κ-Opioid Receptor Activation in Dopamine Neurons Disrupts Behavioral Inhibition

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.

Figure 1

In male C57BL/6 mice, systemic KOR activation disrupted DRL performance. (a) Schematic of the DRL task. Reinforced responses (circle above vertical line) are shown as a positive deflection above the center time line and nonreinforced responses are shown as a vertical line below the center time line. Both reinforced and nonreinforced responses reset the 15 s DRL wait period (gray box), leading to longer wait periods (dashed box) if animals responded before the end of the wait period. Burst responses (horizontal line) were nonreinforced responses that occurred within 1 s of the previous response. (b) To illustrate a typical data set, responses from a single male animal are shown during 60 min DRL sessions with both saline or U50488 pretreatment. Closed circles represent reinforced responses and ‘+’ symbols represent nonreinforced responses. (c) KOR activation by U50488 decreased total number of reinforced responses and increased nonreinforced responses without altering total number of responses. The number of responses made by an individual mouse during a 60 min period following saline pretreatment (▪) were compared with responses following U50488 pretreatment (□) by a paired t-test (*p<0.05). (d) KOR activation significantly increased percent error during the DRL session. (e) A histogram of interresponse times showing the number of responses per 3 s bin. U50488 increased responses occurring within 0–3 s of the previous response. (f) KOR activation significantly increased the number of burst responses (an additional response <1 s after the previous response). (g) Repeated forced-swim stress increased percent error. Pretreatment with nor-BNI (KOR antagonist), but not saline, blocked increases in percent error following a second exposure to repeated forced-swim stress compared with baseline. Error bars indicate SEM. *P<0.05; ***p<0.0001; NS, not significant.

Figure 2

In female C57BL/6 mice, systemic KOR activation disrupted DRL performance. (a) To illustrate a typical data set for female mice, a raster display of responses from a representative animal are shown during 60 min DRL sessions after saline and then U50488 pretreatment. Closed circles represent reinforced responses and ‘+’ symbols represent nonreinforced responses. (b) KOR activation did not affect reinforced responses, but increased nonreinforced and total responses during the DRL session. U50488 significantly increased (c) percent error, (d) the number of responses occurring within 0–3 s and 3–6 s of the previous response, and (e) the number of burst responses (<1 s of the previous response). Error bars indicate SEM. Data were analyzed with paired t-tests; *P<0.05; **p<0.01; ***p<0.0001; NS, not significant.

Figure 3

Systemic KOR activation increased marble burying. U50488 pretreatment did not affect the total number of responses during an (a) FR1 or (b) FR5 session compared with saline pretreatment. (c) There was a significant increase in marble burying following U50488 treatment. Error bars indicate SEM. *P<0.05.

Figure 4

KORs in the VTA and in dopamine neurons are required for KOR-mediated DRL disruptions. (a) Schematic shows microinjection sites for nor-BNI, a long-lasting KOR antagonist. Mice received nor-BNI pretreatment 5 days before DRL test sessions. (b) There was a significant increase in the number of nonreinforced responses in Control and DRN/nor-BNI, but not VTA nor-BNI or PFC/nor-BNI, following U50488 treatment. (c) U50488 caused a significant increase in percent error in Control, DRN/nor-BNI, and PFC/nor-BNI mice, but not VTA/nor-BNI-injected mice. (d) There was a significant effect of U50488 treatment on the number of burst responses, but no significant interaction between treatment and brain region. (e) Schematic shows breeding strategy for generating conditional knockout mice. Dashed boxes indicate parental mice. Parental mice were heterozygous for Cre-recombinase within serotonergic (ePet1) or dopaminergic (DAT) neurons and were heterozygous for a null KOR allele. Cre-recombinase-negative mice with a floxed KOR gene were bred with heterozygous Cre-recombinase mice to produce conditional knockout mice with KOR specifically deleted from serotonergic or dopaminergic neurons or littermate controls. (f) U50488 treatment significantly increased the number of nonreinforced responses but there was no significant interaction between treatment and genotype. KOR activation significantly increased percent error (g) and burst responses (h) in Control and KOR CKO^PET mice, but not KOR CKO^DAT or KOR KO mice. Error bars indicate SEM. *P<0.05; **p<0.01, ***p<0.0001.

Figure 5

KOR-mediated DRL disruptions are arrestin independent. (a) Conditional deletion of the p38α MAPK in dopamine neurons does not prevent KOR-mediated increases in percent error in the DRL task. (b) Global deletion of the GRK3 does not prevent KOR-mediated increases in percent error in the DRL task. (c) The G-biased KOR agonist nalfurafine significantly increases percent error in the DRL task, indicating that DRL disruptions are arrestin independent. Error bars indicate SEM. *P<0.05, ***p<0.0001.