Endogenous kappa opioid activation mediates stress-induced deficits in learning and memory

Abstract

We hypothesized that mice subjected to prolonged stress would demonstrate decreased performance in a learning and memory task attributable to the endogenous activation of the kappa opioid receptor (KOR). C57BL/6J mice were tested using the novel object recognition (NOR) assay at various time points after exposure to repeated forced swim stress (FSS). Unstressed mice demonstrated recognition of the novel object at the end of a procedure using three 10-min object interaction phases, with a recognition index (RI) for the novel object of 71.7+/-3.4%. However, 1 h after exposure to FSS, vehicle-pretreated mice displayed a significant deficit in performance (RI=58.2+/-4.1%) compared with unstressed animals. NOR was still significantly reduced 4 but not 24 h after FSS. Treatment with the KOR-selective antagonist norbinaltorphimine (10 mg/kg, i.p.) prevented the decline in learning and memory performance. Moreover, direct activation of the KOR induced performance deficits in NOR, as exogenous administration of the KOR agonist U50,488 [(+/-)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide] (0.3 mg/kg, i.p.) suppressed NOR (RI=56.0+/-3.9%). The effect of FSS on NOR performance was further examined in mice lacking the gene for the endogenous KOR agonist dynorphin (Dyn). Dyn gene-disrupted mice exposed to FSS did not show the subsequent learning and memory deficits (RI=66.8+/-3.8%) demonstrated by their wild-type littermates (RI=49.7+/-2.9%). Overall, these results suggest that stress-induced activation of the KOR may be both necessary and sufficient to produce subsequent deficits in novel object recognition.

Figure 1.

Schematic of NOR assay and calculation of RI. Images represent testing cages as they were arranged in each phase of the assay. Objects A and B were identical in phases I and II, although object B was moved to a different position during phase II testing. In phase III, object B was replaced with a novel object. In all phases, the percentage of time spent attending to object B was calculated as a percentage recognition index by the formula shown. Unless otherwise noted, initial objects were dice and the novel object was a marble, and each phase lasted 10 min, followed by a 10 min ITI.

Figure 2.

Optimization of conditions for the novel object recognition assay. Data plotted as percentage recognition index ± SEM for each phase. a, Effect of phase duration. Unstressed, vehicle-treated C57BL/6J mice were used in the three phases of the NOR assay. Time in each phase was limited to 1 (black bars), 5 (gray bars), 10 (white bars), or 20 (striped bars) min. Mice demonstrated a significant increase in time spent attending to the novel object in phase III compared with the time spent attending to the equivalent object in phase I, but a peak phase III response was demonstrated with a phase duration of 10 min (n = 15–23 mice per bar; *p < 0.05, significantly different from phase I response, paired-samples t tests). b, Effect of intertrial interval. The duration of the interval between each 10 min trial was varied from 1 (black bar), 10 (white bar), or 20 (gray bar) min. Mice tested with a 10 or 20 min ITI demonstrated significant increases in recognition index for the novel object in phase III compared with matching phase I responses, with a peak response demonstrated in experiments using a 10 min ITI (n = 7–28 mice per bar; *p < 0.05, significantly different from phase I response, paired-samples t tests).

Figure 3.

κ opioid antagonist nor-BNI reduced the time spent immobile during forced swimming. C57BL/6J mice were exposed to repeated trials of forced swimming 1 h after daily pretreatment with vehicle (circles) or nor-BNI (10 mg/kg, i.p., squares). Mice pretreated with nor-BNI demonstrated significantly less time spent immobile throughout all trials compared with vehicle-pretreated littermates. (n = 21–28; *p < 0.05, significant difference from matching trial, vehicle-treated animals, repeated-measures ANOVA with between-groups comparisons using independent samples t tests).

Figure 4.

Novel object recognition performance was reduced up to 24 h after exposure to forced swim stress. Vehicle-pretreated, unstressed (white bars), or FSS-exposed (thatched bars) C57BL/6J mice were tested in the NOR assay 1 (white thatched bars), 4 (light gray thatched bars), or 24 (dark gray thatched bars) h after final exposure to forced swimming. Mice tested 1 or 4 h after exposure to stress showed significantly less recognition of the novel object in phase III compared with unstressed mice, whereas mice tested 24 h after the completion of forced swimming demonstrated a statistically similar response to the unstressed mice. Doubling the ITI did not improve the performance of mice tested 1 h after forced swimming (black bars), suggesting that stress-induced deficits were not the result of insufficient training (n = 20–24; *p < 0.05, significant difference from phase I response, paired-samples t tests; ^†p ≤ 0.05, significant difference from phase III response of unstressed mice, one-way ANOVA with REGWF post hoc testing).

Figure 5.

Forced swim stress affected learning and memory through a κ opioid receptor-dependent mechanism. a, Pretreatment with nor-BNI before forced swimming prevented stress-induced NOR deficits. C57BL/6J mice were pretreated (1 h daily, i.p.) with vehicle (white bars) or the KOR antagonist nor-BNI (10 mg/kg; gray bars) and then returned to home cages (open bars) or exposed to FSS (thatched bars). NOR performance was evaluated 1 h after completion of home cage rest or stress exposure. Unstressed, nor-BNI-pretreated mice (gray open bars) showed no difference in NOR performance from unstressed, vehicle-pretreated mice (open white bars). Both groups demonstrated significant increases in percentage recognition index in phase III compared with the time spent attending to the equivalent object in phase I, which were not produced by vehicle-pretreated, FSS-exposed animals (white thatched bars). However, pretreatment with nor-BNI before FSS prevented stress-induced deficits in phase III novel object recognition (gray thatched bars). b, Antagonism of KOR after exposure to forced swimming also prevented stress-induced deficits in NOR. Vehicle-pretreated mice exposed to FSS and treated immediately thereafter with nor-BNI (10 mg/kg, i.p.) demonstrated significant novel object recognition measured 1 h later (black bars) (n = 17–20; *p < 0.05, significant difference from phase I response, paired-samples t tests; ^†p = 0.05, significant difference from phase III response of unstressed mice, one-way ANOVA with REGWF post hoc testing).

Figure 6.

Direct agonist stimulation of the κ opioid receptor impaired novel object recognition. a, Characterization of dose-dependent deficits of locomotion produced by the κ agonist U50,488. C57BL/6J mice, habituated to the locomotor chambers, were administered vehicle or a single graded dose of U50,488, and the total distance traveled in 60 min was determined (n = 8–16 mice; *p < 0.05, significant difference from distance traveled after saline administration, one-way ANOVA with REGWF post hoc testing). b, U50,488 pretreatment suppressed novel object recognition. U50,488 (0.3 mg/kg; black bars), administered 15 min before object recognition testing, prevented the significant increases in percentage recognition index demonstrated by vehicle-pretreated mice (white bars) in phase III of testing (n = 17–20 mice; *p < 0.05, significant difference from phase I response, repeated-measures ANOVA with paired-samples t tests; ^†p < 0.05, significant difference from phase III response of unstressed mice, one-way ANOVA with REGWF post hoc testing).

Figure 7.

Forced swim stress-induced suppression of novel object recognition is prevented. Prodynorphin gene-disrupted animals (Dyn⁻⁻) and their wild-type littermates (Dyn⁺⁺) were injected with vehicle 1 h before FSS. One hour after stress, mice were tested in the NOR assay. Unstressed Dyn⁻⁻ mice (white bars) and their wild-type littermates (black bars) demonstrated a similar recognition of the novel object in all phases and a significant increase in time spent attending to the novel object in phase III compared with the time spent attending to the equivalent object in phase I. However, whereas wild-type mice showed a significant decrease in NOR in phase III after exposure to stress (black thatched bars), stress-exposed mice lacking the prodynorphin gene (white thatched bars) did not show deficits in learning and memory performance (n = 19–38 mice; *p < 0.05, significant difference from phase I response, paired-samples t tests; ^†p < 0.05, significant difference from phase III response of unstressed mice, REGWF post hoc testing).