Prodynorphin Knockout Mice Research Articles

Increased ethanol intake in prodynorphin knockout mice is associated to changes in opioid receptor function and dopamine transmission

The purpose of this study was to examine the role of the prodynorphin gene in alcohol sensitivity, preference and vulnerability to alcohol consumption. Handling-induced convulsion (HIC) associated to alcohol, alcohol-induced loss of righting reflex (LORR), hypothermic effects in response to acute ethanol challenge, blood ethanol levels (BELs), conditioned place preference, voluntary ethanol consumption and preference, tyrosine hydroxylase (TH), dopamine transporter (DAT) and proenkephalin (PENK) gene expression, and µ-, δ- and κ-opioid agonist-stimulated [S(35) ]- guanosine 5'-triphosphate-binding autoradiography were studied in prodynorphin knockout (PDYN KO) and wild-type (WT) mice. There were no differences in HIC, LORR or the decrease in body temperature in response to acute ethanol challenge between PDYN KO and WT mice. PDYN KO mice presented higher BEL, higher ethanol-conditioned place preference and more ethanol consumption and preference in a two-bottle choice paradigm than WT mice. These findings were associated with lower TH and higher DAT gene expression in the ventral tegmental area and substantia nigra, and with lower PENK gene expression in the caudate-putamen (CPu), accumbens core (AcbC) and accumbens shell (AcbSh) in PDYN KO. The functional activity of the µ-opioid receptor was lower in the CPu, AcbC, AcbSh and cingulate cortex (Cg) of PDYN KO mice. In contrast, δ- and κ-opioid receptor-binding autoradiographies were increased in the CPu and Cg (δ), and in the CPu, AcbC and Cg (κ) of PDYN KO. These results suggest that deletion of the PDYN gene increased vulnerability for ethanol consumption by altering, at least in part, PENK, TH and DAT gene expression, and µ-, δ- and κ-opioid receptor functional activity in brain areas closely related to ethanol reinforcement.

Kappa opioid receptor activation blocks progressive neurodegeneration after kainic acid injection

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.

Lack of a rewarding effect and a locomotor-enhancing effect of the selective μ-opioid receptor agonist amidino-TAPA

Psychological dependence is one of the worst side effects of morphine. It limits the clinical availability of morphine and non-patient morphine users suffer from addiction. An analgesic, which is more potent than morphine but without the liability of psychological dependence, has long been sought in the clinic. We have recently developed a new μ-opioid receptor agonist, N(α)-amidino-Tyr-D-Arg-Phe-β-Ala (amidino-TAPA), as a potent analgesic with an antinociceptive profile that is distinct from morphine, including the release of endogenous κ-opioid peptides. The activation of κ-opioid receptors has been suggested to suppress the development of psychological dependence by μ-opioid receptor agonists. In the present study, the psychological dependence liability and the related locomotor-enhancing effect of amidino-TAPA were evaluated. Amidino-TAPA injected subcutaneously produced an extremely potent and longer lasting antinociception than morphine in ddY mice, prodynorphin-knockout mice, and wild-type C57BL/6J mice. Unlike subcutaneously injected morphine, which had potent locomotor-enhancing and rewarding effects at antinociceptive doses in ddY mice, amidino-TAPA injected subcutaneously did not induce significant locomotor-enhancing and rewarding effects at antinociceptive or even higher doses in ddY mice. In wild-type C57BL/6J mice, amidino-TAPA showed the same pharmacological profile (potent antinociception, lack of locomotor-enhancing and rewarding effects) as in ddY mice. However, amidino-TAPA produced potent locomotor-enhancing and rewarding effects at antinociceptive doses in prodynorphin-knockout mice. The present results suggest that amidino-TAPA is a potent analgesic without the liability of psychological dependence because it releases endogenous κ-opioid peptides.

Prodynorphin gene deletion increased anxiety-like behaviours, impaired the anxiolytic effect of bromazepam and altered GABAA receptor subunits gene expression in the amygdala

This study evaluated the role of prodynorphin gene in the regulation of anxiety and associated molecular mechanisms. Emotional responses were assessed using the light-dark test, elevated plus maze and social interaction tests in prodynorphin knockout and wild-type mice. Corticotrophin releasing factor and proopiomelanocortin gene expressions in the hypothalamus were evaluated after restraint stress using in situ hybridization. The anxiolytic efficacy of bromazepam and GABA(A) receptor subunits gene expression in the amygdala were also assessed in both genotypes. The deletion of prodynorphin increased anxiety-like behaviours and proopiomelanocortin gene expression in the arcuate nucleus (two-fold). Moreover, the anxiolytic action of bromazepam was significantly attenuated in the mutant mice. Decreased GABA(A)γ(2) and increased GABA(A)β(2) gene expression receptor subunits were found in the amygdala of prodynorphin knockout mice. These results indicate that deletion of prodynorphin gene is associated with increased anxiety-like behaviours, enhanced sensibility response to stress stimuli, reduced anxiolytic efficacy of bromazepam and altered expression of the GABA(A) receptor subunits.

Direct Evidence for the Ongoing Brain Activation by Enhanced Dynorphinergic System in the Spinal Cord under Inflammatory Noxious Stimuli

Dynorphin A in the spinal cord is considered to contribute to nociceptive stimuli. However, it has not yet been determined whether activation of the spinal dynorphinergic system under nociceptive stimuli plays a role in direct acceleration of the ascending nociceptive pathway. In this study, the authors investigated the role of spinal dynorphinergic transmission in ongoing brain activation under noxious stimuli in mice. The changes in prodynorphin messenger RNA expression and dynorphin A (1-17)-like immunoreactivity in the mouse spinal cord were determined after the intraplantar injection of complete Freund's adjuvant in mice. The signal intensity in different brain regions after the intraplantar injection of complete Freund's adjuvant or intrathecal injection of dynorphin A (1-17) was measured by a pharmacological functional magnetic resonance imaging analysis. Complete Freund's adjuvant injection produced pain-associated behaviors and induced a dramatic increase in signal intensity in the mouse cingulate cortex, somatosensory cortex, insular cortex, and thalamic nuclei. These effects were not seen in prodynorphin knockout mice. Prodynorphin messenger RNA expression and dynorphin A (1-17)-like immunoreactivity on the ipsilateral side of the spinal cord were markedly increased in complete Freund's adjuvant-injected mice. Furthermore, intrathecal injection of dynorphin A (1-17) at relatively high doses caused pain-associated behaviors and a remarkable increase in the activities of the cingulate cortex, somatosensory cortex, insular cortex, and medial and lateral thalamic nuclei in mice. These findings indicate that spinally released dynorphin A (1-17) by noxious stimuli leads to the direct activation of ascending pain transmission.

Effects of Neonatal Stress and Morphine on Kappa Opioid Receptor Signaling

Background: Critically ill neonates experience multiple stressors during hospitalization. Opioids are commonly prescribed to ameliorate their pain and stress. However, the enduring effects of stress and opioids are not understood. The kappa opioid system is important in the mediation of stress in adults, but little is known about its function in neonates. Objectives: To characterize kappa opioid receptor (KOR) distribution in the neonatal mouse brain and test whether neonatal exposure to morphine, stress, or both, change KOR signaling. Methods: Five groups of wild-type C57BL/6 or prodynorphin (Pdyn) knockout mice were tested: (1) untreated control (dam-reared, no handling), (2) saline-injected control, (3) morphine-injected control, (4) stressed with saline injections and (5) stressed with morphine injections. Mice were treated from postnatal day 5 to postnatal day 9, after which their brains were immunolabeled with a phospho-specific KOR antibody (KOR-P), glial fibrillary acidic protein or glutamic acid decarboxylase. Results: There were no effects of saline or morphine injection on KOR-P immunoreactivity. Neonatal stress increased KOR-P labeling in wild-type brains (p < 0.05), but not in Pdyn^–/– animals. Mice exposed to stress and morphine showed region-specific increases in KOR-P immunoreactivity from 38 to 500% (p < 0.05 to p < 0.001), with marked gliosis. In stressed morphine-treated Pdyn^–/– animals, KOR-P immunoreactivity was absent, but gliosis increased compared to wild-type animals. Conclusions: Neonatal stress increases KOR activation via the dynorphin system. Neonatal stress plus morphine treatment further increased this response and also resulted in hippocampal gliosis. Enhanced gliosis noted in Pdyn^–/– animals suggests that the endogenous dynorphin may play a role in downregulating this inflammatory response.

Influence of estrous cycle on explorative behaviour of wild-type and prodynorphin knockout mice

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.

Behavioural characterization of prodynorphin knockout mice

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 {type:entrez-nucleotide,attrs:{text:U50488,term_id:1277101,term_text:U50488}}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.

Prodynorphin gene disruption increases the sensitivity to nicotine self-administration in mice

The endogenous opioid system has been reported to participate in nicotine behavioural responses. The aim of the study was to determine the contribution of the endogenous peptides derived from prodynorphin in acute and chronic nicotine responses, mainly those related to its addictive properties. Locomotion and nociception were evaluated after acute nicotine administration in prodynorphin knockout mice. In addition, nicotine rewarding properties were investigated in the place-conditioning and the intravenous self-administration paradigms. The somatic signs of nicotine withdrawal were also analysed after the injection of the nicotinic antagonist mecamylamine in nicotine-dependent mice. The hypolocomotor and antinociceptive effects induced by acute nicotine administration were not modified in knockout (KO) animals. Nicotine also produced similar conditioned place preference in both genotypes. However, a shift to the left in the percentage of acquisition of intravenous nicotine-self administration was observed in prodynorphin KO mice. Indeed, a significant increase in the number of KO mice acquiring this operant behaviour was revealed when low doses of nicotine were used. Nicotine physical dependence was similar in wild-type and KO animals. These findings reveal a specific role of endogenous peptides derived from prodynorphin in nicotine self-administration, probably through the modulation of its aversive effects.

Prodynorphin-Derived Peptides Are Critical Modulators of Anxiety and Regulate Neurochemistry and Corticosterone

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 kappa-opioid receptor antagonists GNTI or norbinaltorphimine showed the same effects. Furthermore, treatment of prodynorphin knockout animals with U-50488H, a selective kappa-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 kappa-opioid receptors. The delay in the behavioral response to kappa-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.

Anticonvulsant and neuroprotective actions of endogenous dynorphin

Anticonvulsant actions of dynorphin were proposed since more than 2 decades, however till now, we do not have information on the functions of endogenous dynorphin. Thus, we investigated prodynorphin knockout (KO) mice in different models of seizures and epilepsy. Seizure threshold was investigated by tail-vein infusion of 100 μg pentylenetetrazole (PTZ)/ml at a rate of 100 μl/min until mice displayed generalized clonic seizures. Wild-type mice showed clonic seizures at 39.2 ± 1.88 (mean ± SEM; n = 5) mg PTZ/kg body weight. KOs displayed a significantly reduced seizure threshold of 32.7 ± 1.17 (n = 6) mg PTZ/kg. This phenotype could be rescued entirely by the κ opioid receptor specific agonist U-50488, but not the μ opioid receptor specific agonist DAMGO. The δ opioid receptor specific agonist SNC80 decreased seizure threshold in both genotypes. Pre-treatment with the κ-selective antagonist GNTI completely blocked the rescue effect of U-50488. After injection of kainic acid into stratum radiatum of CA1 of the dorsal hippocampus, KO mice displayed increased neuronal loss along the rostro-caudal axis of the ipsiand partially the contralateral hippocampus at early time points after treatment. Thus, marked neurodegeneration was seen already 1 week after treatment in KO mice. The differences faded with time, being almost gone 5 weeks after kainate. Our data clearly indicate that endogenous prodynorphin derived peptides have anticonvulsant and neuroprotective properties. Acknowledgements This work was supported by the FWF (P20701-B05 and P18471-B05). from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Endogenous dynorphin in epileptogenesis and epilepsy: anticonvulsant net effect via kappa opioid receptors

Neuropsychiatric disorders are one of the main challenges of human medicine with epilepsy being one of the most common serious disorders of the brain. Increasing evidence suggest neuropeptides, particularly the opioids, play an important role in epilepsy. However, little is known about the mechanisms of the endogenous opioid system in epileptogenesis and epilepsy. Therefore, we investigated the role of endogenous prodynorphin-derived peptides in epileptogenesis, acute seizure behaviour and epilepsy in prodynorphin-deficient mice. Compared with wild-type littermates, prodynorphin knockout mice displayed a significantly reduced seizure threshold as assessed by tail-vein infusion of the GABA(A) antagonist pentylenetetrazole. This phenotype could be entirely rescued by the kappa receptor-specific agonist U-50488, but not by the mu receptor-specific agonist DAMGO. The delta-specific agonist SNC80 decreased seizure threshold in both genotypes, wild-type and knockout. Pre-treatment with the kappa selective antagonist GNTI completely blocked the rescue effect of U-50488. Consistent with the reduced seizure threshold, prodynorphin knockout mice showed faster seizure onset and a prolonged time of seizure activity after intracisternal injection of kainic acid. Three weeks after local injection of kainic acid into the stratum radiatum CA1 of the dorsal hippocampus, prodynorphin knockout mice displayed an increased extent of granule cell layer dispersion and neuronal loss along the rostrocaudal axis of the ipsi- and partially also of the contralateral hippocampus. In the classical pentylenetetrazole kindling model, dynorphin-deficient mice showed significantly faster kindling progression with six out of eight animals displaying clonic seizures, while none of the nine wild-types exceeded rating 3 (forelimb clonus). Taken together, our data strongly support a critical role for dynorphin in the regulation of hippocampal excitability, indicating an anticonvulsant role of kappa opioid receptors, thereby providing a potential target for antiepileptic drugs.

Involvement of endogenous opioid peptides in the antinociception induced by the novel dermorphin tetrapeptide analog amidino-TAPA

The antinociceptive effect of i.t. administered N α-amidino-Tyr- d-Arg-Phe-β-Ala (amidino-TAPA), an N-terminal tetrapeptide analog of dermorphin, was characterized in ddY mice. In the opioid receptor ligand-binding assays using mouse brain membranes, amidino-TAPA showed a very high affinity for μ-opioid receptors, a low affinity to δ-opioid receptors and no affinity for κ-opioid receptors. In the mouse tail-flick test, i.t. treatment with amidino-TAPA produced a potent antinociception. The antinociception induced by amidino-TAPA was significantly attenuated by i.t. pretreatment with the μ-opioid receptor antagonist β-funaltrexamine, the κ-opioid receptor antagonist nor-binaltorphimine and the δ-opioid receptor antagonist naltrindole. Moreover, the antinociception induced by amidino-TAPA was significantly attenuated by i.t. pretreatment with antisera against the endogenous κ-opioid peptides dynorphin A, dynorphin B and α-neo-endorphin; and the endogenous δ-opioid peptide [Leu 5]enkephalin. In mice lacking prodynorphin, the precursor of the endogenous κ-opioid peptides, the antinociceptive effect of amidino-TAPA was significantly attenuated compared to that in wild-type C57BL/6J mice. However, there was no difference in G-protein activation by amidino-TAPA in the spinal cord membranes from prodynorphin knockout mice and C57BL/6J mice. The present results suggest that the spinal antinociception induced by the μ-opioid receptor selective peptide amidino-TAPA is mediated in part by the release of endogenous opioid peptides in the spinal cord, which is caused by the direct stimulation of μ-opioid receptors.

Involvement of κ/Dynorphin System in WIN 55,212-2 Self-Administration in Mice

Self-administration procedures have not yet provided evidence that freely moving mice can reliably acquire and maintain an operant behavior to self-administer cannabinoid agonists. The aim of the present work was to establish a model of cannabinoid operant intravenous self-administration in freely moving mice given the relevance of this species for the use of genetically modified animals. In addition, the possible involvement of the kappa/dynorphin system in cannabinoid self-administration was evaluated by using pro-dynorphin knockout mice. Outbred CD1 wild-type mice as well as pro-dynorphin knockout and wild-type mice were trained to self-administer the cannabinoid receptor agonist WIN 55,212-2 under an FR1 schedule of reinforcement. Two cannabinoid training doses (6.25 and 12.5 microg/kg/infusion) were used in the acquisition studies in outbred mice. Animals acquired a reliable operant responding to self-administer WIN 55,212-2 (12.5 microg/kg/infusion), but required as many as 15 sessions to attain this behavior. Interestingly, when a previous injection of WIN 55,212-2 (0.1 mg/kg, i.p.) was administered in the home-cage 24 h before the first session, mice acquired operant responding for cannabinoid self-administration by the fourth session. When the kappa-opioid agonist antagonist nor-binaltorphimine (5 mg/kg s.c.) was administered 4 h before the first session, the time required to acquire a reliable cannabinoid self-administration was also significantly reduced. Finally, a shift to the left in the dose-intake curve to self-administer WIN 55,212-2 was observed in pro-dynorphin knockout mice when compared to wild-type mice. These results indicate that the activation of the kappa/dynorphin opioid system after WIN 55,212-2 administration could counteract cannabinoid rewarding effects.

Prodynorphin knockout mice demonstrate diminished age-associated impairment in spatial water maze performance

Dynorphins, endogenous κ-opioid agonists widely expressed in the central nervous system, have been reported to increase following diverse pathophysiological processes, including excitotoxicity, chronic inflammation, and traumatic injury. These peptides have been implicated in cognitive impairment, especially that associated with aging. To determine whether absence of dynorphin confers any beneficial effect on spatial learning and memory, knockout mice lacking the coding exons of the gene encoding its precursor prodynorphin ( Pdyn) were tested in a water maze task. Learning and memory assessment using a 3-day water maze protocol demonstrated that aged Pdyn knockout mice (13–17 months) perform comparatively better than similarly aged wild-type (WT) mice, based on acquisition and retention probe trial indices. There was no genotype effect on performance in the cued version of the swim task nor on average swim speed, suggesting the observed genotype effects are likely attributable to differences in cognitive rather than motor function. Young (3–6 months) mice performed significantly better than aged mice, but in young mice, no genotype difference was observed. To investigate the relationship between aging and brain dynorphin expression in mice, we examined dynorphin peptide levels at varying ages in hippocampus and frontal cortex of WT 129SvEv mice. Quantitative radioimmunoassay demonstrated that dynorphin A levels in frontal cortex, but not hippocampus, of 12- and 24-month mice were significantly elevated compared to 3-month mice. Although the underlying mechanisms have yet to be elucidated, the results suggest that chronic increases in endogenous dynorphin expression with age, especially in frontal cortex, may adversely affect learning and memory.

Pronociceptive role of dynorphins in uninjured animals: N-ethylmaleimide-induced nociceptive behavior mediated through inhibition of dynorphin degradation

Intrathecal (i.t.) administration into mice of N-ethylmaleimide (NEM), a cysteine protease inhibitor, produced a characteristic behavioral response, the biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank. The behavior induced by NEM was inhibited by the intraperitoneal injection of morphine. We have recently reported that dynorphin A and, more potently big dynorphin, consisting of dynorphins A and B, produce the same type of nociceptive response whereas dynorphin B does not [Tan-No K, Esashi A, Nakagawasai O, Niijima F, Tadano T, Sakurada C, Sakurada T, Bakalkin G, Terenius L, Kisara K. Intrathecally administered big dynorphin, a prodynorphin-derived peptide, produces nociceptive behavior through an N-methyl- d-aspartate receptor mechanism. Brain Res 2002;952:7–14]. The NEM-induced nociceptive behavior was inhibited by pretreatment with dynorphin A- or dynorphin B-antiserum and each antiserum also reduced the nociceptive effects of i.t.-injected synthetic big dynorphin. The characteristic NEM-evoked response was not observed in prodynorphin knockout mice. Naloxone, an opioid receptor antagonist, had no effects on the NEM-induced behavior. Ifenprodil, arcaine and agmatine, antagonists at the polyamine recognition site on the N-methyl- d-aspartate (NMDA) receptor ion-channel complex, and MK-801, an NMDA ion-channel blocker inhibited the NEM-induced effects. Ro25-6981, an antagonist of the NMDA receptor subtype containing NR2B subunit was not active. NEM completely inhibited degradation of dynorphin A by soluble and particulate fractions of mouse spinal cord. Collectively, the results demonstrate that endogenous prodynorphin-derived peptides are pronociceptive in uninjured animals, and required for the NEM-induced behavior. The NEM effects may be mediated through inhibition of the degradation of endogenous dynorphins, presumably big dynorphin that in turn activates the NMDA receptor ion-channel complex by acting on the polyamine recognition site.

Pronociceptive Actions of Dynorphin Maintain Chronic Neuropathic Pain

Whereas tissue injury increases spinal dynorphin expression, the functional relevance of this upregulation to persistent pain is unknown. Here, mice lacking the prodynorphin gene were studied for sensitivity to non-noxious and noxious stimuli, before and after induction of experimental neuropathic pain. Prodynorphin knock-out (KO) mice had normal responses to acute non-noxious stimuli and a mild increased sensitivity to some noxious stimuli. After spinal nerve ligation (SNL), both wild-type (WT) and KO mice demonstrated decreased thresholds to innocuous mechanical and to noxious thermal stimuli, indicating that dynorphin is not required for initiation of neuropathic pain. However, whereas neuropathic pain was sustained in WT mice, KO mice showed a return to baselines by post-SNL day 10. In WT mice, SNL upregulated lumbar dynorphin content on day 10, but not day 2, after injury. Intrathecal dynorphin antiserum reversed neuropathic pain in WT mice at post-SNL day 10 (when dynorphin was upregulated) but not on post-SNL day 2; intrathecal MK-801 reversed SNL-pain at both times. Opioid (mu, delta, and kappa) receptor density and G-protein activation were not different between WT and KO mice and were unchanged by SNL injury. The observations suggest (1) an early, dynorphin-independent phase of neuropathic pain and a later dynorphin-dependent stage, (2) that upregulated spinal dynorphin is pronociceptive and required for the maintenance of persistent neuropathic pain, and (3) that processes required for the initiation and the maintenance of the neuropathic pain state are distinct. Identification of mechanisms that maintain neuropathic pain appears important for strategies to treat neuropathic pain.