsychology of obsessive compulsive disorder: the importance of failures in cognitive and behavioural inhibition as candidate endophenotypic markers. Neurosci Biobehav Rev 29: 399–419.
Chartoff EH, Mavrikaki M (2015). Sex differences in kappa opioid receptor function and their potential impact on addiction. Front Neurosci 9: 466.
Chavkin C (2011). The therapeutic potential of κ-opioids for treatment of pain and addiction. Neuropsychopharmacology 36: 369–370.
Cho YH, Jeantet Y (2010). Differential involvement of prefrontal cortex, striatum, and hippocampus in DRL performance in mice. Neurobiol Learn Mem 93: 85–91.
Cole S, Richardson R, McNally GP (2013). Ventral hippocampal kappa opioid receptors mediate the renewal of fear following extinction in the rat. PLoS ONE 8: e58701.
Dalley JW, Everitt BJ, Robbins TW (2011). Impulsivity, compulsivity, and top-down cognitive control. Neuron 69: 680–694.
Dalley JW, Roiser JP (2012). Dopamine, serotonin and impulsivity. Neuroscience 215: 42–58.
Deacon RMJ (2006). Digging and marble burying in mice: simple methods for in vivo identification of biological impacts. Nat Protoc 1: 122–124.
Ehrich JM, Messinger DI, Knakal CR, Kuhar JR, Schattauer SS, Bruchas MR et al (2015). Kappa opioid receptor-induced aversion requires p38 MAPK activation in VTA dopamine neurons. J Neurosci 35: 12917–12931.
Ehrich JM, Phillips PEM, Chavkin C (2014). Kappa opioid receptor activation potentiates the cocaine-induced increase in evoked dopamine release recorded in vivo in the mouse nucleus accumbens. Neuropsychopharmacology 39: 3036–3048.
Fornaro M, Gabrielli F, Albano C, Fornaro S, Rizzato S, Mattei C et al (2009). Obsessive-compulsive disorder and related disorders: a comprehensive survey. Ann Gen Psychiatry 8: 13.
Goldstein JM, Jerram M, Abbs B, Whitfield-Gabrieli S, Makris N (2010). Sex differences in stress response circuitry activation dependent on female hormonal cycle. J Neurosci 30: 431–438.
Hart AS, Rutledge RB, Glimcher PW, Phillips PEM (2014). Phasic dopamine release in the rat nucleus accumbens symmetrically encodes a reward prediction error term. J Neurosci 34: 698–704.
Holaway RM, Heimberg RG, Coles ME (2006). A comparison of intolerance of uncertainty in analogue obsessive-compulsive disorder and generalized anxiety disorder. J Anxiety Disord 20: 158–174.
Horwood JM, Ripley TL, Stephens DN (2001). DRL performance in mice with deletion of tPA, uPA or PAI-1 genes. Behav Pharmacol 12: 487–496.
Jentsch JD, Taylor JR (1999). Impulsivity resulting from frontostriatal dysfunction in drug abuse: implications for the control of behavior by reward-related stimuli. Psychopharmacology (Berl) 146: 373–390.
Johnson MW, MacLean KA, Reissig CJ, Prisinzano TE, Griffiths RR (2011). Human psychopharmacology and dose-effects of salvinorin A, a kappa-opioid agonist hallucinogen present in the plant Salvia divinorum. Drug Alcohol Depend 115: 150–155.
Kellner M (2010). Drug treatment of obsessive-compulsive disorder. Dialogues Clin Neurosci 12: 187–197.
Kim SW (1998). Opioid antagonists in the treatment of impulse-control disorders. J Clin Psychiatry 59: 159–164.
Knoll AT, Carlezon WA (2010). Dynorphin, stress, and depression. Brain Res 1314: 56–73.
Kramer TJ, Rilling M (1970). Differential reinforcement of low rates: a selective critique. Psychol Bull 74: 225.
Land BB, Bruchas MR, Schattauer S, Giardino WJ, Aita M, Messinger D et al (2009). Activation of the kappa opioid receptor in the dorsal raphe nucleus mediates the aversive effects of stress and reinstates drug seeking. Proc Natl Acad Sci USA 106: 19168–19173.
Liddell MB, Aziz V, Briggs P, Kanakkehewa N, Rawi O (2013). Buprenorphine augmentation in the treatment of refractory obsessive–compulsive disorder. Ther Adv Psychopharmacol 3: 15–19.
Mague SD, Pliakas AM, Todtenkopf MS, Tomasiewicz HC, Zhang Y, Stevens WC et al (2003). Antidepressant-like effects of kappa-opioid receptor antagonists in the forced swim test in rats. J Pharmacol Exp Ther 305: 323–330.
Mansour A, Khachaturian H, Lewis ME, Akil H, Watson SJ (1987). Autoradiographic differentiation of mu, delta, and kappa opioid receptors in the rat forebrain and midbrain. J Neurosci 7: 2445–2464.
Margolis EB, Lock H, Chefer VI, Shippenberg TS, Hjelmstad GO, Fields HL (2006). κ opioids selectively control dopaminergic neurons projecting to the prefrontal cortex. Proc Natl Acad Sci USA 103: 2938–2942.
Matsumoto H, Tian J, Uchida N, Watabe-Uchida M (2016). Midbrain dopamine neurons signal aversion in a reward-context-dependent manner. Elife 5: e17328.
McLaughlin JP, Marton-Popovici M, Chavkin C (2003). Kappa opioid receptor antagonism and prodynorphin gene disruption block stress-induced behavioral responses. J Neurosci 23: 5674–5683.
Mitchell MR, Potenza MN (2015). Importance of sex differences in impulse control and addictions. Front Psychiatry 6: 24.
Murphy FC, Sahakian BJ, Rubinsztein JS, Michael A, Rogers RD, Robbins TW et al (1999). Emotional bias and inhibitory control processes in mania and depression. Psychol Med 29: 1307–1321.
Nemeth CL, Paine TA, Rittiner JE, Béguin C, Carroll FI, Roth BL et al (2010). Role of kappa-opioid receptors in the effects of salvinorin A and ketamine on attention in rats. Psychopharmacology (Berl) 210: 263–274.
O’Donnell JM, Marek GJ, Seiden LS (2005). Antidepressant effects assessed using behavior maintained under a differential-reinforcement-of-low-rate (DRL) operant schedule. Neurosci Biobehav Rev 29: 785–798.
Paine TA, Tomasiewicz HC, Zhang K, Carlezon WA (2007). Sensitivity of the five-choice serial reaction time task to the effects of various psychotropic drugs in Sprague-Dawley rats. Biol Psychiatry 62: 687–693.
Paris JJ, Reilley KJ, McLaughlin JP (2011). Kappa opioid receptor-mediated disruption of novel object recognition: relevance for psychostimulant treatment. J Addict Res Ther Suppl 4:007. doi:10.4172/2155-6105.S4-007.
Pascoli V, Terrier J, Hiver A, Lüscher C (2015). Sufficiency of mesolimbic dopamine neuron stimulation for the progression to addiction. Neuron 88: 1054–1066.
Perreault ML, Seeman P, Szechtman H (2007). Kappa-opioid receptor stimulation quickens pathogenesis of compulsive checking in the quinpirole sensitization model of obsessive-compulsive disorder (OCD). Behav Neurosci 121: 976–991.
Polter AM, Kauer JA (2014). Stress and VTA synapses: implications for addiction and depression. Eur J Neurosci 39: 1179–1188.
Rose JH, Karkhanis AN, Chen R, Gioia D, Lopez MF, Becker HC et al (2016). Supersensitive kappa opioid receptors promotes ethanol withdrawal-related behaviors and reduce dopamine signaling in the nucleus accumbens. Int J Neuropsychopharmacol 19: pyv127.
Russell SE, Rachlin AB, Smith KL, Muschamp J, Berry L, Zhao Z et al (2014). Sex differences in sensitivity to the depressive-like effects of the kappa opioid receptor agonist U-50488 in rats. Biol Psychiatry 76: 213–222.
Schattauer SS, Kuhar JR, Song A, Chavkin C (2017). Nalfurafine is a G-protein biased agonist having significantly greater bias at the human than rodent form of the kappa opioid receptor. Cell Signal 32: 59–65.
Schindler AG, Messinger DI, Smith JS, Shankar H, Gustin RM, Schattauer SS et al (2012). Stress produces aversion and potentiates cocaine reward by releasing endogenous dynorphins in the ventral striatum to locally stimulate serotonin reuptake. J Neurosci 32: 17582–17596.
Schultz W, Dayan P, Montague PR (1997). A neural substrate of prediction and reward. Science 275: 1593–1599.
Selleck RA, Lake C, Estrada V, Riederer J, Andrzejewski M, Sadeghian K et al (2015). Endogenous opioid signaling in the medial prefrontal cortex is required for the expression of hunger-induced impulsiveaction. Neuropsychopharmacology 40: 2464–2474.
Shippenberg TS, Herz A (1986). Differential effects of mu and kappa opioid systems on motivational processes. NIDA Res Monogr 75: 563–566.
Sinden JD, Rawlins JN, Gray JA, Jarrard LE (1986). Selective cytotoxic lesions of the hippocampal formation and DRL performance in rats. Behav Neurosci 100: 320–329.
Sidman M (1955). Technique for assessing the effects of drugs on timing behavior. Science 122: 925.
Sidman M (1956). Time discrimination and behavioral interaction in a free operant situation. J Comp Physiol Psychol 49: 469.
Sokolowski JD, Salamone JD (1994). Effects of dopamine depletions in the medial prefrontal cortex on DRL performance and motor activity in the rat. Brain Res 642: 20–28.
Tejeda HA, Counotte DS, Oh E, Ramamoorthy S, Schultz-Kuszak KN, Bäckman CM et al (2013). Prefrontal cortical kappa-opioid receptor modulation of local neurotransmission and conditioned place aversion. Neuropsychopharmacology 38: 1770–1779.
Tempel A, Zukin RS (1987). Neuroanatomical patterns of the mu, delta, and kappa opioid receptors of rat brain as determined by quantitative in vitro autoradiography. Proc Natl Acad Sci USA 84: 4308–4312.
Thomas A, Burant A, Bui N, Graham D, Yuva-Paylor LA, Paylor R (2009). Marble burying reflects a repetitive and perseverative behavior more than novelty-induced anxiety. Psychopharmacology (Berl) 204: 361–373.
van Hest A, van Haaren F, van de Poll NE (1987). Behavioral differences between male and female Wistar rats on DRL schedules: effect of stimuli promoting collateral activities. Physiol Behav 39: 255–261.
Walker BM, Kissler JL (2013). Dissociable effects of kappa-opioid receptor activation on impulsive phenotypes in Wistar rats. Neuropsychopharmacology 38: 2278–2285.
Acknowledgements
We thank Dr Scott B Evans for help with data acquisition and analysis. We also thank Salina D Johnson and Daniel I Messinger for genotyping and animal care. This work was supported by NIH grants T32DA07278 (to ADA), T32GM007750 (to HMF), RO1DA030074 (to CC), P50MH106428 (to CC), and the NARSAD Young Investigator Award (to BBL).
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Abraham, A., Fontaine, H., Song, A. et al. κ-Opioid Receptor Activation in Dopamine Neurons Disrupts Behavioral Inhibition. Neuropsychopharmacol. 43, 362–372 (2018). https://doi.org/10.1038/npp.2017.133
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DOI: https://doi.org/10.1038/npp.2017.133
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