Kappa opioid receptor antagonism and prodynorphin gene disruption block stress-induced behavioral responses

Abstract

Previous studies have demonstrated that stress may increase prodynorphin gene expression, and kappa opioid agonists suppress drug reward. Therefore, we tested the hypothesis that stress-induced release of endogenous dynorphin may mediate behavioral responses to stress and oppose the rewarding effects of cocaine. C57Bl/6 mice subjected to repeated forced swim testing (FST) using a modified Porsolt procedure at 30 degrees C showed a characteristic stress-induced immobility response and a stress-induced analgesia observed with a tail withdrawal latency assay. Pretreatment with the kappa opioid receptor antagonist nor-binaltorphimine (nor-BNI; 10 mg/kg, i.p.) blocked the stress-induced analgesia and significantly reduced the stress-induced immobility. The nor-BNI sensitivity of the behavioral responses suggests an activation of the kappa opioid receptor by a stress-induced release of dynorphin peptides. Supporting this hypothesis, transgenic mice possessing a disrupted prodynorphin gene showed no increase in immobility or stress-induced analgesia after exposure to repeated FST. Because both stress and the kappa opioid system can modulate the response to drugs of abuse, we tested the effects of forced swim stress on cocaine-conditioned place preference (CPP). FST-exposed mice conditioned with cocaine (15 mg/kg, s.c.) showed significant potentiation of place preference for the drug-paired chamber over the responses of unstressed mice. Surprisingly, nor-BNI pretreatment blocked stress-induced potentiation of cocaine CPP. Consistent with this result, mice lacking the prodynorphin gene did not show a stress-induced potentiation of cocaine CPP, whereas wild-type littermates did. The findings suggest that chronic swim stress may activate the kappa opioid system to produce analgesia, immobility, and potentiation of the acute rewarding properties of cocaine in C57Bl/6 mice.

Figure 1.

Forced swim stress-induced analgesia is blocked by pretreatment with nor-BNI or prodynorphin (Dyn) knock-out. Tail withdrawal latencies presented were obtained 5–9 min after the forced swimming on the first (A) or second (B) day. Mice were tested in the 55°C warm water tail withdrawal assay before (open bars) and after (hatched bars) exposure to the forced swim test, as described in Materials and Methods. On either day, C57Bl/6 mice pretreated with vehicle (0.3 ml/30 gm of body weight) demonstrated a tail withdrawal response that was increased nearly threefold after forced swim stress (A, B, left pair). Pretreatment 60 min before FST with the κ-selective antagonist nor-BNI (10 mg/kg, i.p.) did not significantly change baseline tail withdrawal latencies (A, left center pair) but blocked the increase in stress-induced analgesia produced by forced swimming (A, B, right center pair). Likewise, disruption of the prodynorphin gene prevented forced swim stress-induced analgesia (A, B, right pair). Wild-type littermates of the prodynorphin knock-out mice displayed SIA not significantly different from that of vehicle-treated C57Bl/6 mice (data in Results). *Significantly different from matching preswim latencies; p < 0.05, as determined by ANOVA followed by Student's t test. Bars represent n = 20–24 wild-type animals or 9 each of prodynorphin KO mice in swim test trials and 7 wild-type animals in tests of nor-BNI effect without FST exposure.

Figure 2.

Demonstration of nor-BNI selective antagonism of the κ opioid receptor. Mice were either untreated or pretreated once daily for 2 d with nor-BNI (10 mg/kg, i.p.). On the second day, 30 min after administration of the second dose of nor-BNI, mice were administered the opioid-selective agonists U50,488 (25 mg/kg, i.p., for the κ receptor), morphine sulfate (10 mg/kg, i.p., for the μ receptor), and SNC-80 (100 nmol, i.c.v., for the δ receptor). Preliminary dose–response curves with morphine and SNC-80 demonstrated that the doses used here produced submaximal analgesia comparable with the magnitude produced by U50,488. Animals were then tested in the 55°C warm water tail withdrawal assay 30 min after agonist administration, with the latency of the mouse to withdraw its tail from the water bath taken as the end point. As a control for the SNC-80 experiment, intracerebroventricular administration of vehicle did not produce a significant change in tail withdrawal latency (n = 4; data not shown). Pretreatment with this dose of nor-BNI blocked U50,488- but not morphine- or SNC-80-induced antinociception, suggesting that nor-BNI under these dosing conditions did not significantly occupy μ or δ opioid receptors. *Significantly different from baseline response; p < 0.05, as determined by Student's t test. Bars represent n = 4–10 mice.

Figure 3.

Immobility response to forced swim stress is reduced on the second day of testing by pretreatment with nor-BNI or by disruption of the prodynorphin gene. The time mice spent immobile during the last 4 min of the forced swim test was measured during multiple trials over 2 d. A, Mice received either vehicle (open bars) or nor-BNI (10 mg/kg, i.p., filled bars) in a bolus of 0.3 ml/30 gm of body weight 1 hr before daily swimming. Mice exposed to a single 15-min forced swim demonstrated no difference in immobility response on the first day, regardless of pretreatment (left-most bars). However, mice pretreated with nor-BNI spent significantly less time immobile in the FST on the second day during the four 6 min trials than vehicle-treated mice. Bars represent n = 20–24 animals. Note that mice did not demonstrate significant differences in body temperature 10 min after forced swimming on either day from unstressed mice, regardless of pretreatment. B, Wild-type, littermates (open bars) or prodynorphin gene knock-out mice (filled bars) were pretreated with vehicle in a bolus of 0.3 ml/30 gm of body weight 1 hr before daily swimming. Mice exposed to a single 15 min forced swim demonstrated no difference in immobility response on the first day, regardless of genotype. In contrast, on the second day, mice lacking dynorphin peptides through disruption of the prodynorphin gene spent significantly less time immobile in the last two 6 min trials of the FST than wild-type littermates. *Significant difference between immobility responses of stress-exposed vehicle-treated and nor-BNI treated mice (A) or between immobility responses of wild-type and prodynorphin gene-disrupted mice (B); p < 0.05, as determined by ANOVA followed by Student's t test. Bars represent n = 9 animals.

Figure 4.

Exposure to forced swim stress produces a nor-BNI-sensitive potentiation of cocaine-conditioned place preference.A, Schematic of training paradigm. The CPP protocol was as described in Materials and Methods. Preference testing allowed mice to move freely for 30 min in the morning to measure preconditioning and subsequent responses for either of two conditioning chambers, as described in Materials and Methods (represented here by triangles). After assessment of preconditioning preference, mice were exposed to repeated forced swim stress over the next 24 hr, as detailed in Materials and Methods (diamonds), or allowed to remain in home cages without swimming. Within 10 min after forced swim testing on day 2, mice were administered cocaine (15 mg/kg, s.c.) and confined to the drug-paired box for a 30 min conditioning session (squares). Four hours later, mice were administered vehicle and confined to the vehicle-paired box for a 30 min conditioning session (circles). Cocaine and saline conditioning was repeated the next day, separated again by 4 hr (represented by joined square and circle, day 3). On day 4, the final preference test was performed blind to determine the effect of treatment and conditioning on place preference. B, Preference test data demonstrating a nor-BNI-sensitive, FST-induced potentiation of cocaine CPP. Preferences are given as the difference between time spent in the drug-paired chamber and time in the saline-paired chamber during the 30 min trial. A positive value represents time spent in the drug-paired chamber. Mice were divided into three groups. The first group was unstressed, remaining in home cages and not exposed to swim stress before 2 d of cocaine and saline conditioning, as described in Materials and Methods (black bar). The second group was administered vehicle and exposed to the forced swim stressor before 2 d of cocaine and saline conditioning (light gray bar). The third group was administered nor-BNI, exposed to the forced swim stressor as described above, and then conditioned over 2 d with cocaine and saline (dark gray bar). After conditioning, all three groups demonstrated an increase in time spent in the cocaine-paired chamber that was significantly greater than the time spent in that chamber before conditioning, an example of conditioned place preference. Control unstressed mice and nor-BNI-treated, FST-exposed mice demonstrated an equivalent degree of cocaine CPP. In contrast, vehicle-treated mice exposed to FST demonstrated a significant potentiation over the unstressed animals responses. Note that mice did not demonstrate significant differences in body temperature from unstressed mice immediately after forced swimming, immediately before place conditioning, or 30 min after cocaine administration. *Significant difference in cocaine CPP compared with CPP for both unstressed and nor-BNI-treated mice; p<0.05, as determined by ANOVA followed by Student's t test. Bars represent n = 11–16 mice.

Figure 5.

Exposure to forced swim stress results in rapid, long-lasting nor-BNI-sensitive potentiation of cocaine conditioned place preference. A, Schematic of modified CPP paradigm. The CPP protocol was modified to allow one preference test and one conditioning session per day, alternating between cocaine (15 mg/kg, s.c.; squares) and saline (circles), such that the study extended over 6 d. Preference testing allowed mice to move freely for 30 min in the morning to measure preconditioning and subsequent responses for either of two conditioning chambers, as described in Materials and Methods (represented here by triangles). After assessing preconditioning preferences, some of the mice were exposed to repeated forced swim stress over the next 24 hr, as detailed in Materials and Methods (diamonds). On day 2, unstressed mice or FST-treated mice within 10 min after forced swim were administered cocaine (15 mg/kg, s.c.; squares), confined to the drug-paired box for a 30 min conditioning session, and then returned to the home cage overnight. Preference testing followed the next day (triangle, day 3), with animals then administered vehicle and confined to the vehicle-paired box for a 30 min conditioning session (circle). The monitoring of cocaine CPP acquisition continued on days 4 and 5 to ascertain a steady-state response, with one more cocaine-conditioning session (day 4) and vehicle-conditioning session (day 5) preceding the final preference test on day 6. B, Daily preference test data demonstrating acquisition of cocaine CPP and nor-BNI-sensitive potentiation by exposure to FST. Summarized results of daily preference tests (represented by triangles in A) are plotted in seconds to highlight time spent on the drug-paired side of the apparatus. Grouped mice on day 1 demonstrate a 94-sec preconditioning preference for the chamber that would subsequently be vehicle-paired. Mice were then divided into three groups. The first group was administered vehicle and exposed to the forced swim stressor (open circles); the second group was administered nor-BNI 60-min before FST (open squares); and the third group was returned to home cages and not exposed to swim stress (filled circles). All mice were subsequently used in cocaine CPP testing, with daily preference testing done blind to monitor the acquisition of cocaine CPP as detailed in A. Both control, unstressed mice and swim-stressed mice pretreated with nor-BNI demonstrated rapid acquisition of cocaine CPP on day 3, indicated by a time spent in the drug-paired chamber that was significantly (p < 0.05) greater after cocaine conditioning than before. Moreover, the preferences shown in subsequent tests with these animals were not significantly different from the day 3 response (filled circles). Vehicle-treated mice exposed to FST demonstrated the same rapid acquisition of cocaine CPP, also reaching a stable, peak response by day 5, but the response on each day was significantly potentiated twofold to fourfold over the unstressed animals responses. *Significant difference between cocaine CPP mice and preconditioning preference, as determined by ANOVA followed by Student's t test. Points represent means for six to eight animals.

Figure 6.

Control experiments. A, nor-BNI has no effect on cocaine CPP in the absence of stress Mice were pretreated twice over 2 d with vehicle or nor-BNI (10 mg/kg, i.p.) and either exposed to the forced swim stressor as detailed in Material and Methods or allowed to sit in their home cages before the development of cocaine-conditioned place preference as described in Figure 5. By the final day of preference testing, nor-BNI pretreatment of unstressed mice showed cocaine CPP that was not significantly different from the cocaine CPP of unstressed, untreated control mice. *Significant difference in matching cocaine CPP response of unstressed mice; p < 0.05 for all, as determined by ANOVA followed by Student's t test. B, Swim stress alone does not produce place preference in the absence of cocaine. Three sets of mice were pretreated with vehicle and either exposed to the forced swim stressor or left in their home cages. Conditioned place preference testing was performed as described in Figure 5A, but vehicle was substituted for cocaine in all conditioning sessions for the two sets of mice. Mice conditioned with saline on both sides of the apparatus did not show place preference significantly different from the animal's preconditioning responses. *Significant difference in time spent on the drug paired side compared with time spent on the drug paired side by saline-conditioned animals; p < 0.05 for all as determined by ANOVA followed by Student's t test. Bars represent n = 3–8 animals.

Figure 7.

Disruption of the prodynorphin gene prevents the forced swim stress-induced potentiation of cocaine-conditioned place preference. Prodynorphin gene knock-out or wild-type littermate mice were exposed to 2 d forced swim stress or left in their home cages and then used in cocaine-conditioned place preference assays as detailed in Figure 5A and Materials and Methods. All animals demonstrated cocaine CPP by day 6 that was significantly different from matching preconditioning responses displayed on day 1. However, FST-exposed wild-type littermates demonstrated cocaine CPP on day 6 that was double the preference responses of the unstressed wild-type littermates or swim-stressed prodynorphin knock-out mice. The horizontal dashed line designates for comparison the cocaine CPP response of unstressed C57Bl/6 mice obtained on the same testing day (see Fig. 6). *Significant difference in time spent on the drug paired side after cocaine conditioning compared with baseline response; ^ζsignificant difference in matching cocaine CPP response of FST-exposed prodynorphin wild-type versus unstressed wild-type or FST-exposed knock-out mice; p<0.05 for all as determined by ANOVA followed by Student's t test. Bars represent n = 4–10 animals.