Stress-induced p38 mitogen-activated protein kinase activation mediates kappa-opioid-dependent dysphoria

Abstract

The molecular mechanisms mediating stress-induced dysphoria in humans and conditioned place aversion in rodents are unknown. Here, we show that repeated swim stress caused activation of both kappa-opioid receptor (KOR) and p38 mitogen-activated protein kinase (MAPK) coexpressed in GABAergic neurons in the nucleus accumbens, cortex, and hippocampus. Sites of activation were visualized using phosphoselective antibodies against activated kappa receptors (KOR-P) and against phospho-p38 MAPK. Surprisingly, the increase in P-p38-IR caused by swim-stress exposure was completely KOR dependent; P-p38-IR did not increase in KOR(-/-) knock-out mice subjected to the same swim-paradigm or in wild-type mice pretreated with the KOR antagonist norbinaltorphimine. To understand the relationship between p38 activation and the behavioral effects after KOR activation, we administered the p38 inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (i.c.v.)] and found that it selectively blocked the conditioned place aversion caused by the kappa agonist trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide (U50488) and the KOR-dependent swim stress-induced immobility while not affecting kappa-opioid analgesia or nonselectively affecting associative learning. We found that the mechanism linking KOR and p38 activation in vivo was consistent with our previous in vitro data suggesting that beta-arrestin recruitment is required; mice lacking G-protein-coupled receptor kinase 3 also failed to increase p-p38-IR after KOR activation in vivo, failed to show swim stress-induced immobility, or develop conditioned place aversion to U50488. Our results indicate that activation of p38 MAPK signaling by the endogenous dynorphin-kappa-opioid system likely constitutes a key component of the molecular mechanisms mediating the aversive properties of stress.

Figure 1.

Repeated swim-stress results in KOR-dependent P-p38 staining on GABAergic cells in the nucleus accumbens. A, Representative images of phospho-p38 (P-p38; green) in NAc after repeated swim-stress exposure, in stressed or nonstressed KOR wild-type (KOR +/+) and stressed knock-out mice (KOR −/−). B, Representative images of phospho-p38 (P-p38; green) and phospho-KOR (KORp; red) colabeling in NAc after repeated swim-stress exposure in stressed KOR (+/+) mice. C, Top row, Representative images of NAc phospho-KOR (KORp; green) and GAD (red) staining after repeated swim-stress exposure. Middle row, Higher magnification of phospho-p38 (green) costaining with the GABA marker (GAD; red) in NAc after repeated swim-stress exposure, in wild-type mice. Bottom row, Show representative high-magnification image of colabeled phospho-p38 (green) staining, GAD (red), and nuclear stain, DAPI (blue). All images are representative of three to four independent experiments.

Figure 2.

Repeated swim-stress exposure induces KOR-dependent p38 phosphorylation in the mouse striatum. A, Representative images of dorsal striatum, caudate-putamen tissue sections from control (no stress; left panels), and repeated swim-stress exposed mice (swim-stress; right panels); white boxed area outlines higher-magnification image below. B, Representative Western blots of phospho-p38 (P-p38) in wild-type mouse striatum after single (Single Swim) or multiple swim-stress (WT-Repeated Swim) exposure (C, no-swim saline; wt, swim saline; norBNI, swim plus pretreatment with 10 mg/kg norBNI). For WT-repeated swim, blots show no swim (C) compared with 10 min postswim (10) and 30 min postswim (30). KOR (−/−) repeated swim are blots taken from KOR gene deleted mice under the same conditions as their wild-type littermates. Below each P-p38 image is the protein loading control showing similar levels of β-actin. C, The mean data (±SEM) taken from tissue sections where P-p38 staining was quantified as the total positive p38 cells/200 μm. Data show that norBNI pretreatment (10 mg/kg; 1 h) before stress exposure blocks P-p38 staining in both the dorsal and ventral striatal (n = 4; p < 0.01, t test). D, Data are the means ± SEM of the quantified band intensities taken from multiple swim stress-induced phospho-p38 activity in WT mice, mice pretreated with norBNI (10 mg/kg), or mice lacking KOR (−/−) compared with the saline-treated no swim-stress comparable group (basal; dashed line). *p < 0.05, for WT multiple swim stress versus saline control, t test. n = 3–7, where each n is a separate animal.

Figure 3.

Inhibition of p38 MAPK attenuates swim stress-induced immobility. A, Time spent immobile during the last 5 min of the forced swim stress measured during multiple trials over 2 d after intracerebroventricular injection (30 min before swim) of saline or 0.5 nmol/mouse of SB203580. Mice were injected a second and third time with 0.5 nmol/mouse between swims (SW) 1 and 2 and 2 and 3. As expected, SB203580 had no significant effect on time spent immobile on the first day of testing (swim 1; black bar). However, on the second day of forced swim, inhibition of p38 MAPK with SB203580 caused a significant decrease in time spent immobile on the second and third exposures (swim 3 and swim 4; n = 19–21, with each n taken as a separate animal; *p < 0.05, as determined by ANOVA followed by the Bonferroni's post hoc test). B, To confirm lowered P-p38 activity in SB203580-treated mice, we measure the SB203580 effect on P-p38 in mouse striatum as determined by Western blot analysis. Top, Representative Western blot of P-p38 after swim stress and SB203580 treatment. Lanes are as follows: no swim plus saline group (NS + SA), swim plus saline (SW + SA), and swim plus SB0203580 (0.5 nmol/mouse). Each lane was taken from one striatum of one animal. β-Actin staining was used to confirm equal protein loading. Bottom, Bar graph data are the mean ± SEM of P-p38 compared with control (No Swim + Saline group) for each of the treatments designated below the graph. The dashed line denotes basal (control; n = 4–8, with each n taken as a separate animal; *p < 0.05, compared with control, saline-injected mice; t test). C, Data are the means ± SEM of mouse locomotor activity (distance traveled in centimeters vs time in minutes) before and after intracerebroventricular injection of 0.5 nmol of SB203580 or saline. Mice were injected 1 h after initial exposure to locomotor chamber as indicated.

Figure 4.

U50488-mediated CPA, but not cocaine CPP, is mediated by p38 MAPK (A). Timeline for CPA studies: mice were pretested on day 1, trained with drug [U50488 (2.5 mg/kg, i.p.), saline (i.p.), or cocaine (15 mg/kg)] on days 2 and 3, and posttested for preference or aversion on day 4 (see Materials and Methods). B, Mean CPA data: U50488 (2.5 mg/kg, i.p.) caused a significant CPA as measured by time spent posttest minus pretest in the drug-paired side. U50488-mediated CPA was significantly blocked by injection of p38 inhibitor SB203580 (SB) (0.5 nmol, i.c.v., 30 min before U50488, i.p.) Saline training (i.p.) did not produce CPA in either SB203580 (i.c.v.)-injected group or saline (i.c.v.)-injected groups. Cocaine CPP was unaffected by p38 inhibition; there was no significant difference between saline (i.c.v.)-injected and SB203580-injected place preference scores in cocaine-trained mice. *p < 0.05, t test (n = 6–9). C, Data are the means ± SEM of mouse conditioned taste aversion scores (Saccharin preference score = amount of saccharin consumed/total consumption) after intracerebroventricular injection of 0.5 nmol of SB203580 or saline (see Materials and Methods). There was no difference between SB203580 and saline-treated mice taste aversion scores. n = 6–7, where each n is a separate animal.

Figure 5.

GRK3 is required for KOR-mediated p38 activation, KOR-mediated CPA, and KOR-mediated swim stress-induced immobility. A, Top, Representative Western blots of P-p38 after administration of U50488 (U50) (20 mg/kg, i.p.) (duplicate lanes) or saline (S) in GRK3 wild-type (+/+) and GRK3(−/−) mice showing that U50488 increased P-p38 in a GRK3-dependent manner. Bottom, Protein-loading control Western blot for β-actin. B, Top, Representative Western blots of pERK1/2 after administration of U50488 (20 mg/kg) or saline in GRK3(−/−) (in duplicate lanes) mice showing that U50488 caused a robust increase in pERK1/2-IR in GRK3(−/−) mice. C, Mean P-p38 and pERK1/2 data taken from Western blotting experiments comparing the effect of GRK3(−/−) on U50488-induced MAPK activity. Data show that the U50488-induced increase in pERK was insensitive to GRK3 deletion but that U50-induced P-p38 activity was not evident in GRK3(−/−) mice. *p < 0.05, t test; n = 6–8. D, Data are the means ± SEM forced swim stress-induced immobility data taken from wild-type (GRK3+/+) and GRK3(−/−) mice. Data show that over swim trials (SW), wild-type mice show a characteristic increase in swim stress-induced immobility; however, GRK3(−/−) mice show a significantly reduced immobility response. *p < 0.05, **p < 0.01, one-way ANOVA followed by Bonferroni's post hoc test; n = 9–10.

Figure 6.

Repeated swim stress-induced upregulation of the transcription factor zif268 is KOR and p38 MAPK dependent. A, Representative Western blots of zif268 and pCREB in mouse striatum of WT and KOR(−/−) from no swim (NS) and swim (SW) groups. Total CREB (T-CREB) and actin are shown below to confirm equal protein loading. Data are representative of three to four independent experiments. B, Representative image of zif268-IR staining (red; nuclear staining) in the CPu in control (nonstressed) versus stressed wild-type (KOR +/+). C, Data are means ± SEM of the zif268 and pCREB levels obtained from Western blotting experiments 20–30 min after repeated swim-stress exposure compared with the no-swim control group (dashed line). *p < 0.05 versus control no-swim levels, t test; n = 3–4, where each n was taken from an individual experiment and animal. D, p38 inhibition by SB203580 attenuated the swim stress-induced upregulation of zif268 but not pCREB. Data are the mean ± SEM of zif268 and pCREB levels obtained from Western blots after repeated swim stress in mice preinjected with either 0.5 nmol of SB203580 (SB; i.c.v.) or saline (SAL). *p < 0.05 versus SB203580 group t test; n = 5–6, where each n was from an individual animal.