Brain Dynorphin Research Articles

The dynorphin/kappa opioid receptor mediates adverse immunological and behavioral outcomes induced by repetitive blast trauma

BackgroundAdverse pathophysiological and behavioral outcomes related to mild traumatic brain injury (mTBI), posttraumatic stress disorder (PTSD), and chronic pain are common following blast exposure and contribute to decreased quality of life, but underlying mechanisms and prophylactic/treatment options remain limited. The dynorphin/kappa opioid receptor (KOR) system helps regulate behavioral and inflammatory responses to stress and injury; however, it has yet to be investigated as a potential mechanism in either humans or animals exposed to blast. We hypothesized that blast-induced KOR activation mediates adverse outcomes related to inflammation and affective behavioral response.MethodsC57Bl/6 adult male mice were singly or repeatedly exposed to either sham (anesthesia only) or blast delivered by a pneumatic shock tube. The selective KOR antagonist norBNI or vehicle (saline) was administered 72 h prior to repetitive blast or sham exposure. Serum and brain were collected 10 min or 4 h post-exposure for dynorphin A-like immunoreactivity and cytokine measurements, respectively. At 1-month post-exposure, mice were tested in a series of behavioral assays related to adverse outcomes reported by humans with blast trauma.ResultsRepetitive but not single blast exposure resulted in increased brain dynorphin A-like immunoreactivity. norBNI pretreatment blocked or significantly reduced blast-induced increase in serum and brain cytokines, including IL-6, at 4 h post exposure and aversive/anxiety-like behavioral dysfunction at 1-month post-exposure.ConclusionsOur findings demonstrate a previously unreported role for the dynorphin/KOR system as a mediator of biochemical and behavioral dysfunction following repetitive blast exposure and highlight this system as a potential prophylactic/therapeutic treatment target.

CRF2 receptor-deficiency eliminates opiate withdrawal distress without impairing stress coping

The opiate withdrawal syndrome is a severe stressor that powerfully triggers addictive drug intake. However, no treatment yet exists that effectively relieves opiate withdrawal distress and spares stress-coping abilities. The corticotropin-releasing factor (CRF) system mediates the stress response, but its role in opiate withdrawal distress and bodily strategies aimed to cope with is unknown. CRF-like signaling is transmitted by two receptor pathways, termed CRF(1) and CRF(2). Here, we report that CRF(2) receptor-deficient (CRF(2)(-/-)) mice lack the dysphoria-like and the anhedonia-like states of opiate withdrawal. Moreover, in CRF(2)(-/-) mice opiate withdrawal does not increase the activity of brain dynorphin, CRF and periaqueductal gray circuitry, which are major substrates of opiate withdrawal distress. Nevertheless, CRF(2) receptor-deficiency does not impair brain, neuroendocrine and autonomic stress-coping responses to opiate withdrawal. The present findings point to the CRF(2) receptor pathway as a unique target to relieve opiate withdrawal distress without impairing stress-coping abilities.

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.

Effects of neonatal handling on nociceptin/orphanin FQ and opioid peptide levels in female rats

Animals exposed to short periods of handling during the critical period of development, i.e., the first 21 days of life in rats, show attenuated neuroendocrine responses to stress in adult life. We have previously reported long-term changes in brain dynorphin (DYN) peptide levels in male Sprague–Dawley rats after neonatal handling. The purpose of this study was to investigate whether neonatal handling, 15-min individual separation from the mother during postnatal days 1–21, can induce long-term changes in DYNB, Met-enkephalin Arg 6Phe 7 (MEAP) and nociceptin/orphanin FQ (N/OFQ) immunoreactive (ir) levels in female Sprague–Dawley rats. The peptides were measured in brain and pituitary gland 2 months after the handling procedure. The results reveal that handled (H) rats had increased ir levels of N/OFQ, DYNB and MEAP in the periaqueductal gray (PAG) as compared to nonhandled (NH) controls. Furthermore, H rats had decreased ir levels of DYNB in the frontal cortex and in the amygdala. In contrast to previous findings in male rats, DYNB levels were unaffected in areas related to the hypothalamo–pituitary–adrenal (HPA)-axis. The results indicate that a manipulation early in life can induce persistent neurochemical changes in the N/OFQ and opioid peptide system in female Sprague–Dawley rats.

Role of Brain Dynorphin in Nitrous Oxide Antinociception in Mice

Earlier studies indicate that nitrous oxide antinociception is mediated by opioid receptors, and we have hypothesized that nitrous oxide stimulates a neuronal release of an endogenous opioid peptide (EOP) that stimulates opioid receptors. To further test this hypothesis, male NIH Swiss mice were pretreated intracerebroventricularly with rabbit antisera to opioid peptides or with various inhibitors of peptidases involved in the degradation of EOPs. Mice were subsequently exposed to three different concentrations of nitrous oxide in oxygen, and their antinociceptive responsiveness was measured using the acetic acid abdominal constriction test. Nitrous oxide antinociception was significantly attenuated by 24-h pretreatment with antisera to various fragments of dynorphin (DYN) but not by antisera against methionine-enkephalin (ME) or β-endorphin (β-EP). In other experiments, nitrous oxide antinociception was significantly enhanced by 30-min pretreatment with phosphoramidon, an inhibitor of endopeptidase 24.11, which has been implicated in DYN degradation, but not bestatin or captopril, which inhibit aminopeptidase and angiotensin-converting enzyme, respectively. The latter enzymes have been implicated in degradation of certain EOPs albeit not DYN. These findings support the hypothesis that nitrous oxide antinociception in the mouse abdominal constriction test is mediated by endogenous DYN acting in the central nervous system.

Dynorphin A(1-8): stability and implications for in vitro opioid activity

The opioid binding profile and in vitro activity of the endogenous opioid peptide dynorphin A(1-8) have been studied. At opioid receptors in guinea-pig brain dynorphin A(1-8) was nonselective, although with some preference for the delta receptor (Ki 4.6 nM) over µ (Ki 18 nM) and kappa (Ki 40 nM) receptors. However, a high degree of metabolism was observed, with less than 10% of added dynorphin A(1-8) remaining at the end of the binding assay. In the presence of peptidase inhibitors to prevent breakdown of the N- and C-termini and the Gly3-Phe4 bond the major metabolite was [Leu5]enkephalin (representing 49% recovered material). This was reduced by inclusion of an inhibitor of endopeptidase EC 3.4.24.15. In the presence of all the peptidase inhibitors the affinity for kappa receptors (Ki 0.5 nM) relative to µ and delta receptors increased, but no selectivity of binding was observed. This lack of selectivity was confirmed using membranes from C6 glioma cells expressing rat opioid receptors. The agonist effect of dynorphin A(1-8) in the mouse vas deferens (EC50 116 nM) and guinea-pig ileum (EC50 38 nM) was mediated through the kappa receptor as evidenced by the rightward shifts afforded by the kappa -selective antagonist norbinaltorphimine. In the presence of peptidase inhibition potency was improved 2-fold in the mouse vas deferens and 20-fold in the guinea-pig ileum, but this agonist activity was mediated through delta receptors in the vas deferens and µ receptors in the ileum, as a result of the formation and stabilization of [Leu5]enkephalin. The results confirm the absence of receptor selectivity of dynorphin A(1-8) in binding assays but show that its agonist effects, at least in vitro, are mediated exclusively through the kappa opioid receptor.Key words: dynorphin A(1-8), opioid receptors, peptide metabolism, mouse vas deferens, guinea-pig ileum.

Brain dynorphin and enkephalin systems in Fischer and Lewis rats: effects of morphine tolerance and withdrawal

Lewis rats are more likely to self-administer various drugs of abuse than Fischer rats. Here these two strains of rats were compared with regard to basal brain opioid peptide levels and the response to chronic morphine treatment and to naloxone-precipitated withdrawal. Lewis rats had lower basal dynorphin peptides in the substantia nogra, striatum (not Leu-enkephalinArg 6) and VTA (not dynorphin B) and the pituitary gland. Leu-enkephalinArg 6 levels were also lower in these structures (with the exception of striatum which had higher levels) and in the nucleus accumbens. There were also strain differences in the response to chronic morphine treatment; in the nucleus accumbens, morphine treatment increased dynorphin A levels in Fischer rats only, in the ventral tegmental area effects were opposite with increased dynorphin levels in Fischer and decreased levels in Lewis rats, in the hippocampus dynorphin levels were markedly reduced in Lewis rats only. In Fischer rats, chronic morphine strongly affected peptide levels in the substantia nigra and striatum, whereas Lewis rats responded less in these areas. Leu-enkephalin, which derives from both prodynorphin and proenkephalin, and Met-enkephalin, which derives from proenkephalin, were effected by chronic morphine mainly in Fischer rats, increasing levels in most of the brain areas examined. The results in this study show (1) strain differences in basal levels of prodynorphin-derived opioid peptides, (2) the prodynorphin system to be differently influenced by morphine in Lewis rats than in Fischer rats and 3) the proenkephalin system to be influenced by chronic morphine in brain areas related to reward processes only in Fischer rats.

Dynorphin A content in the rat brain and spinal cord after a localized trauma to the spinal cord and its modification with p-chlorophenylalanine : An experimental study using radioimmunoassay technique

The distribution of dynorphin A in the spinal cord and brain of normal rats and of rats subjected to a focal injury of the spinal cord was examined in a rat model using a radioimmunoassay (RIA) technique. The validity of RIA was checked by high performance liquid chromatography (HPLC). Furthermore, the possibility that the peptide is somehow functionally related with endogenous 5-hydroxytryptamine (5-HT, serotonin), was also evaluated using a pharmacological approach. In normal animals, the peptide content was very similar in the spinal cord segments (T 9, T 10–11, and T 12) examined whereas, the dynorphin content of the whole brain was about two-fold higher compared with that in the spinal cord. A focal injury to the spinal cord in the right dorsal horn (about 1.5 mm deep, 2.5 mm long and 1.5 mm to the right of the midline) of the lower thoracic cord (T 10–11) in urethane anaesthetised animals significantly altered the peptide content in the whole brain as well as in the spinal cord. Thus, a decrease in the peptide level in whole brain, T 10–11 and in the T 12 segments of the spinal cord was observed 1 and 2 h after trauma. At 5 h, the peptide had accumulated markedly in the T 9 segment (about a two-fold increase) as compared with the controls. At this time, the peptide content had been restored in the T 10–11 and T 12segments. On the other hand, the whole brain dynorphin level continued to remain low (by 55%) as compared to the control group. Pretreatment with p-chlorophenylalanine (p-CPA, a 5-HT synthesis inhibitor) prevented the increase of the peptide in the T 9 segment of the cord seen after 5 h injury. The peptide level of the other two spinal cord samples and the whole brain was similar to that of untreated traumatised rats. Furthermore, p-CPA treatment alone (in untraumatised animals) significantly reduced the whole brain dynorphin content (45% decrease) without having any effect on the spinal cord peptide level. Our results, for the first time, provide biochemical evidence that (i) dynorphin actively participates in the tissue response to a spinal cord trauma, (ii) its concentration is widely altered in the spinal cord and brain after a focal trauma to the cord, and (iii) suggest that a functional interaction between serotonin and dynorphin exists in the central nervous system.

Central mu, delta, and kappa opioid binding sites, and brain and pituitary beta-endorphin and met-enkephalin in genetically obese (ob/ob) and lean mice

The equilibrium dissociation constants and maximal binding capacities of 3H-dihydromorphine (DHM), 3H-D-Ala 2-D-leu 3 -enkephalin (DADL), and 3H-dynorphin A(1–8) for their respective mu, delta, and kappa opiate binding sites were studied in brain membrane preparations from lean and genetically obese-hyperglycaemic (Aston ob/ob) mice. The concentration of kappa binding sites was 2.7 fold higher in obese compared with lean mouse brain ( 231 ±44.6 versus 83.3±10.3 fmoles 3 H-dynorphin/mg protein respectively. mean ± SEM ). The concentration of delta binsing sites in obese was 1.6 fold that in lean mouse brain ( 94.5±8.6 vesus 59.5±6.5 fmoles 3 H-DADL/mg protein ). In contrast, the concentration of brain mu receptors was 40% lower in obese compared with lean mice ( 20.8±2.19 and 34.8±3.1 fmoles 3 H-DHM/mg protein respectively ). Binding affinities of delta and kappa sites for their respective ligands were not significantly different inlean V. obese mice. However, for mu sites, lean mouse binding data showed two affinities, one was not significantly different from obese (0.35nM) the second was lower (1.18nM) for DHM. Increases of approximately 5 fold and 3 fold in the brain content of beta-endorphin and met-enkephalin respectively, and no differences in brain dynorphin levels, were demonstrated in obese mice compared with lean controls. In obese mice, pituitary beta-endorphin content was 9 fold higher, met-enkephalin 4 fold higher and dynorphin 12 fold higher than in lean mice. The striking differences in opioid binding-site characteristics and in endogenous opioid peptide levels in obese compared with lean mice may contribute to the hyperphagia and, directly or indirectly, to the development of hyperglycaemia and hyperinsulinaemia in obese mice.

Levels of immunoreactive dynorphin in brain and pituitary of hyperthyroid and hypothyroid rats

Dynorphin is a potent opioid peptide. Using a highly sensitive radioimmunoassay we have measured the levels of immunoreactive (ir) dynorphin in the cortex, hypothalamus, anterior and posterior pituitary in hyperthyroid and hypothyroid rats. Ir dynorphin levels were increased in the cortex of hypothyroid animals and decreased in the hypothalamus of hyperthyroid rats.

Levels of immunoreactive dynorphin in brain and pituitary of Brattleboro rats

The very potent opioid peptide, dynorphin, has recently been shown by radioimmunoassay and immunocytochemical techniques to be selectively concentrated in the pars nervosa of pituitary, suggesting a possible association of dynorphin with the neurohypophyseal hormones, vasopressin and oxytocin. In this study, we report that pituitary and brain contents of immunoreactive dynorphin in homozygote Brattleboro rats (unable to synthesize vasopressin) are similar to those in heterozygote animals and in normal Long-Evans rats. Thus, the syntheses of immunoreactive dynorphin and vasopressin in the hypothalamo-hypophyseal tract appear to be independently regulated.