Stress and anxiety are mainly regulated by amygdala and hypothalamic circuitries involving several neurotransmitter systems and providing physiological responses to peripheral organs via the hypothalamic-pituitary-adrenal axis and other pathways. The role of endogenous opioid peptides in this process is largely unknown. Here we show for the first time that anxiolytic parameters of explorative behavior in mice lacking prodynorphin were increased 2-4-fold in the open field, the elevated plus maze and the light-dark test. Consistent with this, treatment of wild-type mice with selective k-opioid receptor antagonists GNTI or norbinaltorphimine showed the same effects. Furthermore, treatment of prodynorphin knockout animals with U-50488H, a selective k-opioid receptor agonist, fully reversed their anxiolytic phenotype. These behavioral data are supported by an approximal 30% reduction in corticotropin-releasing hormone (CRH) mRNA expression in the hypothalamic paraventricular nucleus and central amygdala and an accompanying 30-40% decrease in corticosterone serum levels in prodynorphin knockout mice. Although stress-induced increases in corticosterone levels were attenuated in prodynorphin knockout mice, they were associated with minor increases in depression-like behavior in the tail suspension and forced swim tests. Taken together, our data suggest a pronounced impact of endogenous prodynorphin-derived peptides on anxiety, but not stress coping ability and that these effects are mediated via k-opioid receptors. The delay in the behavioral response to k-opioid receptor agonists and antagonist treatment suggests an indirect control level for the action of dynorphin, probably by modulating the expression of CRH or neuropeptide Y, and subsequently influencing behavior.

We recently demonstrated that endogenous prodynorphin-derived peptides mediate anticonvulsant, antiepileptogenic and neuroprotective effects via kappa opioid receptors (KOP). Here we show acute and delayed neurodegeneration and its pharmacology after local kainic acid injection in prodynorphin knockout and wild-type mice and neuroprotective effect(s) of KOP activation in wild-type mice. Prodynorphin knockout and wild-type mice were injected with kainic acid (3 nmoles in 50 nl saline) into the stratum radiatum of CA1 of the right dorsal hippocampus. Knockout mice displayed significantly more neurodegeneration of pyramidal cells and interneurons than wild-type mice 2 days after treatment. This phenotype could be mimicked in wild-type animals by treatment with the KOP antagonist GNTI and rescued in knockout animals by the KOP agonist U-50488. Minor differences in neurodegeneration remained 3 weeks after treatment, mostly because of higher progressive neurodegeneration in wild-type mice compared with prodynorphin-deficient animals. In wild-type mice progressive neurodegeneration, but not acute neuronal loss, could be mostly blocked by U-50488 treatment. Our data suggest that endogenous prodynorphin-derived peptides sufficiently activate KOP receptors during acute seizures, and importantly in situations of reduced dynorphinergic signaling-like in epilepsy-the exogenous activation of KOP receptors might also have strong neuroprotective effects during excitotoxic events.

Dynorphin, with a high selectivity for kappa opioid receptors, is a member of the opioid peptide family. Application of opioid agonists and antagonists revealed crucial effects of the opioid system in numerous physiological functions. However, data obtained are often contradictive, reflecting the complex situation of the cross-activation of 3 receptors through a peptide derived from 3 different precursors. Therefore we investigated prodynorphin knockout mice (DynKO) to establish the effect of prodynorphin deficiency on explorative behaviour in mice. DynKO exhibited higher ambulation in the open field test. Thus, center distance, center time and number of center entries were increased about 2-fold, and number of center rearings about 3-fold. DynKO mice showed also more visits (~2-fold) and more time (~3-fold) spent on open arms of the elevated plus maze test. Significantly higher numbers of entries, distance and time spent in open lit area (ca. 30% higher values) in the light-dark test were observed in DynKO as compared to wild type mice (WT). In contrast to increased explorative behaviour of DynKO under aversive conditions, no differences in motor activity or circadian rhythms were observed in the home cage. The anxiolytic phenotype of DynKO could be mimicked by injection of the selective kappa antagonists norBNI (10 mg/kg, i.p.) or GNTI (3 nmol, i.c.) in WT. Applying the specific kappa agonist U50488 H (2.5 mg/ kg, i.p.) entirely reversed the anxiolytic phenotype of DynKO. Taken together our data clearly show an anxiolytic phenotype of male DynKO mice. These data are in line with reduced CRH expression in the PVN and attenuated corticosteron serum levels.

No abstract

Since several years dynorphin, a member of the opioid peptide family, was suggested to play a regulatory role in numerous functional pathways of the brain. In line with its localization in hippocampus, amygdala, hypothalamus and striatum these functions resemble learning and memory, emotional control and stress response. Male prodynorphin-deficient (Dyn KO) mice display an anxiolytic phenotype [see abstract A5]. However, emotional control and stress response depend on hormonal state and differ between sexes. In female rodents mood is influenced also on the estrous state. We now analysed the explorative behaviour in correlation to the estrous cycle in female wild-type (WT) and prodynorphin knockout mice. In the open field test WT mice showed significantly more anxiety during the estrous. This effect is abolished in Dyn KO mice. No differences were seen throughout the other stages. In the elevated plus maze test Dyn KO mice showed a significant anxiolytic phenotype regarding time spent, distance traveled and entries in the open arm at all estrous stages compared to WT mice. In addition, WT mice showed a significant decrease in all parameters during proestrous and estrous. This increased anxiety is attenuated by prodynorphin deficiency. In the light-dark test WT mice showed a decrease in time spent and distance traveled in the lit area during proestrous. In contrast, KO mice behaved relatively stable throughout the stages. Our data indicate an anxiolytic phenotype also in female Dyn KO mice. However, the phenotype is less prominent than in male mice and appears to be dependent on estrous stage and paradigm. It is interesting to note that the influence of the estrous stage appears to be abolished by the prodynorphin deficiency. The functional and pharmacological background will be investigated in further experiments.