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KNDy neurons co-expressing kisspeptin (KP), neurokinin B (NKB) and dynorphin A (DYN A) in the arcuate nucleus of the hypothalamus (ARC) are key regulators of reproduction. Their activity is influenced by metabolic and hormonal signals. Previously, we have shown that orchidectomy alters the KP-, NKB-, and DYN A-immunoreactivity in the high-fat diet-induced (HFD) obesity and diabetes type 2 (DM2) models. Considering the potential sex difference in the response of KNDy neurons, we have hypothesized that ovariectomy (OVX) and post-ovariectomy replacement with estradiol (OVX+E2) or estradiol and progesterone (OVX+E2+P4) will also affect these neurons in HFD and DM2 females. Thus, each of these treatment protocols were employed for control, HFD, and DM2 groups of rats leading to nine experimental conditions within which we have determined the number of KP-, NKB-, or DYN-immunoreactive (-ir) neurons and assessed the metabolic and hormonal profiles of the animals. Accordingly: (1) no effects of group and surgery were observed on the number of KP-ir neurons; (2) the overall number of NKB-ir neurons was higher in the OVX+E2+P4 and OVX+E2 animals compared to OVX; (3) overall, the number of DYN A-ir neurons was higher in DM2 vs. control group, and surgery had an effect on the number of DYN A-ir neurons; (4) the metabolic and hormonal profiles were altered in HFD and DM2 animals compared to controls. Current data together with our previously published results indicate sex-specific differences in the response of KNDy neurons to DM2.

The opioid peptides are involved in the regulation of neuroendocrine functions in vertebrates. Nonetheless, the influence of an opioid peptide, dynorphin A (DYN), on reproduction in fish is understudied. The aim of this work was to study the influence of DYN on the pituitary-ovary axis in Oreochromis mossambicus. Daily injections (ip) of 250 μg DYN kg-1 body weight for 22 days during the ovarian cycle caused a reduction in the intensity and the per cent area of luteinizing hormone (LH) immunoreactive content in the proximal pars distalis region of the pituitary gland compared with an intense immunostaining in time-matched controls. In the ovary, DYN treatment caused a decrease in the number of stage I (previtellogenic) follicles compared with time-matched controls. No difference was observed in the number of stage IV (vitellogenic) follicles among different experimental groups, whereas the numbers of stage II and stage III follicles (previtellogenic) were higher in DYN-treated fish than in time-matched controls. Nonetheless, there was a reduction in the number of stage V (preovulatory) follicles in DYN-treated fish compared with time-matched controls. Taken together, these results indicate that DYN exerts an inhibitory effect on follicular recruitment at the late vitellogenic stage, through the suppression of LH secretion in fish.

Increasing evidence accumulated during the past two decades has demonstrated that the then-novel kisspeptin, which was discovered in 2001, the known neuropeptides neurokinin B and dynorphin A, which were discovered in 1983 and 1979, respectively, and their G-protein-coupled receptors, serve as key molecules that control reproduction in mammals. The present review provides a brief historical background and a summary of our recent understanding of the roles of hypothalamic neurons expressing kisspeptin, neurokinin B, and dynorphin A, referred to as KNDy neurons, in the central mechanism underlying gonadotropin-releasing hormone (GnRH) pulse generation and subsequent tonic gonadotropin release that controls mammalian reproduction.

Major depression disorder is a severe and recurrent neuropsychological disorder characterized by lowered mood and social activity and cognitive impairment. Owing to unclear molecular mechanisms of depression, limited interventions are available in clinic. In this study we investigated the role of dynorphin/κ opioid receptor system in the development of depression. Mice were subjected to chronic social defeat stress for 14 days. Chronic social defeat stress induced significant social avoidance in mice characterized by decreased time duration in the interaction zone and increased time duration in the corner zone. Pre-administration of a κ opioid receptor antagonist norBNI (10 mg/kg, i.p.) could prevent the development of social avoidance induced by chronic social defeat stress. Social avoidance was not observed in κ opioid receptor knockout mice subjected to chronic social defeat stress. We further revealed that social defeat stress activated c-fos and ERK signaling in the amygdala without affecting the NAc, hippocampus and hypothalamus, and ERK activation was blocked by systemic injection of norBNI. Finally, the expression of dynorphin A, the endogenous ligand of κ opioid receptor, was significantly increased in the amygdala following social defeat stress; microinjection of norBNI into the amygdala prevented the development of depressive-like behaviors caused by social defeat stress. The present study demonstrates that upregulated dynorphin/κ opioid receptor system in the amygdala leads to the emergence of depression following chronic social defeat stress, and sheds light on κ opioid receptor antagonists as potential therapeutic agents for the prevention and treatment of depression following chronic stress.

The contents of Dynorphin A(1-8) decreased gradually in ischemic cortices in rats and an intracerebroventricular administration of synthetic Dynorphin A(1-8) reduced the volume of cerebral infarction in our previous research. However, the specific protective mechanism is unclear and Dynorphin A(1-8) is unlikely to cross the blood-brain barrier (BBB) by noninvasive oral or intravenous administration as a macromolecule neuropeptide. In this study, intranasal administration was used to middle cerebral artery occlusion(MCAO) rats to assessed the therapeutic effects of Dynorphin A(1-8) by evaluating behavior, volume of cerebral infarct, cerebral edema ratio, histological observation. Then apoptosis neuron rate was detected by TUNEL staining. Immunohistochemical staining was carried out to explore the alteration of Bcl-2, Bax and Caspase-3. Finally, κ-opioid receptor antagonist and N-methyl-d-aspartate(NMDA) receptor antagonist were used to explore its possible mechanism. We found that MCAO rats under intranasal administration of Dynorphin A(1-8) showed better behavioral improvement, higher extent of Bcl-2, activity of SOD along with much lower level of infarction volume, brain water content, number of cell apoptosis, extent of Bax and Caspase-3, and concentration of MDA compared with those in MCAO model group and intravenous Dynorphin A(1-8) group. Administration of nor-BNI or MK-801 reversed these neuroprotective effects of intranasal Dynorphin A(1-8). In summary, Dynorphin A(1-8), with advantages of intranasal administration, could be effectively delivered to central nervous system(CNS). Dynorphin A(1-8) inhibited oxidative stress and apoptosis against cerebral ischemia/reperfusion injury, affording neuroprotection through NMDA receptor and κ-opioid receptor channels.

Pulsatile gonadotropin-releasing hormone (GnRH) secretion is indispensable for reproduction in mammals. Kisspeptin neurons in the hypothalamic arcuate nucleus (ARC), referred to as KNDy neurons because of the coexpression of neurokinin B and dynorphin A, are considered as components of the GnRH pulse generator that produces rhythmic GnRH secretion. The present study aimed to investigate if peripheral administration of PF-4455242, a κ-opioid receptor (KOR, a dynorphin A receptor) antagonist, facilitates pulsatile luteinizing hormone (LH) secretion and GnRH pulse generator activity in estrogen-treated ovariectomized Shiba goats to determine the possibility of using KOR antagonists to artificially control ovarian activities. PF-4455242 was intravenously infused for 4 h (1 or 10 μmol/kg body weight/4 h) or as a single subcutaneous injection (1 or 10 μmol/kg body weight). In a separate experiment, the same KOR antagonist (10 μmol/kg body weight/4 h) was intravenously infused during the recording of multiple unit activity (MUA) in the ARC that reflects the activity of the GnRH pulse generator to test the effects of KOR antagonist administration on GnRH pulse generator activity. Intravenous infusion and single subcutaneous injection of the KOR antagonist significantly increased the frequency of LH pulses compared with controls. Intravenous infusion of KOR antagonist also significantly increased the frequency of episodic bursts in the MUA. The present study demonstrates that peripherally administered KOR antagonist stimulates pulsatile LH secretion by acting on the GnRH pulse generator, and peripheral administration of PF-4455242 can be used to facilitate pulsatile LH secretion, which in turn facilitates ovarian activities in farm animals.

Follicular development and ovulation are profoundly suppressed during lactation in mammals. This suppression is suggested to be mainly due to the suckling-induced inhibition of kisspeptin gene (Kiss1) expression in the arcuate nucleus (ARC) and consequent inhibition of pulsatile GnRH/LH release. We examined whether central somatostatin (SST) signaling mediates the suckling-induced suppression of pulsatile LH secretion. SST has been reported to be expressed in the posterior intralaminar thalamic nucleus (PIL), where the suckling stimulus is postulated to be relayed to the hypothalamus during lactation. SST inhibitory receptors (SSTRs) are abundantly expressed in the ARC, where kisspeptin/neurokinin B/dynorphin A (KNDy) neurons are located. Histological and quantitative studies revealed that the suckling stimulus increased the number of SST-expressing cells in the PIL, and Sstr2 expression in the ARC. Furthermore, a central injection of an SSTR2 antagonist caused a significant increase in pulsatile LH release in lactating rats. Double labeling of Sstr2 and the neurokinin B gene, as a marker for ARC KNDy neurons, showed Sstr2 expression was abundantly detected in the ARC, but few KNDy neurons coexpressed Sstr2 in lactating rats. Taken together, these findings suggest the suckling-induced activation of SST-SSTR2 signaling mediates, at least in part, the suppression of pulsatile LH secretion during lactation in rats, probably via the indirect effects of SST on KNDy neurons. These results provide a new aspect on the role of central SST-SSTR signaling in understanding the mechanism underlying lactational anestrus.

The brain mechanism responsible for the pulsatile secretion of gonadotropin-releasing hormone (GnRH) is important for maintaining reproductive function in mammals. Accumulating evidence suggests that kisspeptin/neurokinin B/dynorphin A (KNDy) neurons in the hypothalamic arcuate nucleus (ARC) play a critical role in the regulation of pulsatile GnRH and subsequent gonadotropin secretion. Dynorphin A (Dyn) and its receptor, kappa-opioid receptor (KOR, encoded by Oprk1), have been shown to be involved in the suppression of pulsatile GnRH/luteinizing hormone (LH) release. On the other hand, it is still unclear whether the inhibitory Dyn signaling affects KNDy neurons or KOR-expressing non-KNDy cells in the ARC or other brain regions. We therefore aimed to clarify the role of ARC-specific Dyn-KOR signaling in the regulation of pulsatile GnRH/LH release by the ARC specific cell deletion of KOR-expressing cells using Dyn-conjugated-saporin (Dyn-SAP). Estrogen-primed ovariectomized female rats were administered Dyn-SAP to the ARC. In situ hybridization of Oprk1 showed that ARC Dyn-SAP administration significantly decreased the number of Oprk1-expressing cells in the ARC, but not in the ventromedial hypothalamic nucleus and paraventricular nucleus. The frequency of LH pulses significantly increased in animals bearing the ARC Dyn-SAP administration. The number of Kiss1-expressing cells in the ARC was not affected by ARC Dyn-SAP treatment. Dyn-KOR signaling within the ARC seems to mediate the suppression of the frequency of pulsatile GnRH/LH release, and ARC non-KNDy KOR neurons may be involved in the mechanism modulating GnRH/LH pulse generation.

Bulleyaconitine A (BAA), a C19-diterpenoid alkaloid, has been prescribed as a nonnarcotic analgesic to treat chronic pain over four decades in China. The present study investigated its inhibition in morphine-induced withdrawal symptoms, conditioned place preference (CPP) and locomotor sensitization, and then explored the underlying mechanisms of actions. Multiple daily injections of morphine but not BAA up to 300 μg/kg/day into mice evoked naloxone-induced withdrawal symptoms (i.e., shakes, jumps, genital licks, fecal excretion and body weight loss), CPP expression, and locomotor sensitization. Single subcutaneous BAA injection (30–300 μg/kg) dose-dependently and completely attenuated morphine-induced withdrawal symptoms, with ED50 values of 74.4 and 105.8 μg/kg in shakes and body weight loss, respectively. Subcutaneous BAA (300 μg/kg) also totally alleviated morphine-induced CPP acquisition and expression and locomotor sensitization. Furthermore, subcutaneous BAA injection also specifically stimulated dynorphin A expression in microglia but not astrocytes or neurons in nucleus accumbens (NAc) and hippocampal, measured for gene and protein expression and double immunofluorescence staining. In addition, subcutaneous BAA-inhibited morphine-induced withdrawal symptoms and CPP expression were totally blocked by the microglial metabolic inhibitor minocycline, dynorphin A antiserum, or specific KOR antagonist GNTI, given intracerebroventricularly. These results, for the first time, illustrate that BAA attenuates morphine-induced withdrawal symptoms, CPP expression, and locomotor sensitization by stimulation of microglial dynorphin A expression in the brain, suggesting that BAA may be a potential candidate for treatment of opioids-induced physical dependence and addiction.

Significance Reports suggest that at least 25% of women suffering from reproductive disorders are due to hypothalamic dysfunction. Pulsatile gonadotropin-releasing hormone (GnRH) regimens are used for infertile women. Thus, elucidation of the GnRH/gonadotropin pulse generator is warranted to improve therapeutic approaches for these disorders. Normal pulsatile gonadotropin release and folliculogenesis were rescued by transfecting the kisspeptin gene ( Kiss1 ) into the hypothalamic neurokinin B (NKB) neurons in global Kiss1 knockout infertile female rats but not by transfecting outside the NKB neurons. Our results demonstrate that kisspeptin/NKB/dynorphin A (KNDy) neurons serve as the GnRH pulse generator, and >20% KNDy neurons are enough to maintain GnRH/gonadotropin pulses and folliculogenesis in female rats. The findings provide a potential therapeutic aspect for hypothalamic reproductive disorders.

Intestinal neuropeptides and neurotrophins as endocrine messengers play a key role in the bidirectional gut-brain interaction both in health and disease status. Their alterations in several neurological disorders have been reported, but whether a remarkable change occurs in Parkinson disease (PD) remains unexplored. In this study, we aimed to investigate the levels of 13 neuropeptides and 4 neurotrophins in the intestine of neurotoxin-induced PD mice. The PD mice were obtained by chronic injection of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) or MPTP/probenecid (MPTP/p). The levels of mRNA and protein expression in mouse intestines were measured by using real-time reverse transcription polymerase chain reaction and Western blotting, respectively. We found that the mRNA expression of 2 neuropeptides (cholecystokinin [CCK] and dynorphin A [Dyn A]) and 2 neurotrophins (brain-derived neurotrophic factor [BDNF] and neurotrophin-5) was significantly decreased in the colon of MPTP group compared to the vehicle-treated group. The protein levels of CCK, Dyn A, and BDNF were reduced in the colon of MPTP- or MPTP/p-treated mice compared to those of the vehicle-treated group. These data suggest that the intestinal expression of CCK, Dyn A, and BDNF was significantly reduced in PD animal models, and may play a role in the gut-brain axis in PD.

Malnutrition suppresses reproductive functions in mammals, which is considered to be mostly due to the inhibition of pulsatile gonadotropin-releasing hormone (GnRH)/gonadotropin secretion. Accumulating evidence suggests that kisspeptin neurons in the arcuate nucleus (ARC) play a critical role in the regulation of pulsatile GnRH/gonadotropin release. The present study aimed to examine if the hypothalamic dynorphin A (Dyn) neurons mediate the suppression of GnRH/luteinizing hormone (LH) pulses during malnutrition. Ovariectomized rats treated with a negative feedback level of estradiol-17β-treated (OVX+E2) were administered with intravenous (iv) or fourth cerebroventricle (4V) 2-deoxy-D-glucose (2DG), an inhibitor of glucose utilization, to serve as a malnutrition model. Central administration of a Dyn receptor antagonist blocked the iv- or 4V-2DG-induced suppression of LH pulses in OVX+E2 rats. The 4V 2DG administration significantly increased the number of Pdyn (Dyn gene)-positive cells co-expressing fos in the paraventricular nucleus (PVN), but not in the ARC and supraoptic nucleus (SON), and the iv 2DG treatment significantly increased the number of fos and Pdyn-co-expressing cells in the PVN and SON, but decreased it in the ARC. The E2 treatment significantly increased Pdyn expression in the PVN, but not in the ARC and SON. Double in situ hybridization for Kiss1 (kisspeptin gene) and Oprk1 (Dyn receptor gene) revealed that around 60% of ARC Kiss1-expressing cells co-expressed Oprk1. These results suggest that the PVN Dyn neurons, at least in part, mediate LH pulse suppression induced by the hindbrain or peripheral glucoprivation, and Dyn neurons may directly suppress the ARC kisspeptin neurons in female rats.

Kisspeptin (KISS1; encoded by Kiss1) neurons in the arcuate nucleus (ARC) coexpress tachykinin 3 (TAC3; also known as neurokinin B) and dynorphin A (PDYN). Accordingly, they are termed KNDy neurons and considered to be crucial in generating pulsatile release of gonadotropin-releasing hormone. Accumulating evidence suggests that Kiss1 and Tac3 are negatively regulated by estrogen. However, it has not been fully determined whether and how estrogen modulates Pdyn and PDYN. Here, we examined the expression of Pdyn mRNA and PDYN by in situ hybridization and immunohistochemistry, respectively, in the ARC of female rats after ovariectomy (OVX) and OVX plus low- or high-dose beta-estradiol (E2) replacement. We also investigated the effect of E2 on expression of Kiss1, KISS1, Tac3, and TAC3. Furthermore, colocalization of PDYN and estrogen receptor alpha (ESR1) was determined. Subsequently, we found that low-dose E2 treatment had no effect on Pdyn mRNA-expressing cells, but increased PDYN-immunoreactive (ir) cell numbers. In contrast, high-dose E2 treatment resulted in prominent reductions in both Pdyn mRNA-expressing and PDYN-ir cell numbers. Changes induced by low or high doses of E2 were similarly observed in the expression of Kiss1, KISS1, Tac3, and TAC3. The majority of PDYN-ir neurons coexpressed ESR1 in all groups. Our results indicate that E2 regulates the expression of PDYN, as well as KISS1 and TAC3, with regulation by E2 differing according to its levels.

Our group has earlier demonstrated that three enzymes sensitive to peptidase inhibitors (PIs), amastatin (A)-, captopril (C)-, and phosphoramidon (P), played an important role in inactivation of enkephalins at the spinal level. Dynorphin-converting enzyme (DCE) hydrolyzes dynorphin (Dyn) A (1-17) or Dyn A (1-13) mainly at the Arg6-Arg7 bond. Dynorphin A and its derived peptides interact with opioid and glutamate receptors at their N- and C-terminals, respectively. The purpose of the present study was to evaluate the antinociceptive potency and toxicity of intrathecal administered Dyn A (1-17), Dyn A (1-13), or Dyn A (1-6) under pretreatment with ACP and/or the DCE inhibitor p-hydroxymercuribenzoate (PHMB). The effect of these PIs on Dyn A (1-17)-induced inhibition of electrically-evoked contractions in mouse vas deferens was also investigated. The inhibitory potency of Dyn A (1-17) on electrically-evoked contractions in mouse vas deferens under pretreatment with ACP was higher than that with AC, AP, or CP. Pretreatment with ACP augmented Dyn A (1-17) or (1-13)-induced antinociception by approximately 50- or 30-fold with no sign of allodynia when administered intrathecally at low doses. Pretreatment with ACP and PHMB induced neuropathy. These findings showed that intrathecal administration of low-dose Dyn A (1-17) or DynA (1-13) increased antinociception under pretreatment with ACP, but without signs of allodynia in rat.

Pubertal onset is thought to be timed by an increase in pulsatile gonadotropin-releasing hormone (GnRH)/gonadotropin secretion in mammals. The underlying mechanism of pubertal onset in mammals is still an open question. Evidence accumulated in the last 15 years suggests that kisspeptin/neurokinin B/dynorphin A (KNDy) neurons in the hypothalamic arcuate nucleus play a key role in pubertal onset by triggering pulsatile GnRH/gonadotropin secretin in mammals. Specifically, KNDy neurons are now considered a part of GnRH pulse generator, in which neurokinin B facilitates and dynorphin A inhibits, the synchronized discharge of KNDy neurons in autocrine and/or paracrine manners. Kisspeptin serves as a potent secretagogue of GnRH secretion and thus its release is fundamental to pubertal increase in GnRH/gonadotropin secretion in mammals. Proposed mechanisms inhibiting Kiss1 (kisspeptin gene) expression during childhood to juvenile varies from species to species: we envisage that negative feedback action of estrogen plays a key role in the inhibition of Kiss1 expression in KNDy neurons in rodents and sheep, whereas estrogen-independent inhibition of kisspeptin secretion by γ-amino butyric acid or neuropeptide Y are suggested to be responsible for the pre-pubertal suppression of GnRH/gonadotropin secretion in primates. Taken together, the timing of pubertal onset is postulated to be controlled by upstream regulators for kisspeptin biosynthesis and secretion in mammals.

Understanding molecular alterations associated with peripheral inflammation is a critical factor in selectively controlling acute and persistent pain. The present report employs in situ hybridization of the 2 opioid precursor mRNAs coupled with quantitative measurements of 2 peptides derived from the prodynorphin and proenkephalin precursor proteins: dynorphin A 1-8 and [Met5]-enkephalin-Arg6-Gly7-Leu8. In dorsal spinal cord ipsilateral to the inflammation, dynorphin A 1-8 was elevated after inflammation, and persisted as long as the inflammation was sustained. Qualitative identification by high performance liquid chromatography and gel permeation chromatography revealed the major immunoreactive species in control and inflamed extracts to be dynorphin A 1-8. In situ hybridization in spinal cord after administration of the inflammatory agent, carrageenan, showed increased expression of prodynorphin (Pdyn) mRNA somatotopically in medial superficial dorsal horn neurons. The fold increase in preproenkephalin mRNA (Penk) was comparatively lower, although the basal expression is substantially higher than Pdyn. While Pdyn is not expressed in the dorsal root ganglion (DRG) in basal conditions, it can be induced by nerve injury, but not by inflammation alone. A bioinformatic meta-analysis of multiple nerve injury datasets confirmed Pdyn upregulation in DRG across different nerve injury models. These data support the idea that activation of endogenous opioids, notably dynorphin, is a dynamic indicator of persistent pain states in spinal cord and of nerve injury in DRG. PERSPECTIVE: This is a systematic, quantitative assessment of dynorphin and enkephalin peptides and mRNA in dorsal spinal cord and DRG neurons in response to peripheral inflammation and axotomy. These studies form the foundational framework for understanding how endogenous spinal opioid peptides are involved in nociceptive circuit modulation.

The present review summarizes the state of knowledge of endogenous opioids in birds. Endogenous opioid peptides acts in a neuromodulatory, hormonal and paracrine manner to mediate analgesic and other physiological functions. These peptides act through specific G-protein coupled receptors. Opioid receptors consist of a family of four closely-related proteins. The three types of opioid receptors are the mu (MOR or μ), delta (DOR or δ), and kappa (KOR or κ) opioid receptor proteins. The role of the fourth member of the opioid receptor family, the nociceptin or orphanin FQ receptor (ORL), is not clear. The ligands for opioid receptors are: β –endorphin (MOR), Met- enkephalin, Leu-enkephalin (DOR) and dynorphin (KOR), together with probably endomorphins 1 and 2. In spite of long history of research on endogenous opioid peptides, there are no studies of endogenous opioids per se in wild birds and few in poultry species. β-endorphin is present in all birds investigated and there is close agreement between the structures of β-endorphin in different birds. Plasma concentrations of β-endorphin are increased by ether stress in geese. There is evidence that β-endorphin plays a role in the control of luteinizing hormone release in chickens. Met-enkephalin is present in tissues such as the retina, hypothalamus, pituitary gland, and adrenals together with circulation of birds. Stresses such as crowding and withholding water increase circulating concentrations of Met-enkephalin in chickens. The structures of chicken dynorphin A and B have been deduced from cDNA. What is missing are comprehensive studies of plasma concentrations and expression of the full array of endogenous opioids in multiple avian species under different situations. Also, what is not known is the extent to which circulating or locally released or intra-cellular Met-enkephalin influence physiological process in birds. Thus, there is considerable scope for investigation of the physiology of endogenous opioids in birds.

KNDy neurons are named for their co-expression of three neuropeptides, kisspeptin, neurokinin B (NKB) and dynorphin A (DynA). These cells, located in the hypothalamic arcuate nucleus (ARC), are associated with generation of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) pulses to control follicular growth and steroidogenesis. However, subcellular sorting mechanisms for secretory vesicles containing these neuropeptides have not been elucidated. In this study, we analyzed the localization pattern of kisspeptin, NKB and DynA in the ARC of the ovariectomized rat immediately treated with estrogen using immunoelectron microscopy. First, we identified neuropeptides by dual-labeled fluorescence immunohistochemistry, with results indicating all three neuropeptides co-express within individual ARC cells in female rats. Next, we investigated the subcellular localization pattern of kisspeptin, NKB, and/or DynA using post-embedding double immunoelectron microscopy, indicating that each type of neuropeptide is contained within separate and individual neurosecretory vesicles. This suggests sorting and packaging of kisspeptin, NKB and DynA is differentially regulated within KNDy neurons. Our findings facilitate understanding of regulatory mechanisms underlying kisspeptin secretion in KNDy neurons, and generation of GnRH/LH pulses induced by kisspeptin in the ARC.

The pulsatile secretion of hormones is an efficient way of coding a large variety of chemical messages. The GnRH pulse pattern determines which gonadotropin is released when and at what concentration, prescribing a detailed set of instructions to the gonads that produce changes in the steroid hormone milieu. Although GnRH neurons possess some inherent rhythmicity, they are diffusely situated within the hypothalamus and in isolation are only capable of generating physiologically irrelevant messages, hence a synchronization module exists upstream. The identity of the neural unit comprising the GnRH pulse generator is now generally thought to include KNDy neurons in the arcuate nucleus. These neurons coexpress the neuropeptides kisspeptin, neurokinin B and dynorphin A, as well as other transmitters, and are in intimate contact with the GnRH network. The GnRH pulse generator’s function is the precise control of GnRH neuron excitability, coordinated activation, stimulation of neurosecretory events, modulation of gene transcription and the mediation of the negative feedback effect of gonadal steroids. Additionally, the GnRH pulse generator is an ideal venue for the integration of various sensory and homeostatic cues that regulate reproductive functions. In this chapter we provide a historical perspective of the elegant science that sparked interest in the central mechanisms underlying the functions of the reproductive system, explain how hypotheses surrounding GnRH pulse generation have evolved and describe the current state of knowledge within the dynamic field of GnRH pulse generator research.

Purpose The opioid system role in anorexia nervosa (AN) pathophysiology is still unclear since conflicting results were reported on peripheral and cerebrospinal fluid opioids levels. The study main aim was to evaluate cerebral AN opiate receptor availability by using [11C] diprenorphine, a ligand with non-selective binding. Methods In vivo [11C]diprenorphine cerebral non-displaceable binding potential (BPND) evaluated by PET imaging was compared between three groups : 17 undernourished restrictive-type AN patients (LeanAN), 15 AN patients having regained normal weight (RecAN) and 15 controls. A lower BPND may account for an increased opioid tone and vice versa. Serum hormones and endogenous opioids levels, eating-related and unspecific psychological traits were also evaluated. Results Compared to controls, LeanAN and RecAN patients had decreased [11C]diprenorphine BPND in middle frontal gyrus, temporo-parietal cortices, anterior cingulate cortex and in left accumbens nucleus. Hypothalamo-pituitary (H-P), left amygdala and insula BPND was found decreased only in LeanAN and that of putamen only in RecAN. LeanAN presented higher dynorphin A and enkephalin serum levels than in controls or RecAN. Inverse correlations were found in total group between : 24 h mean serum cortisol levels and anterior cingulate gyrus or insula BPND; eating concern score and left amygdala BPND. Positive correlation were found between leptin and hypothamus BPND; LH and pituitary BPND. Conclusions Low opiate receptor availability may be interpreted as an increased opioid tone in areas associated with both reward/aversive system in both AN groups. The relationship between the opioid receptors activity and hypercorticism or specific psychometric scores in some of these regions suggests adaptive mechanisms facing anxiety but also may play a role in the disease perpetuation.

Olfactory bulbectomized (OBX) mice are characterized by impaired performance in the passive avoidance test and decreased number of cholinergic neurons in the hippocampus. Several studies have reported that κ-opioid receptor agonists improve cognitive function in mice. However, their influence on OBX-induced cognitive dysfunction remains unclear. To address this question, we evaluated the effects of the endogenous κ-opioid receptor agonist dynorphin A (Dyn A) and the selective agonist trans-(-)-U-50488 on the behavior of OBX mice in the passive avoidance test. The cognitive dysfunction of OBX mice was significantly recovered by the intracerebroventricular administration of Dyn A or trans-(-)-U-50488. The effects of these two agonists were counteracted by the selective κ-opioid receptor antagonist nor-binaltorphimine or the inhibitor of acetylcholine release β-bungarotoxin. These findings suggest that κ-opioid receptor agonists produce anti-dementia effects through activation of cholinergic neurons in OBX mice.

Experiments on rats showed that the selective opioid κ receptor agonist dynorphin A (1-13) had marked antioxidant actions in immobilization stress of different durations, apparent as reductions in lipid peroxidation products in liver tissues. The effects of this peptide on the activities of the antioxidant defense enzymes superoxide dismutase and catalase depended on the duration of stress. While the peptide had a notable stimulatory effect on catalase activity in animals subjected to immobilization for 3 h, this effect was significantly smaller in rats exposed to stress for 6 or 12 h. These studies demonstrated that dynorphin A (1-13) has a stimulatory action on superoxide dismutase activity in animals exposed to immobilization for 6 or 12 h, this being seen at all study time points. Peptide had no effect on the activity of this enzyme in rats exposed to immobilization stress for 3 h.

Infraorbital nerve constriction (CION) causes hypersensitivity to facial mechanical, heat and cold stimulation in rats and mice and is a reliable model to study trigeminal neuropathic pain. In this model there is evidence that mechanisms operated by kinin B1 and B2 receptors contribute to heat hyperalgesia in both rats and mice. Herein we further explored this issue and assessed the role of kinin receptors in mechanical hyperalgesia after CION. Swiss and C57Bl/6 mice that underwent CION or sham surgery or dynorphin A (1-17) administration were repeatedly submitted to application of either heat stimuli to the snout or mechanical stimuli to the forehead. Treatment of the animals on the fifth day after CION surgery with DALBK (B1 receptor antagonist) or HOE-140 (B2 receptor antagonist), both at 0.01-1μmol/kg (i.p.), effectively reduced CION-induced mechanical hyperalgesia. Knockout mice for kinin B1, B2 or B1/B2 receptors did not develop heat or mechanical hyperalgesia in response to CION. Subarachnoid dynorphin A (1-17) delivery (15nmol/5μL) also resulted in orofacial heat hyperalgesia, which was attenuated by post-treatment with DALBK (1 and 3μmol/kg, i.p.), but was not affected by HOE-140. Additionally, treatment with an anti-dynorphin A antiserum (200μg/5μL, s.a.) reduced CION-induced heat hyperalgesia for up to 2h. These results suggest that both kinin B1 and B2 receptors are relevant in orofacial sensory nociceptive changes induced by CION. Furthermore, they also indicate that dynorphin A could stimulate kinin receptors and this effect seems to contribute to the maintenance of trigeminal neuropathic pain.

It has been shown that under chronic pain or nerve injury conditions, up-regulated dynorphin A (Dyn A) interacts with bradykinin receptors (BRs) to cause hyperalgesia in the spinal cord. Thus BRs antagonist can modulate hyperalgesia by blocking Dyn A’s interaction with the BRs in the central nervous system. In our earlier structure–activity relationship (SAR) study, [des-Arg 7 ]-Dyn A-(4–11) 13 was discovered as a minimum pharmacophore for rat brain BRs with its antagonist activity (anti-hyperalgesic effect) in in vivo tests using naive or injured animals. We have pursued further modification on the [des-Arg 7 ]-Dyn A analogues and identified a key insight into the pharmacophore of the rat brain BRs: amphipathicity.

Guinea pigs are important zoo animals and have been recommended for animal-assisted activities or therapy, however there are problems concerning testosterone inducing aggressive or sexual behaviors in male guinea pigs. Testicular testosterone secretion is regulated by pulsatile gonadotropin releasing hormone (GnRH)/luteinizing hormone (LH) release in mammals. The mechanism generating GnRH/LH pulses is thought to be governed by kisspeptin neurons, which coexpress neurokinin B (NKB) and dynorphin A (Dyn), in the arcuate nucleus (ARC). Kisspeptin neurons in the ARC are frequently referred to as KNDy neurons. The purpose of this study was to examine whether the antagonization of NKB-neurokinin-3 receptor (NK3R) signaling can manipulate testosterone secretion in male guinea pigs. A single subcutaneous administration or 7 days of oral administration of an NK3R-selective antagonist, SB223412 (50 mg/body), significantly decreased plasma testosterone levels in male guinea pigs. In vitro binding assays confirmed that SB223412 has a high affinity to guinea pig NK3R. These results suggest that SB223412 could be used as an orally-available compound to suppress testosterone levels in male guinea pigs. Double labeling in situ hybridization of kisspeptin and either NKB or Dyn showed that kisspeptin-expressing neurons contained NKB (77.9%) or Dyn (62.3%) in the ARC, suggesting the presence of KNDy neurons in the ARC of guinea pigs. In conclusion, the present study shows that SB223412 could be a candidate compound to suppress testosterone secretion in male guinea pigs for controlling sexual and aggressive behaviors in the species.

Neurons expressing kisspeptin, neurokinin B, and dynorphin A, located in the arcuate nucleus of the hypothalamus (ARC), are important regulators of reproduction. Their functions depend on metabolic and hormonal status. We hypothesized that male rats with high-fat diet-induced obesity (DIO) and/or streptozotocin-induced diabetes mellitus type 1 (DM1) and type 2 (DM2) will have: 1) alterations in numbers of immunoreactive (ir) cells: kisspeptin-ir and/or neurokinin B-ir, and dynorphin A-ir neurons in the ARC in the sham condition; 2) orchidectomy alone (ORX) and with testosterone treatment (ORX+T) will unmask possible deficits in the response of these neurons in DIO, and/or DM1 and DM2 rats. Rats were assigned to four groups: a control (C) and one diabetic group (DM1) were fed a regular chow diet, while the obese group (DIO) and the other diabetic group (DM2) were fed a high-fat diet. To induce diabetes, streptozotocin was injected. After 6 weeks, each group was divided into three subgroups: ORX, ORX+T, and sham. After another 2 weeks, metabolic and hormonal profiles were assessed, and immunocytochemistry was performed. We found that: 1) under sham condition: i) DM1 and DM2 animals had higher numbers of kisspeptin-ir cells than controls; ii) DM2 rats had increased numbers of neurokinin B-ir and dynorphin A-ir cells, compared to C animals; 2) ORX and ORX+T treatments unmasked deficits of the studied neurons in DM1 and DM2 but not in DIO animals; 4) DIO, DM1, and DM2 rats had altered metabolic and hormonal profiles, in particular decreased levels of testosterone. We concluded that alterations in numbers of kisspeptin-ir and neurokinin B-ir neurons in the ARC and their response to ORX and ORX+T may account for disruptions of metabolic and reproductive functions in diabetic but not in obese rats. This article is protected by copyright. All rights reserved.

Key points Dynorphin A inhibits anorexigenic hypothalamic proopiomelanocortin neurons by activating a potassium conductance. Although dynorphin A is considered selective for κ-opioid receptors, the present data show that the dynorphin A-induced potassium conductance is reversed by a μ-opioid receptor-selective antagonist and is absent in mice lacking functional μ-opioid receptors. Thus, μ-opioid receptors mediate the inhibition of proopiomelanocortin neurons caused by dynorphin A, consistent with other studies showing that κ-opioid receptor-selective agonists and antagonists can act at the μ-opioid receptor when used at sufficiently high concentrations. The results indicate that if dynorphin A inhibits proopiomelanocortin neurons to increase food intake, it does so either by activating μ-opioid receptors on these neurons or by inhibiting κ-opioid receptors located in the presynaptic compartment of proopiomelanocortin neurons. Abstract It has recently been shown that dynorphin A (Dyn A), an endogenous agonist of the κ-opioid receptor (KOR), directly inhibits proopiomelanocortin (POMC) neurons in the hypothalamus through activation of G-protein-coupled inwardly rectifying K+ channels (GIRKs). This effect has been proposed to be mediated by the putative κ2-opioid receptor (KOR-2), and has been suggested as a possible mechanism for the orexigenic actions of KOR agonists. Using whole-cell voltage clamp recordings in brain slice preparations, the present study demonstrates that Dyn A (1 or 5 μm) induces an outward current in POMC neurons that is reversed by the highly selective μ-opioid receptor (MOR) antagonist CTAP and is absent in mice lacking MORs. Additionally, the KOR-2-selective agonist GR89696 binds MORs on POMC neurons but fails to induce an outward current. Similar to Dyn A, the KOR-selective antagonist nor-binaltorphimine (nor-BNI) lacked specificity when used at sufficiently high concentrations. Maximal concentrations of the MOR-selective agonist DAMGO induced outward currents in POMC neurons that were completely reversed by a relatively high concentration of nor-BNI. Experiments using a half-maximal concentration of DAMGO demonstrate that nor-BNI must be used at concentrations

Background and Purpose Crotalphine is an antinociceptive peptide that, despite its opioid-like activity, does not induce some of the characteristic side effects of opioids, and its amino acid sequence has no homology to any known opioid peptide. Here, we evaluated the involvement of the peripheral cannabinoid system in the crotalphine effect and its interaction with the opioid system. Experimental Approach Hyperalgesia was evaluated using the rat paw pressure test. Involvement of the cannabinoid system was determined using a selective cannabinoid receptor antagonist. Cannabinoid and opioid receptor activation were evaluated in paw slices by immunofluorescence assays using conformation state-sensitive antibodies. The release of endogenous opioid peptides from skin tissue was measured using a commercial enzyme immunoassay (EIA). Key Results Both p.o. (0.008–1.0 μg·kg−1) and intraplantar (0.0006 μg per paw) administration of crotalphine induced antinociception in PGE2-induced hyperalgesia. Antinociception by p.o. crotalphine (1 μg·kg−1) was blocked by AM630 (50 μg per paw), a CB2 receptor antagonist, and by antiserum anti-dynorphin A (1 μg per paw). Immunoassay studies confirmed that crotalphine increased the activation of both κ-opioid (51.7%) and CB2 (28.5%) receptors in paw tissue. The local release of dynorphin A from paw skin was confirmed by in vitro EIA and blocked by AM630. Conclusions and Implications Crotalphine-induced antinociception involves peripheral CB2 cannabinoid receptors and local release of dynorphin A, which is dependent on CB2 receptor activation. These results enhance our understanding of the mechanisms involved in the peripheral effect of crotalphine, as well as the interaction between the opioid and cannabinoid systems.

Acute systemic stress disrupts reproductive function by inhibiting pulsatile gonadotropin secretion. The underlying mechanism involves stress-induced suppression of the GnRH pulse generator, the functional unit of which is considered to be the hypothalamic arcuate nucleus kisspeptin/neurokinin B/dynorphin A neurons. Agonists of the neurokinin B (NKB) receptor (NK3R) have been shown to suppress the GnRH pulse generator, in a dynorphin A (Dyn)-dependent fashion, under hypoestrogenic conditions, and Dyn has been well documented to mediate several stress-related central regulatory functions. We hypothesized that the NKB/Dyn signaling cascade is required for stress-induced suppression of the GnRH pulse generator. To investigate this ovariectomized rats, iv administered with Escherichia coli lipopolysaccharide (LPS) following intracerebroventricular pretreatment with NK3R or κ-opioid receptor (Dyn receptor) antagonists, were subjected to frequent blood sampling for hormone analysis. Antagonism of NK3R, but not κ-opioid receptor, blocked the suppressive effect of LPS challenge on LH pulse frequency. Neither antagonist affected LPS-induced corticosterone secretion. Hypothalamic arcuate nucleus NKB neurons project to the paraventricular nucleus, the major hypothalamic source of the stress-related neuropeptides CRH and arginine vasopressin (AVP), which have been implicated in the stress-induced suppression of the hypothalamic-pituitary-gonadal axis. A separate group of ovariectomized rats was, therefore, used to address the potential involvement of central CRH and/or AVP signaling in the suppression of LH pulsatility induced by intracerebroventricular administration of a selective NK3R agonist, senktide. Neither AVP nor CRH receptor antagonists affected the senktide-induced suppression of the LH pulse; however, antagonism of type 2 CRH receptors attenuated the accompanying elevation of corticosterone levels. These data indicate that the suppression of the GnRH pulse generator by acute systemic stress requires hypothalamic NKB/NK3R signaling and that any involvement of CRH therewith is functionally upstream of NKB.

In this study we investigated whether κ-opioid receptor stimulation by dynorphin A (1-13), a potent fragment of endogenous peptide, attenuated repeated stress-induced behavioral impairments in mice. In order to reduce the motivation to escape, mice were preexposed to inescapable electric footshock (day 0), and then dynorphin A (1-13) was administered to mice prior to the stress from the next day for 4 d (days 1-4). Dynorphin A (1-13) (1500 pmol/5 µL intracerebroventricular (i.c.v.)) attenuated the repeated stress-induced escape failures from the shock, and this improvement was inhibited by the pretreatment of nor-binaltorphimine (4.9 nmol/kg subcutaneously (s.c.)), a κ-opioid receptor antagonist. In the neurochemical experiments, we detected an increase in 5-hydroxyindoleacetic acid (5-HIAA) content, but not in serotonin (5-HT) content, and an increase in the 5-HIAA/5-HT ratio was observed in the amygdala of the group with footshock compared with the group without shock. Additionally, the changes in 5-HIAA content and the ratio were reversed by dynorphin A (1-13). However, there were no differences in 5-HT or 5-HIAA content or their ratios in the hippocampus among the three groups. These results suggest that dynorphin might alleviate the stress-induced behavioral impairments accompanied by regulation of the 5-HTergic system in the brain.

The KNDy neuropeptides, kisspeptin, neurokinin B (NKB) and dynorphin A (Dyn), have been implicated in regulating pulsatile luteinising hormone (LH) secretion. Studies of the interactions between KNDy signalling systems, however, are currently few. Although the stimulatory effect of kisspeptin and the inhibitory effect of Dyn on the gonadotropin-releasing hormone pulse generator are widely accepted, the effects of NKB in rodents are variable and sometimes controversial. Literature describing increased LH secretion in response to NKB receptor agonism predominates and is in line with human physiology, as well as the pathophysiology of pubertal failure associated with disruption of NKB signalling. However, the robust suppression of the LH pulse, induced by the same treatment under hypoestrogenic conditions, may hold clues as to the mechanisms of reproductive inhibition under pathological conditions. This review discusses the recent evidence for this paradox and outlines a revised working model incorporating the mechanisms by which KNDy neuropeptides modulate the reproductive axis.

In this study, we provide evidence for the first time that membrane-bound endopeptidase neurolysin is up-regulated in different parts of mouse brain affected by focal ischemia-reperfusion in a middle cerebral artery occlusion model of stroke. Radioligand binding, enzymatic and immunoblotting experiments in membrane preparations of frontoparietal cortex, striatum, and hippocampus isolated from the ischemic hemisphere of mouse brain 24 h after reperfusion revealed statistically significant increase (≥ twofold) in quantity and activity of neurolysin compared with sham-operated controls. Cerebellar membranes isolated from the ischemic hemisphere served as negative control supporting the observations that up-regulation of neurolysin occurs in post-ischemic brain regions. This study also documents sustained functional up-regulation of neurolysin in frontoparietal cortical membranes for at least 7 days after stroke, which appears not to be transcriptionally or translationally regulated, but rather depends on translocation of cytosolic neurolysin to the membranes and mitochondria. Considering diversity of endogenous neurolysin substrates (neurotensin, bradykinin, angiotensins I/II, substance P, hemopressin, dynorphin A(1-8), metorphamide, somatostatin) and the well-documented role of these peptidergic systems in pathogenesis of stroke, resistance to ischemic injury and/or post-stroke brain recovery, our findings suggest that neurolysin may play a role in processes modulating the brain's response to stroke and its recovery after stroke. We provide evidence that peptidase neurolysin is up-regulated in the mouse brain membranes after stroke. Neurolysin metabolizes several neuropeptides in the brain (neurotensin, bradykinin, angiotensins I and II, substance P, hemopressin, dynorphin A(1-8), metorphamide, somatostatin), which have various functions in stroke pathophysiology. We hypothesize that neurolysin plays a key role in processes modulating the brain's response to stroke and its recovery after stroke.

Apart from the primary metabolic symptoms of obesity and/or diabetes, there are numerous secondary problems, including disruptions of the reproductive system. The KNDy neurones, which express kisspeptin, neurokinin B and dynorphin A and are located in the arcuate nucleus of the hypothalamus (ARC), are important regulators of reproduction. Their functions are highly influenced by metabolic and hormonal status. We have previously shown that, in male rats with experimentally-induced diabetes type 2 (but not with high-fat diet-induced obesity), there are alterations in the number of these cells. In the present study, we hypothesised that a high-fat diet (HFD) and/or diabetes type 2 (DM2) in female rats affect the oestrous cycle, hormonal profiles and the number of kisspeptin-immunoreactive, neurokinin B-immunoreactive and/or dynorphin A-immunoreactive neurones in the ARC. Rats were assigned to one of three groups: a control group fed a regular chow diet, a high-fat diet group (HFD) and a diabetic group (DM2), with both of the latter two groups receiving a high calorie diet (50% of energy from lard). The third group was additionally treated with streptozotocin to induce DM2. Their oestrous cycles was monitored and their metabolic and hormonal status were assessed. We found that HFD and DM2 female rats, despite having significant alterations in their metabolic and hormonal profiles, as well as disruptions of the oestrous cycle, showed no changes in the number of the kisspeptin-immunoreactive, neurokinin B-immunoreactive and/or dynorphin A-immunoreactive neurones in the ARC. However, slight differences in the rostrocaudal distribution of these neurones among groups were reported. In conclusion, the data from the present study, together with our previously published results in males, indicate sex differences in the response of KNDy neurones to DM2 but not to HFD conditions.

Pain relief is necessary and essential for dental treatments. Recently, the relationships of pain and emotion were studied, and electrical stimulation applied to the amygdala depressed the nociceptive response in the anterior cingulate cortex (ACC). Thus, the antinociceptive effects of the amygdala are elucidated, but its mechanism is not yet clarified. The present study was performed to investigate whether endogenous opioid system is related to the depression, and the quantitative changes of endogenous opioids induced by electrical stimulation to the amygdala. We investigated immunohistologically c-Fos expression to confirm the activated neurons, as well as the distribution and the amount of endogenous opioids (β-endorphin, enkephalin and dynorphin A) in the brain using male Wistar rats, when electrical stimulation was applied to the central nucleus of the amygdala (CeA) or noxious stimulation was delivered to the peripheral tissue. c-Fos expression in the ipsilateral ACC was increased by electrical stimulation to the CeA. However, only a small amount of endogenous opioids was observed in the ACC when noxious stimulation or electrical stimulation was applied. In contrast, the amount of dynorphin A in the periaqueductal gray (PAG) was increased by electrical stimulation to the CeA, and the amount of β-endorphin in the PAG was increased by noxious stimulation to the peripheral tissue. The results suggest that dynorphin A in the PAG induced by electrical stimulation to the CeA activate the descending antinociceptive system, and suggest that the nociceptive response in the ACC is depressed indirectly.

The discovery of a novel non-AT1, non-AT2 binding site for angiotensins in the rodent brain and testis that is unmasked by the organomercurial compound para-chloromercuribenzoic acid (PCMB) has catalyzed efforts to purify and characterize this protein. We recently reported that this protein is neurolysin and now report upon the specificity of this binding site for various neuropeptides. Competition binding assays in rat brain and testis used (125)I-Sar(1), Ile(8) angiotensin II (Ang II) as the radioligand in the presence of saturating concentrations of AT1 and AT2 receptor antagonists and 100 μM parachloromercuribenzoate. Primary screening of 36 peptides and other compounds at 10 μM concentration revealed seven peptides that inhibited specific binding50 %: ghrelin, Tyr(1) S36057 (a melanin-concentrating hormone receptor ligand), orphanin FQ and its congeners (Tyr(1) and Tyr(14)), Dynorphin A (1-8), and Ang (1-9). The selective neurolysin inhibitor Proline-Isoleucine dipeptide was inactive at 1 mM. These results suggest that the ability of PCMB to unmask high affinity binding of Ang II to neurolysin is a pharmacological effect and that neurolysin may significantly affect the activity of the renin-angiotensin system.

Previous research suggests that a failure of opioid inhibition may contribute to chronic bladder pain. We determined how acute adult and/or prior early in life exposure to bladder inflammation alters the adult content of endogenous opioid peptides in the bladder, spinal cord and blood.Inflammation was induced by intravesical administration of zymosan. Female Sprague-Dawley® rats were exposed to anesthesia only or zymosan early in life (postnatal days 14 to 16) and anesthesia only or zymosan as adults (ages 12 to 17 weeks). Thoracolumbar and lumbosacral segments of the spinal cord, and blood and bladders were collected 24 hours after adult treatment. Opioid peptide content was measured using enzyme-linked immunosorbent assay.Early in life bladder inflammation alone produced a chronic increase in dynorphin A (1-17) in the lumbosacral spinal cord. When early in life inflammation was followed by adult re-inflammation, spinal cord dynorphin remained unchanged but bladder dynorphin was decreased. In addition, early in life inflammation combined with adult bladder inflammation decreased endomorphin-2 content in the thoracolumbar spinal cord. Neither early in life nor adult bladder inflammation affected thoracolumbar dynorphin, serum dynorphin, lumbosacral endomorphin-2 or plasma β-endorphin.Several opioid peptides were measured using enzyme-linked immunosorbent assay following early in life and adult bladder inflammation. The changes observed are consistent with the view that early in life bladder inflammation alone can chronically alter spinal cord peptide content. When coupled with adult re-inflammation, these changes could set the neurochemical stage to support bladder hypersensitivity.

Neurons in the arcuate nucleus (ARC) that concomitantly express kisspeptin, neurokinin B (NKB) and dynorphin A are termed KNDy neurons and are likely candidates for the intrinsic gonadotropin-releasing hormone (GnRH) pulse generator. Our hypothesis is that KNDy neurons are functionally and anatomically interconnected to generate discrete neural signals that govern pulsatile GnRH secretion. Our goal was to address this hypothesis using electrophysiological and anatomical experiments in goats. Bilateral electrodes targeting KNDy neurons were implanted into ovariectomized goats, and GnRH pulse generator activity, represented by characteristic increases in multiple-unit activity (MUA volleys), was measured. Spontaneous and pheromone- or senktide (an NKB receptor agonist)-induced MUA volleys were simultaneously recorded from both sides of the ARC. An anterograde tracer, biotinylated dextran amine (BDA), was also injected unilaterally into the ARC of castrated male goats, and the distribution of fibers containing both BDA and NKB was examined using dual-labeling histochemistry. The results showed that MUA volleys, regardless of origin (spontaneous or experimentally induced), occur simultaneously between the right and left sides of the ARC. Tract tracing indicated that axons projecting from NKB neurons in the ARC were directly apposed to other NKB neuronal cells located bilaterally in the ARC. These results demonstrate that GnRH pulse generator activity occurs synchronously between both sides of the ARC in goats and that KNDy neurons are bilaterally interconnected in the ARC via NKB-containing fibers. Taken together, the results suggest that KNDy neurons form a neuronal circuit to synchronize burst activity among KNDy neurons and thereby generate discrete neural signals that govern pulsatile GnRH secretion.

Dynorphin A 1–17 (Dyn A 1–17) is an endogenous neuropeptide known to act at the kappa opioid receptor; it has been implicated in a number of neurological disorders, including neuropathic pain, stress, depression, and Alzheimer’s and Parkinson’s diseases. The investigation of Dyn A 1–17 metabolism at the blood-brain barrier (BBB) is important since the metabolites exhibit unique biological functions compared to the parent compound. In this work, Dyn A 1–6 is identified as a metabolite of Dyn A 1–17 in the presence of bovine brain microvessel endhothelial cells (BBMECs), using LC-MS/MS. The transport of Dyn A 1–6 at the BBB was examined using this in vitro cell culture model of the BBB. Furthermore, the permeation of the BBB by the low molecular weight, permeability marker fluorescein was characterized in the presence and absences of Dyn A 1–6.

Previously study showed κ-opioid receptor stimulation with exogenous κ-opioid receptor agonist elicited a protective effect against hypoxic pulmonary hypertension (HPH). However, the effect of endogenous κ-opioid receptor agonist dynorphin A on HPH remains unclear. This study was to determine the role of dynorphin in HPH. Hypoxia for 2 weeks induced HPH. Compared with the HPH group, the HPH + nor-BNI (a selective κ-opioid receptor antagonist) group showed a significant increase in mean pulmonary arterial pressure (mPAP). Exogenous treatment with dynorphin A 1-13 significantly decreased mPAP in HPH rat. In addition, we evaluated the effect of exogenous κ-opioid receptor agonist U50,488H on mPAP. The anti-HPH effect of dynorphin A was less than that of U50,488H. Meanwhile, level of dynorphin A in serum and lung was increased during hypoxia for 2 weeks, while it decreased after hypoxia for 4 weeks. In addition, both the level of ET-1 and AngII were increased during hypoxia. Dynorphin A 1-13 and U50,488H time-dependently relaxed pulmonary artery from both normal and HPH rats. The relaxation of dynorphin A was less than that of U50,488H. Dynorphin A 1-13 inhibited the proliferation of pulmonary artery smooth muscle cells (PASMCs) during hypoxia, which was blocked by nor-BNI. κ-opioid receptor expression increased in PASMCs in both normoxia exposed to dynorphin A 1-13 and during hypoxia. Hypoxia-induced increase was enhanced by dynorphin A 1-13 and abolished by nor-BNI. In conclusion, endogenous dynorphin A released in the early stage of hypoxia plays a protective effect against HPH via stimulation of κ-opioid receptor.

Dynorphins are important neuropeptides with a central role in nociception and pain alleviation. Many mechanisms regulate endogenous dynorphin concentrations, including proteolysis. Proprotein convertases (PCs) are widely expressed in the central nervous system and specifically cleave at C-terminal of either a pair of basic amino acids, or a single basic residue. The proteolysis control of endogenous big dynorphin (BDyn) and dynorphin A (Dyn A) levels has a profound impact on pain perception and the role of PCs remain unclear. The objective of this study was to decipher the role of PC1 and PC2 in the proteolysis control of BDyn and Dyn A levels using cellular fractions of spinal cords from wild-type (WT), PC1(-/+) and PC2(-/+) animals and mass spectrometry. Our results clearly demonstrate that both PC1 and PC2 are involved in the proteolysis regulation of BDyn and Dyn A with a more important role for PC1. C-terminal processing of BDyn generates specific peptide fragments dynorphin 1-19, dynorphin 1-13, dynorphin 1-11 and dynorphin 1-7, and C-terminal processing of Dyn A generates dynorphin 1-13, dynorphin 1-11 and dynorphin 1-7, all these peptide fragments are associated with PC1 or PC2 processing. Moreover, the proteolysis of BDyn leads to the formation of Dyn A and Leu-Enk, two important opioid peptides. The rate of formation of both is significantly reduced in cellular fractions of spinal cord mutant mice. As a consequence, even the partial inhibition of PC1 or PC2 may impair the endogenous opioid system.

Opioids are the most powerful analgesics. As pain is driven by sensory transmission and opioid receptors couple to inhibitory G proteins, according to the classical concept, opioids alleviate pain by activating receptors on neurons and blocking the release of excitatory mediators (e.g., substance P). Here we show that analgesia can be mediated by opioid receptors in immune cells. We propose that activation of leukocyte opioid receptors leads to the secretion of opioid peptides Met-enkephalin, β-endorphin and dynorphin A (1-17), which subsequently act at local neuronal receptors, to relieve pain. In a mouse model of neuropathic pain induced by a chronic constriction injury of the sciatic nerve, exogenous agonists of δ-, μ- and κ-opioid receptors injected at the damaged nerve infiltrated by opioid peptide- and receptor-expressing leukocytes, produced analgesia, as assessed with von Frey filaments. The analgesia was attenuated by pharmacological or genetic inactivation of opioid peptides, and by leukocyte depletion. This decrease in analgesia was restored by the transfer of wild-type, but not opioid receptor-lacking leukocytes. Ex vivo, exogenous opioids triggered secretion of opioid peptides from wild-type immune cells isolated from damaged nerves, which was diminished by blockade of Gαi/o or Gβγ (but not Gαs) proteins, by chelator of intracellular (but not extracellular) Ca(2+), by blockers of phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP3) receptors, and was partially attenuated by protein kinase C inhibitor. Similarly, the leukocyte depletion-induced decrease in exogenous opioid analgesia was re-established by transfer of immune cells ex vivo pretreated with extracellular Ca(2+) chelator, but was unaltered by leukocytes pretreated with intracellular Ca(2+) chelator or blockers of Gαi/o and Gβγ proteins. Thus, both ex vivo opioid peptide release and in vivo analgesia were mediated by leukocyte opioid receptors coupled to the Gαi/o-Gβγ protein-PLC-IP3 receptors-intracellular Ca(2+) pathway. Our findings suggest that opioid receptors in immune cells are important targets for the control of pathological pain.

The role of Neurokinin B (NKB) and Dynorphin A (Dyn) in the regulation of the hypothalamic pituitary axis is an important area of recent investigation. These peptides are critical for the rhythmic release of GnRH, which subsequently stimulates the secretion of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The present study utilized the gonadotroph cell line LβT2 and the somatolactotroph GH3 cell line to examine the possible role of these peptides in pituitary hormone secretion. The NKB receptor (NK3R) and the Dyn receptor (the κ-opiate receptor (KOR)) were both detected in LβT2 cells and GH3 cells. NKB, by itself, failed to increase gonadotropin LHβ and FSHβ promoter activities and did not modulate the effects of GnRH on gonadotropin promoter activity. In GH3 cells, NKB significantly increased TRH-induced PRL promoter activity although NKB alone did not have an effect on basal PRL promoter activity. Dyn had no effect on gonadotropin promoters alone or in combination with GnRH stimulation. PRL promoters stimulated by TRH were not significantly changed by Dyn. TRH-induced PRL promoter activity was further increased in the presence of higher concentrations of NKB, whereas Dyn did not have a significant effect on the PRL promoter even at a high concentration. In addition, TRH-induced ERK (Extracelluar signal-regulated kinase) activation was enhanced in the presence of NKB. Our current study demonstrated that NKB had a stimulatory effect on PRL expression in a PRL-producing cell, but had no effect on gonadotropin secretion from a gonadotroph cell line.

Periaqueductal gray (PAG) plays a very important role in pain modulation through endogenous opiate peptides including leucine-enkephalin (L-Ek), methionine-enkephalin (M-Ek), β-endorphin (β-Ep) and dynorphin A(1-13) (DynA(1-13)). Our pervious study has demonstrated that intra-PAG injection of oxytocin (OXT) increases the pain threshold, and local administration of OXT receptor antagonist decreases the pain threshold, in which the antinociceptive role of OXT can be reversed by pre-PAG administration of OXT receptor antagonist. The experiment was designed to investigate the effect of OXT on endogenous opiate peptides in the rat PAG during the pain process. The results showed that (1) the concentrations of OXT, L-Ek, M-Ek and β-Ep, not DynA(1-13) in the PAG perfusion liquid were increased after the pain stimulation; (2) the concentrations of L-Ek, M-Ek and β-Ep, not DynA(1-13) in the PAG perfusion liquid were decreased by the OXT receptor antagonist; (3) the increased pain threshold induced by the OXT was attenuated by naloxone, an opiate receptor antagonist; and (4) the concentrations of L-Ek, M-Ek and β-Ep, not DynA(1-13) in the PAG perfusion liquid were increased by exogenous OXT administration. The data suggested that OXT in the PAG could influence the L-Ek, M-Ek and β-Ep rather than DynA(1-13) to participate in pain modulation, i.e. OXT in the PAG participate in pain modulation by influencing the L-Ek, M-Ek and β-Ep rather than DynA(1-13).

Methadone (Mtd) is a widely used opioid drug associated with the side effect of hyperprolactinemia. The mechanism of how Mtd induces prolactin secretion remains unclear. The effects of Mtd and its two main metabolites (EDDP: (±)-2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolinium percholarate and EMDP: 2-ethyl-5-methyl-3,3-dipnehyl-1-pyrroline) on ion currents were investigated in GH₃ pituitary tumor cells. Hyperpolarization-elicited K+ currents in GH₃ cells bathed in a high-K(+), Ca(2+)-free solution were studied to evaluate the effects of Mtd and other related compounds on the ether-a-go-go-related-gene (erg) K(+) current (I(K(erg))). Mtd suppressed the amplitude of I(K(erg)) in a concentration-dependent manner with an IC(50) value of 10.4 μM. With the aid of a minimal binding scheme, the inhibitory action of Mtd on I(K(erg)) was estimated with a dissociation constant of 8.2 μM. Mtd tended to increase the rate of I(K(erg)) deactivation in a voltage-dependent fashion. EDDP (10 μM) had no effect on I(K(erg)), while EMDP (10μM) slightly suppressed it. In GH₃ cells incubated with naloxone (30 μM), the Mtd-induced inhibition of I(K(erg)) remained unaltered. Under cell-attached voltage-clamp recordings, Mtd increased the frequency of spontaneous action currents with no change in current amplitude. Similarly, Mtd can suppress I(K(erg)) in differentiated NG108-15 cells; dynorphin A(1-13) did not reverse Mtd-induced inhibition of I(K(erg)). This study shows that Mtd has a depressant effect on I(K(erg)), and suggests its ability to affect membrane excitability and prolactin secretion. The cyclization of Mtd, in which EDDP and EMDP are formed, tends to be critical in removal of the Mtd binding to erg K+ channel.

Background Dysregulation of excitatory synaptic input to nucleus accumbens (NAc) medium spiny neurons (MSNs) underlies a key pathophysiology of drug addiction and addiction-associated emotional and motivational alterations. Dynorphin peptides, which exhibit higher affinity to κ type opioid receptors, are upregulated within the NAc upon exposure to cocaine administration, and the increased dynorphin-signaling in the NAc has been critically implicated in negative mood observed in cocaine- or stress-exposed animals. Despite such apparent behavioral significance of the NAc dynorphins, the understanding of how dynorphins regulate excitatory synaptic transmission in the NAc remains incomplete. Methods We used electrophysiological recording in brain slices to examine the effects of dynorphins on excitatory synaptic transmission in the NAc. Results We focused on two key dynorphins, dynorphin A and B. Our current results show that dynorphin A and B differentially regulated excitatory postsynaptic currents (EPSCs) in NAc MSNs. Whereas perfusions of both dynorphin A and B to NAc slices decreased EPSCs in MSNs, the effect of dynorphin A but not dynorphin B was completely reversed by the κ receptor-selective antagonist nor-binaltorphimine. These results implicate κ receptor-independent mechanisms in dynorphin B-mediated synaptic effects in the NAc. Furthermore, repeated exposure to cocaine (15 mg/kg/day via intraperitoneal injection for 5 days, with 1, 2, or 14 days withdrawal) completely abolished dynorphin A-mediated modulation of EPSCs in NAc MSNs, whereas the effect of dynorphin B remained largely unchanged. Conclusions Given the quantitatively higher abundance of dynorphin B in the NAc, our present results suggest that the dynorphin B-mediated, κ receptor-independent pathways predominate in the overall effect of dynorphins in cocaine-pretreated animals and potentially in cocaine-induced alterations in mood.

Kisspeptin (Kiss1) and neurokinin B (NKB) (encoded by the Kiss1 and Tac2 genes, respectively) are indispensable for reproduction. In the female of many species, Kiss1 neurons in the arcuate nucleus (ARC) coexpress dynorphin A and NKB. Such cells have been termed Kiss1/NKB/Dynorphin (KNDy) neurons, which are thought to mediate the negative feedback regulation of GnRH/LH secretion by 17β-estradiol. However, we have less knowledge about the molecular physiology and regulation of Kiss1/Kiss1-expressing neurons in the ARC of the male. Our work focused on the adult male mouse, where we sought evidence for coexpression of these neuropeptides in cells in the ARC, assessed the role of Kiss1 neurons in negative feedback regulation of GnRH/LH secretion by testosterone (T), and investigated the action of NKB on KNDy and GnRH neurons. Results showed that 1) the mRNA encoding Kiss1, NKB, and dynorphin are coexpressed in neurons located in the ARC; 2) Kiss1 and dynorphin A mRNA are regulated by T through estrogen and androgen receptor-dependent pathways; 3) senktide, an agonist for the NKB receptor (neurokinin 3 receptor, encoded by Tacr3), stimulates gonadotropin secretion; 4) KNDy neurons express Tacr3, whereas GnRH neurons do not; and 5) senktide activates KNDy neurons but has no discernable effect on GnRH neurons. These observations corroborate the putative role for KNDy neurons in mediating the negative feedback effects of T on GnRH/LH secretion and provide evidence that NKB released from KNDy neurons is part of an auto-feedback loop that generates the pulsatile secretion of Kiss1 and GnRH in the male.

Both immune/inflammatory and psychosocial stressors have profound effects on suppressing reproduction. Suppressing reproduction in times of stress, such as when fighting infection, or due to other environmental factors such as overcrowding, is an important shift of survival functions for a species, especially those with longer gestation periods or parental investment in rearing of offspring (1). Among other effects on reproduction, stress acts through glucocorticoids to decrease LH pulsatile secretion (2, 3). Immune/inflammatory stress, such as that induced by lipopolysaccharide (LPS) derived from Gramnegative bacteria, suppresses the GnRH and LH surge during the early preovulatory estradiol rise in sheep (4), and this suppression is independent of prostaglandins (5). In contrast, prostaglandins are mediators of LPS-induced suppression of GnRH and LH pulsatile secretion during the luteal phase in ewes (6). Cortisol treatment mimics the suppressive effects of LPS, but blocking cortisol production does not prevent LPS-induced suppression of LH pulses (7). Until now, it has not been clear which neural pathways mediate stress-induced suppression of GnRH and LH. Neurons in the arcuate nucleus of the hypothalamus coexpress kisspeptin, neurokinin B (NKB), and dynorphin A (8). Of the peptides produced by the arcuate kisspeptin, NKB, dynorphin (KNDy) neurons, NKB appears unique in differentially modulating GnRH and LH secretion based on the steroid milieu. Both NKB and a selective neurokinin-3 receptor (NK3R) agonist have differential effects on GnRH and LH secretion, depending on the gonadal state of the subjects treated (9). For example, in female gonadal-intact, estradiol-treated rats and sheep, and pubertal rats, NKB or NK3R agonist treatment increases GnRH and LH secretion, whereas in ovariectomized rats, these same treatments suppress GnRH or LH secretion (reviewed in Reference 9). During fasting, another type of stressor, the increase of LH in response to NK3R agonist treatment is even greater in pubertal rats compared with ad libitum feeding (10). In contrast, responses to kisspeptin or dynorphin or agonists that bind to their cognate receptors, G protein-coupled receptor 54 and -opioid receptor (KOR), respectively, are more consistent across the reproductive cycle (8). In this issue, Grachev et al (11) report the results of a series of experiments that link the suppression of reproduction by immune/inflammatory stress with the NKB/ NK3R pathway in female rats. They first determined whether NK3R or KOR, the receptor for which dynorphin has the greatest affinity, were necessary for the LPS-induced suppression of LH pulses or increases in corticosterone (CORT) concentrations (11). Administration of the KOR antagonist nor-BNI did not reverse the effects of LPS on either LH pulsatility or CORT secretion, whereas the NK3R antagonist (SB222200) did reverse the suppression of LH pulsatility, although CORT remained elevated (11). This suggests that immune/inflammatory stress acts upstream of NKB to increase CORT secretion, and the effects of CORT on LH pulsatility are at least in part mediated through NK3R-containing neurons (11). Administration of LPS stimulates other factors involved in the immune/inflammatory response, including prostaglandins and ILs (12). In sheep, blocking prostaglandin synthesis reverses the LPS-induced suppression of LH in the luteal (6) but not the follicular phase (5). In women with polycystic ovary syndrome, a disorder including failure to ovulate and development of large cystic

The Wistar Kyoto (WKY) rat strain is a putative genetic model of comorbid depression and anxiety. Previous research showing increased kappa-opioid receptor (KOR) gene expression in the brains of WKY rats, combined with studies implicating the KOR in animal models of depression and anxiety, suggests that alterations in the KOR system could have a role in the WKY behavioral phenotype. Here, the effects of KOR antagonists in the forced swim test (FST) were compared with the WKY and the Sprague-Dawley (SD) rat strains. As previously reported, WKY rats showed more immobility behavior than SD rats. The KOR antagonists selectively produced antidepressant-like effects in the WKY rats. By contrast, the antidepressant desipramine reduced immobility in both strains. Brain regions potentially underlying the strain-specific effects of KOR antagonists in the FST were identified using c-fos expression as a marker of neuronal activity. The KOR antagonist nor-binaltorphimine produced differential effects on the number of c-fos-positive profiles in the piriform cortex and nucleus accumbens shell between SD and WKY rats. The piriform cortex and nucleus accumbens also contained higher levels of KOR protein and dynorphin A peptide, respectively, in the WKY strain. In addition, local administration of nor-binaltorphimine directly into the piriform cortex produced antidepressant-like effects in WKY rats further implicating this region in the antidepressant-like response to KOR antagonists. These results support the use of the WKY rat as a model of affective disorders potentially involving KOR overactivity and provide more evidence that KOR antagonists could potentially be used as novel antidepressants.

Morphine-3-glucuronide (M3G) administered centrally produces dose-dependent neuro-excitatory behaviours in rodents via a predominantly non-opioid mechanism. The endogenous opioid peptide, dynorphin A (Dyn A) (1-17), is rapidly cleaved in vivo to the relatively more stable fragment Dyn A(2-17) which also produces excitatory behaviours in rodents via a non-opioid mechanism. This study investigated the possible contribution of Dyn A(2-17) to the neuro-excitatory behaviours evoked by supraspinally and spinally administered M3G in male Sprague-Dawley (SD) rats. Marked qualitative differences in behaviours were apparent following administration of M3G and Dyn A(2-17). Administration of 11 nmol i.c.v. doses of M3G produced intermittent myoclonic jerks, tonic-clonic convulsions, and ataxia, as well as postural changes, whereas i.c.v. Dyn A(2-17) at 15 nmol produced effects on body posture alone. Administration of 11 nmol i.t. doses of M3G produced intermittent explosive motor activity, and touch-evoked agitation, as well as postural changes, whereas i.t. Dyn A(2-17) at 15 nmol produced postural changes, touch-evoked agitation, and paralysis. Pre-treatment with Dyn A antiserum (200 microg) markedly attenuated total behavioural excitation following i.c.v. and i.t. administration of Dyn A(2-17) by approximately 94% and 78%, respectively. However, total behavioural excitation following i.c.v. and i.t. administration of M3G was less markedly attenuated (both approximately 27%) by pre-treatment with Dyn A antiserum, with reductions in tonic-clonic convulsions ( approximately 43%), explosive motor behaviour ( approximately 28%), and touch-evoked agitation ( approximately 22%). The present findings discount a major role for Dyn A in mediating the neuro-excitatory effects of M3G, although it may contribute to maintaining some individual neuro-excitatory behaviours.

We investigated whether acute treatment with agonists affected the subcellular distribution of kappa opioid receptor (KOPR) in the dorsal horn of the rat lumbar spinal cord by using immunoelectron microscopy. Rats were injected intrathecally (i.t.) with U50,488H (100 nmole), dynorphin A(1-17) (15 nmole), or vehicle. The doses chosen have been shown to induce antinociception. Rats were perfused transcardially 30 min later, and lumbar spinal cords were removed and processed for electron microscopic analysis. KOPR was stained with KT-2, a specific polyclonal antibody against the rat/mouse KOPR(371-380) peptide, followed by gold-labeled secondary antibody and silver intensification. The silver grains were present in axons, terminals, dendrites, and somata, and the association with plasma membranes was quantified in dendrites, because KOPR immunoreactivity was most frequently observed in these profiles. In vehicle-treated rats, approximately 27% of KOPR immunoreactivity was associated with plasma membranes. U50,488H, i.t., did not cause a significant change in the percentage of KOPR present on plasma membranes, whereas dynorphin A, i.t., significantly decreased cell surface KOPR to approximately 19%. In summary, these data indicate that U50,488H and dynorphin A differentially regulate the subcellular distribution of endogenous KOPR.