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The pulsatile activity of gonadotropin-releasing hormone neurons (GnRH neurons) is a key factor in the regulation of reproductive hormones. This pulsatility is orchestrated by a network of neurons that release the neurotransmitters kisspeptin, neurokinin B, and dynorphin (KNDy neurons), and produce episodic bursts of activity driving the GnRH neurons. We show in this computational study that the features of coordinated KNDy neuron activity can be explained by a neural network in which connectivity among neurons is modular. That is, a network structure consisting of clusters of highly-connected neurons with sparse coupling among the clusters. This modular structure, with distinct parameters for intracluster and intercluster coupling, also yields predictions for the differential effects on synchronization of changes in the coupling strength within clusters versus between clusters., Competing Interests: The authors declare that there are no competing interests., (Copyright: © 2024 Blanco et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Dynorphin is an endogenous opiate localized in many brain regions and spinal cord, but the activity of dynorphin neurons during sleep is unknown. Dynorphin is an inhibitory neuropeptide that is coreleased with orexin, an excitatory neuropeptide. We used microendoscopy to test the hypothesis that, like orexin, the dynorphin neurons are wake-active. Dynorphin-cre mice (n = 3) were administered rAAV8-Ef1a-Con/Foff 2.0-GCaMP6M into the zona incerta-perifornical area, implanted with a GRIN lens (gradient reflective index), and electrodes to the skull that recorded sleep. One month later, a miniscope imaged calcium fluorescence in dynorphin neurons during multiple bouts of wake, non-rapid-eye movement (NREM), and rapid-eye movement (REM) sleep. Unbiased data analysis identified changes in calcium fluorescence in 64 dynorphin neurons. Most of the dynorphin neurons (72%) had the highest fluorescence during bouts of active and quiet waking compared to NREM or REM sleep; a subset (20%) were REM-max. Our results are consistent with the emerging evidence that the activity of orexin neurons can be classified as wake-max or REM-max. Since the two neuropeptides are coexpressed and coreleased, we suggest that dynorphin-cre-driven calcium sensors could increase understanding of the role of this endogenous opiate in pain and sleep., (Published by Oxford University Press on behalf of Sleep Research Society (SRS) 2024.)

Neurones in the arcuate nucleus co-expressing kisspeptin, neurokinin B (NKB) and dynorphin (KNDy) play a critical role in the control of gonadotrophin-releasing hormone (GnRH) and luteinising hormone (LH) secretion. In sheep, KNDy neurones mediate both steroid-negative- and -positive-feedback during pulsatile and preovulatory surge secretions of GnRH/LH, respectively. In addition, KNDy neurones receive glutamatergic inputs expressing vGlut2, a glutamate transporter that serves as a marker for those terminals, from both KNDy neurones and other populations of glutamatergic neurones. Previous work reported higher numbers of vGlut2-positive axonal inputs onto KNDy neurones during the LH surge than in luteal phase ewes. In the present study, we further examined the effects of the ovarian steroids progesterone (P) and oestradiol (E 2 ) on glutamatergic inputs to KNDy neurones. Ovariectomised (OVX) ewes received either no further treatment (OVX) or steroid treatments that mimicked the luteal phase (low E 2  + P), and early (low E 2 ) or late follicular (high E 2 ) phases of the oestrous cycle (n = 4 or 5 per group). Brain sections were processed for triple-label immunofluorescent detection of NKB/vGlut2/synaptophysin and analysed using confocal microscopy. We found higher numbers of vGlut2 inputs onto KNDy neurones in high E 2 compared to the other three treatment groups. These results suggest that synaptic plasticity of glutamatergic inputs onto KNDy neurones during the ovine follicular phase depend on increasing levels of E 2 required for the preovulatory GnRH/surge. These synaptic changes likely contribute to the positive-feedback action of oestrogen on GnRH/LH secretion and thus the generation of the preovulatory surge in the sheep., (© 2021 British Society for Neuroendocrinology.)

Hypothalamic kisspeptin neurons serve as the nodal regulatory centre of reproductive function. These neurons are subjected to a plethora of regulatory factors that ultimately affect the release of kisspeptin, which modulates gonadotropin-releasing hormone (GnRH) release from GnRH neurons to control the reproductive axis. The presence of sufficient energy reserves is critical to achieve successful reproduction. Consequently, metabolic factors impose a very tight control over kisspeptin synthesis and release. This Review offers a synoptic overview of the different steps in which kisspeptin neurons are subjected to metabolic regulation, from early developmental stages to adulthood. We cover an ample array of known mechanisms that underlie the metabolic regulation of KISS1 expression and kisspeptin release. Furthermore, the novel role of kisspeptin neurons as active players within the neuronal circuits that govern energy balance is discussed, offering evidence of a bidirectional role of these neurons as a nexus between metabolism and reproduction.

Fertility critically depends on the gonadotropin-releasing hormone (GnRH) pulse generator, a neural construct comprised of hypothalamic neurons coexpressing kisspeptin, neurokoinin-B and dynorphin. Here, using mathematical modeling and in vivo optogenetics we reveal for the first time how this neural construct initiates and sustains the appropriate ultradian frequency essential for reproduction. Prompted by mathematical modeling, we show experimentally using female estrous mice that robust pulsatile release of luteinizing hormone, a proxy for GnRH, emerges abruptly as we increase the basal activity of the neuronal network using continuous low-frequency optogenetic stimulation. Further increase in basal activity markedly increases pulse frequency and eventually leads to pulse termination. Additional model predictions that pulsatile dynamics emerge from nonlinear positive and negative feedback interactions mediated through neurokinin-B and dynorphin signaling respectively are confirmed neuropharmacologically. Our results shed light on the long-elusive GnRH pulse generator offering new horizons for reproductive health and wellbeing. SIGNIFICANCE STATEMENT The gonadotropin-releasing hormone (GnRH) pulse generator controls the pulsatile secretion of the gonadotropic hormones LH and FSH and is critical for fertility. The hypothalamic arcuate kisspeptin neurons are thought to represent the GnRH pulse generator, since their oscillatory activity is coincident with LH pulses in the blood; a proxy for GnRH pulses. However, the mechanisms underlying GnRH pulse generation remain elusive. We developed a mathematical model of the kisspeptin neuronal network and confirmed its predictions experimentally, showing how LH secretion is frequency-modulated as we increase the basal activity of the arcuate kisspeptin neurons in vivo using continuous optogenetic stimulation. Our model provides a quantitative framework for understanding the reproductive neuroendocrine system and opens new horizons for fertility regulation., (Copyright © 2019 the authors.)

Neural circuits that enable an organism to protect itself by promoting escape from immediate threat and avoidance of future injury are conceptualized to carry an "aversive" signal. One of the key molecular elements of these circuits is the kappa opioid receptor (KOR) and its endogenous peptide agonist, dynorphin. In many cases, the aversive response to an experimental manipulation can be eliminated by selective blockade of KOR function, indicating its necessity in transmitting this signal. The dopamine system, through its contributions to reinforcement learning, is also involved in processing of aversive stimuli, and KOR control of dopamine in the context of aversive behavioral states has been intensely studied. In this review, we have discussed the multiple ways in which the KORs regulate dopamine dynamics with a central focus on dopamine neurons and projections from the ventral tegmental area. At the neuronal level, KOR agonists inhibit dopamine neurons both in the somatodendritic region as well as at terminal release sites, through various signaling pathways and ion channels, and these effects are specific to different synaptic sites. While the dominant hypotheses are that aversive states are driven by decreases in dopamine and increases in dynorphin, reported exceptions to these patterns indicate these ideas require refinement. This is critical given that KOR is being considered as a target for development of new therapeutics for anxiety, depression, pain, and other psychiatric disorders., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This review recounts the origins and development of the concept of the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator. It starts in the late 1960s when striking rhythmic episodes of luteinizing hormone secretion, as reflected by circulating concentrations of this gonadotropin, were first observed in monkeys and ends in the present day. It is currently an exciting time witnessing the application, primarily to the mouse, of contemporary neurobiological approaches to delineate the mechanisms whereby Kiss1/NKB/Dyn (KNDy) neurons in the arcuate nucleus of the hypothalamus generate and time the pulsatile output of kisspeptin from their terminals in the median eminence that in turn dictates intermittent GnRH release and entry of this decapeptide into the primary plexus of the hypophysial portal circulation. The review concludes with an examination of questions that remain to be addressed., Competing Interests: No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.

Negative affective states are prevalent symptoms in a plethora of neuropsychiatric disorders, including depression and drug addiction. Dysfunction of mesocorticolimbic dopamine systems has been implicated in negative affective states in neuropsychiatric disorders. The dynorphin/kappa-opioid receptor system is a powerful effector of stress-related behavior and is highly enriched within the mesocorticolimbic dopamine system. Dysfunction of dynorphin/KOR signaling within the mesocorticolimbic dopamine system is implicated in promoting symptoms in neuropsychiatric disorders. As such, the kappa-opioid receptor system provides an important therapeutic target to treat negative affective states associated with psychiatric disorders. In this review, we provide a comprehensive overview of the dynorphin/kappa-opioid receptor system and its role in regulating the mesocorticolimbic dopamine system, motivation, and emotional behavior. Furthermore, we highlight unresolved issues in the field and offer some insights for future research., (Copyright © 2018. Published by Elsevier B.V.)

Dynorphin is a neuropeptide involved in pain, addiction and mood regulation. It exerts its activity by binding to the kappa opioid receptor (KOP) which belongs to the large family of G protein-coupled receptors. The dynorphin peptide was discovered in 1975, while its receptor was cloned in 1993. This review will describe: (a) the activities and physiological functions of dynorphin and its receptor, (b) early structure-activity relationship studies performed before cloning of the receptor (mostly pharmacological and biophysical studies of peptide analogues), (c) structure-activity relationship studies performed after cloning of the receptor via receptor mutagenesis and the development of recombinant receptor expression systems, (d) structural biology of the opiate receptors culminating in X-ray structures of the four opioid receptors in their inactive state and structures of MOP and KOP receptors in their active state. X-ray and EM structures are combined with NMR data, which gives complementary insight into receptor and peptide dynamics. Molecular modeling greatly benefited from the availability of atomic resolution 3D structures of receptor-ligand complexes and an example of the strategy used to model a dynorphin-KOP receptor complex using NMR data will be described. These achievements have led to a better understanding of the complex dynamics of KOP receptor activation and to the development of new ligands and drugs., (© 2019 Elsevier Inc. All rights reserved.)

The organization of estrogenic signaling in the CNS is exceedingly complex. It is comprised of peripherally and centrally synthesized estrogens, and a plethora of types of estrogen receptor that can localize to both the nucleus and the plasma membrane. Moreover, CNS estrogen receptors can exist independent of aromatase (aka estrogen synthase) as well as oligomerize with it, along with a host of other membrane signaling proteins. This ability of CNS estrogen receptors to either to physically pair or exist separately enables locally produced estrogens to act on multiple spatial levels, with a high degree of gradated regulation and plasticity, signaling either in-phase or out-of phase with circulating estrogens. This complexity explains the numerous contradictory findings regarding sex-dependent pain processing and sexually dimorphic opioid antinociception. This review highlights the increasing awareness that estrogens are major endogenous arbiters of both opioid analgesic actions and the mechanisms used to achieve them. This behooves us to understand, and possibly intercede at, the points of intersection of estrogenic signaling and opioid functionality. Factors that integrate estrogenic actions at subcellular, synaptic, and CNS regional levels are likely to be prime drug targets for novel pharmacotherapies designed to modulate CNS estrogen-dependent opioid functionalities and possibly circumvent the current opioid epidemic., (© 2019 Elsevier Inc. All rights reserved.)

In this review, the effects of stress on alcohol drinking are discussed. The interactions between biological stress systems and alcohol drinking are examined, with a focus on the hypothalamic pituitary adrenal axis, corticotropin releasing factor, dynorphin, neuropeptide Y, and norepinephrine systems. Findings from animal models suggest that these biological stress systems may be useful targets for medications development for alcohol use disorder and co-occurring stress-related disorders in humans., Competing Interests: Financial Disclosure Dr. Gilpin owns shares in Glauser Life Sciences, a company with interest in developing therapeutics for mental health disorders. There is no direct link between those interests and the work contained herein.

Women during perimenopausal period experience a range of symptoms, which interfere with physical, sexual, and social life. About 65-75% of symptoms connected with postmenopausal period are vasomotor symptoms (VMS), such as hot flushes and night sweats. Hot flushes are subjective sensation of heat associated with cutaneous vasodilatation and drop in core temperature. It is suspected that VMS are strongly correlated with pulsatile oversecretion of gonadotropin-releasing hormone (GnRH) and subsequently luteinizing hormone (LH). Evidence has accumulated in parallel showing that lack of negative feedback of steroid hormones synthesized in ovary causes overactivation of hypertrophied kisspeptin/neurokinin B/dynorphin (KNDy) neurons, located in infundibular nucleus. Oversecretion of both kisspeptin (KISS1) and neurokinin B (NKB), as well as downregulation of dynorphin, plays dominant role in creation of GnRH pulses. This in turn causes VMS. Administration of senktide, highly potent and selective NK3R agonist, resulted in increase of serum LH concentration, induction of VMS, increase in heart rate, and skin temperature in postmenopausal women. These finding suggest that modulation of KNDy neurons may become new therapeutic approach in the treatment of VMS.

Early work in ewes provided a wealth of information on the physiological regulation of pulsatile gonadotropin-releasing hormone (GnRH) secretion by internal and external inputs. Identification of the neural systems involved, however, was limited by the lack of information on neural mechanisms underlying generation of GnRH pulses. Over the last decade, considerable evidence supported the hypothesis that a group of neurons in the arcuate nucleus that contain kisspeptin, neurokinin B and dynorphin (KNDy neurons) are responsible for synchronizing secretion of GnRH during each pulse in ewes. In this review, we describe our current understanding of the neural systems mediating the actions of ovarian steroids and three external inputs on GnRH pulsatility in light of the hypothesis that KNDy neurons play a key role in GnRH pulse generation. In breeding season adults, estradiol (E 2 ) and progesterone decrease GnRH pulse amplitude and frequency, respectively, by actions on KNDy neurons, with E 2 decreasing kisspeptin and progesterone increasing dynorphin release onto GnRH neurons. In pre-pubertal lambs, E 2 inhibits GnRH pulse frequency by decreasing kisspeptin and increasing dynorphin release, actions that wane as the lamb matures to allow increased pulsatile GnRH secretion at puberty. Less is known about mediators of undernutrition and stress, although some evidence implicates kisspeptin and dynorphin, respectively, in the inhibition of GnRH pulse frequency by these factors. During the anoestrus, inhibitory photoperiod acting via melatonin activates A15 dopaminergic neurons that innervate KNDy neurons; E 2 increases dopamine release from these neurons to inhibit KNDy neurons and suppress the frequency of kisspeptin and GnRH release., (© 2018 Society for Reproduction and Fertility.)

Understanding the neural systems that drive alcohol motivation and are disrupted in alcohol use disorders is of critical importance in developing novel treatments. The dynorphin and orexin/hypocretin neuropeptide systems are particularly relevant with respect to alcohol use and misuse. Both systems are strongly associated with alcohol-seeking behaviors, particularly in cases of high levels of alcohol use as seen in dependence. Furthermore, both systems also play a role in stress and anxiety, indicating that disruption of these systems may underlie long-term homeostatic dysregulation seen in alcohol use disorders. These systems are also closely interrelated with one another - dynorphin/kappa opioid receptors and orexin/hypocretin receptors are found in similar regions and hypocretin/orexin neurons also express dynorphin - suggesting that these two systems may work together in the regulation of alcohol seeking and may be mutually disrupted in alcohol use disorders. This chapter reviews studies demonstrating a role for each of these systems in motivated behavior, with a focus on their roles in regulating alcohol-seeking and self-administration behaviors. Consideration is also given to evidence indicating that these neuropeptide systems may be viable targets for the development of potential treatments for alcohol use disorders.

Key Points: Both endogenous opioids and opiate drugs of abuse modulate learning of habitual and goal-directed actions, and can also modify long-term plasticity of corticostriatal synapses. Striatal projection neurons of the direct pathway co-release the opioid neuropeptide dynorphin which can inhibit dopamine release via κ-opioid receptors. Theta-burst stimulation of corticostriatal fibres produces long-term potentiation (LTP) in striatal projection neurons when measured using whole-cell patch recording. Optogenetic activation of direct pathway striatal projection neurons inhibits LTP while reducing dopamine release. Because the endogenous release of opioids is activity dependent, this modulation of synaptic plasticity represents a negative feedback mechanism that may limit runaway enhancement of striatal neuron activity in response to drugs of abuse., Abstract: Synaptic plasticity in the striatum adjusts behaviour adaptively during skill learning, or maladaptively in the case of addiction. Just as dopamine plays a critical role in synaptic plasticity underlying normal skill learning and addiction, endogenous and exogenous opiates also modulate learning and addiction-related striatal plasticity. Though the role of opioid receptors in long-term depression in striatum has been characterized, their effect on long-term potentiation (LTP) remains unknown. In particular, direct pathway (dopamine D1 receptor-containing; D1R-) spiny projection neurons (SPNs) co-release the opioid neuropeptide dynorphin, which acts at presynaptic κ-opioid receptors (KORs) on dopaminergic afferents and can negatively regulate dopamine release. Therefore, we evaluated the interaction of co-released dynorphin and KOR on striatal LTP. We optogenetically facilitate the release of endogenous dynorphin from D1R-SPNs in brain slice while using whole-cell patch recording to measure changes in the synaptic response of SPNs following theta-burst stimulation (TBS) of cortical afferents. Our results demonstrate that TBS evokes corticostriatal LTP, and that optogenetic activation of D1R-SPNs during induction impairs LTP. Additional experiments demonstrate that optogenetic activation of D1R-SPNs reduces stimulation-evoked dopamine release and that bath application of a KOR antagonist provides full rescue of both LTP induction and dopamine release during optogenetic activation of D1R-SPNs. These results suggest that an increase in the opioid neuropeptide dynorphin is responsible for reduced TBS LTP and illustrate a physiological phenomenon whereby heightened D1R-SPN activity can regulate corticostriatal plasticity. Our findings have important implications for learning in addictive states marked by elevated direct pathway activation., (Published 2017. This article is a U.S. Government work and is in the public domain in the USA.)

Pulsatile secretion of gonadotrophin-releasing hormone (GnRH)/luteinising hormone is indispensable for the onset of puberty and reproductive activities at adulthood in mammalian species. A cohort of neurones expressing three neuropeptides, namely kisspeptin, encoded by the Kiss1 gene, neurokinin B (NKB) and dynorphin A, localised in the hypothalamic arcuate nucleus (ARC), so-called KNDy neurones, comprises a putative intrinsic source of the GnRH pulse generator. Synchronous activity among KNDy neurones is considered to be required for pulsatile GnRH secretion. It has been reported that gap junctions play a key role in synchronising electrical activity in the central nervous system. Thus, we hypothesised that gap junctions are involved in the synchronised activities of KNDy neurones, which is induced by NKB-NK3R signalling. We determined the role of NKB-NK3R signalling in Ca 2+ oscillation (an indicator of neuronal activities) of KNDy neurones and its synchronisation mechanism among KNDy neurones. Senktide, a selective agonist for NK3R, increased the frequency of Ca 2+ oscillations in cultured Kiss1-GFP cells collected from the mediobasal hypothalamus of the foetal Kiss1-green fluorescent protein (GFP) mice. The senktide-induced Ca 2+ oscillations were synchronised in the Kiss1-GFP and neighbouring glial cells. Confocal microscopy analysis of these cells, which have shown synchronised Ca 2+ oscillations, revealed close contacts between Kiss1-GFP cells, as well as between Kiss1-GFP cells and glial cells. Dye coupling experiments suggest cell-to-cell communication through gap junctions between Kiss1-GFP cells and neighbouring glial cells. Connexin-26 and -37 mRNA were found in isolated ARC Kiss1 cells taken from adult female Kiss1-GFP transgenic mice. Furthermore, 18β-glycyrrhetinic acids and mefloquine, which are gap junction inhibitors, attenuated senktide-induced Ca 2+ oscillations in Kiss1-GFP cells. Taken together, these results suggest that NKB-NK3R signalling enhances synchronised activities among neighbouring KNDy neurones, and that both neurone-neurone and neurone-glia communications via gap junctions possibly contribute to synchronised activities among KNDy neurones., (© 2017 British Society for Neuroendocrinology.)

Pulsatile secretion of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) decreases during aging. Kisspeptin (encoded by Kiss1) neurons in the arcuate nucleus coexpress neurokinin B (Tac3) and dynorphin (Pdyn) and are critical for regulating the GnRH/LH pulse. We therefore examined kisspeptin neurons by histochemistry and pulsatile LH release in rats aged 2-3 (Young), 12-13 (Young-Middle), 19-22 (Late-Middle), and 24-26 (Old) months. Total LH concentrations, sampled for 3 hours, decreased in both sexes with aging. In females, numbers of Tac3 and Pdyn neurons were significantly reduced in all aging rats, and numbers of Kiss1 neurons were significantly reduced in Late-Middle and Old rats. In males, numbers of all 3 neuron-types were significantly decreased in all aging rats. GnRH agonist induced LH release in all animals; however, the increased LH concentration in all aging rats was less than that in Young rats. These results suggest that expression of each gene in kisspeptin neurons may be controlled individually during aging, and that reduction of their expression or change in pituitary responsiveness may cause attenuated pulsatile LH secretion., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Endogenous dynorphin signaling via the kappa-opioid receptor (KOR) in the nucleus accumbens (NAcc) powerfully mediates negative affective states and stress reactivity. Excitatory inputs from the hippocampus and amygdala play a fundamental role in shaping the activity of both NAcc D1 and D2 MSNs, which encode positive and negative motivational valences, respectively. However, a circuit-based mechanism by which KOR modulation of excitation-inhibition balance modifies D1 and D2 MSN activity is lacking. Here, we provide a comprehensive synaptic framework wherein presynaptic KOR inhibition decreases the excitatory drive of D1 MSN activity by the amygdala, but not the hippocampus. Conversely, presynaptic inhibition by KORs of inhibitory synapses on D2 MSNs enhances integration of excitatory drive by the amygdala and hippocampus. In conclusion, we describe a circuit-based mechanism showing differential gating of afferent control of D1 and D2 MSN activity by KORs in a pathway-specific manner., (Published by Elsevier Inc.)

Puberty onset involves increased gonadotrophin-release (GnRH) release as a result of decreased sensitivity to oestrogen (E 2 )-negative feedback. Because GnRH neurones lack E 2 receptor α, this pathway must contain interneurones. One likely candidate is KNDy neurones (kisspeptin, neurokinin B, dynorphin). The overarching hypothesis of the present study was that the prepubertal hiatus in luteinising hormone (LH) release involves reduced kisspeptin and/or heightened dynorphin input. We first tested the specific hypothesis that E 2 would reduce kisspeptin-immunopositive cell numbers and increase dynorphin-immunopositive cell numbers. We found that kisspeptin cell numbers were higher in ovariectomised (OVX) lambs than OVX lambs treated with E 2 (OVX+ E 2 ) or those left ovary-intact. Very few arcuate dynorphin cells were identified in any group. Next, we hypothesised that central blockade of κ-opioid receptor (KOR) would increase LH secretion at a prepubertal (6 months) but not postpubertal (10 months) age. Luteinising hormone pulse frequency and mean LH increased during infusion of a KOR antagonist, norbinaltorphimine, in OVX + E 2 lambs at the prepubertal age but not in the same lambs at the postpubertal age. We next hypothesised that E 2 would increase KOR expression in GnRH neurones or alter synaptic input to KNDy neurones in prepubertal ewes. Oestrogen treatment decreased the percentage of GnRH neurones coexpressing KOR (approximately 68%) compared to OVX alone (approximately 78%). No significant differences in synaptic contacts per cell between OVX and OVX + E 2 groups were observed. Although these initial data are consistent with dynorphin inhibiting pulsatile LH release prepubertally, additional work will be necessary to define the source and mechanisms of this inhibition., (© 2016 British Society for Neuroendocrinology.)

Kappa-opioid receptor (KOR) antagonists are currently being considered for the treatment of a variety of neuropsychiatric conditions, including depressive, anxiety, and substance abuse disorders. A general ability to mitigate the effects of stress, which can trigger or exacerbate these conditions, may explain their putative efficacy across such a broad array of conditions. The discovery of their potentially therapeutic effects evolved from preclinical research designed to characterize the molecular mechanisms by which experience causes neuroadaptations in the nucleus accumbens (NAc), a key element of brain reward circuitry. This research established that exposure to drugs of abuse or stress increases the activity of the transcription factor CREB (cAMP response element binding protein) in the NAc, which leads to elevated expression of the opioid peptide dynorphin that in turn causes core signs of depressive- and anxiety-related disorders. Disruption of KORs-the endogenous receptors for dynorphin-produces antidepressant- and anxiolytic-like actions in screening procedures that identify standard drugs of these classes, and reduces stress effects in tests used to study addiction and stress-related disorders. Although interest in this target is high, prototypical KOR antagonists have extraordinarily persistent pharmacodynamic effects that complicate clinical trials. The development of shorter acting KOR antagonists together with more rapid designs for clinical trials may soon provide insight on whether these drugs are efficacious as would be predicted by preclinical work. If successful, KOR antagonists would represent a unique example in psychiatry where the therapeutic mechanism of a drug class is understood before it is shown to be efficacious in humans., (© 2016 Wiley Periodicals, Inc.)

Spinocerebellar ataxia type 23 (SCA23) is caused by missense mutations in prodynorphin, encoding the precursor protein for the opioid neuropeptides α-neoendorphin, Dynorphin (Dyn) A and Dyn B, leading to neurotoxic elevated mutant Dyn A levels. Dyn A acts on opioid receptors to reduce pain in the spinal cord, but its cerebellar function remains largely unknown. Increased concentration of or prolonged exposure to Dyn A is neurotoxic and these deleterious effects are very likely caused by an N-methyl-d-aspartate-mediated non-opioid mechanism as Dyn A peptides were shown to bind NMDA receptors and potentiate their glutamate-evoked currents. In the present study, we investigated the cellular mechanisms underlying SCA23-mutant Dyn A neurotoxicity. We show that SCA23 mutations in the Dyn A-coding region disrupted peptide secondary structure leading to a loss of the N-terminal α-helix associated with decreased κ-opioid receptor affinity. Additionally, the altered secondary structure led to increased peptide stability of R6W and R9C Dyn A, as these peptides showed marked degradation resistance, which coincided with decreased peptide solubility. Notably, L5S Dyn A displayed increased degradation and no aggregation. R6W and wt Dyn A peptides were most toxic to primary cerebellar neurons. For R6W Dyn A, this is likely because of a switch from opioid to NMDA- receptor signalling, while for wt Dyn A, this switch was not observed. We propose that the pathology of SCA23 results from converging mechanisms of loss of opioid-mediated neuroprotection and NMDA-mediated excitotoxicity., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Food intake and physical activity are regulated by multiple neuropeptides, including orexin and dynorphin (DYN). Orexin-A (OXA) is one of two orexin peptides with robust roles in regulation of food intake and spontaneous physical activity (SPA). DYN collectively refers to several peptides, some of which act through opioid receptors (opioid DYN) and some whose biological effects are not mediated by opioid receptors (non-opioid DYN). While opioid DYN is known to increase food intake, the effects of non-opioid DYN peptides on food intake and SPA are unknown. Neurons that co-express and release OXA and DYN are located within the lateral hypothalamus. Limited evidence suggests that OXA and opioid DYN peptides can interact to modulate some aspects of behaviors classically related to orexin peptide function. The paraventricular hypothalamic nucleus (PVN) is a brain area where OXA and DYN peptides might interact to modulate food intake and SPA. We demonstrate that injection of des-Tyr-dynorphin (DYN-A(2-17), a non opioid DYN peptide) into the PVN increases food intake and SPA in adult mice. Co-injection of DYN-A(2-17) and OXA in the PVN further increases food intake compared to DYN-A(2-17) or OXA alone. This is the first report describing the effects of non-opioid DYN-A(2-17) on food intake and SPA, and suggests that DYN-A(2-17) interacts with OXA in the PVN to modulate food intake. Our data suggest a novel function for non-opioid DYN-A(2-17) on food intake, supporting the concept that some behavioral effects of the orexin neurons result from combined actions of the orexin and DYN peptides., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Opioids are traditionally associated with pain, analgesia and drug abuse. It is now clear, however, that the opioids are central players in mood. The implications for mood disorders, particularly clinical depression, suggest a paradigm shift from the monoamine neurotransmitters to the opioids either alone or in interaction with monoamine neurons. We have a special interest in dynorphin, the last of the major endogenous opioids to be isolated and identified. Dynorphin is derived from the Greek word for power, dynamis, which hints at the expectation that the neuropeptide held for its discoverers. Yet, dynorphin and its opioid receptor subtype, kappa, has always taken a backseat to the endogenous b-endorphin and the exogenous morphine that both bind the mu opioid receptor subtype. That may be changing as the dynorphin/ kappa system has been shown to have different, often opposite, neurophysiological and behavioral influences. This includes major depressive disorder (MDD). Here, we have undertaken a review of dynorphin/ kappa neurobiology as related to behaviors, especially MDD. Highlights include the unique features of dynorphin and kappa receptors and the special relation of a plant-based agonist of the kappa receptor salvinorin A. In addition to acting as a kappa opioid agonist, we conclude that salvinorin A has a complex pharmacologic profile, with potential additional mechanisms of action. Its unique neurophysiological effects make Salvinorina A an ideal candidate for MDD treatment research.

While the DSM 5 has formalized the terminology "Alcohol Use Disorders" (AUD) or "disorders of the use of alcohol" (UAW French translation in progress), the term "alcohol dependence" still used in ICD-10, apriority in the future ICD-11 and above in clinical practice. Addiction to alcohol is the cause of mortality and major morbidity. In terms of therapeutic strategies for its management, alongside the maintenance of abstinence after withdrawal (with a high rate of relapse), the reduction of alcohol consumption below certain thresholds of intake is emerging in order to reduce risk, improve health and regain control of consumption even be an intermediate step towards abstinence. The role of the endogenous opioid system in the modulation of the activity of dopaminergic neurons from the circuit of reward and motivation is well established. An unsteadiness of this system has been described in the alcohol dependence. Indeed, a hypofunction of the endorphin pathway and its mu receptor and a hyperactivity of the dynorphin pathway and its kappa receptor participate in the alcohol reinforcing effects (especially positive and negative). The development of active molecules in this system allows better management of alcohol dependence. Besides naltrexone (mu antagonist) allowed in the maintenance of abstinence after withdrawal, another molecule (nalmefene) with modulating properties of μ and κ opioid receptors is the first drug having obtained an MA in reducing consumption in adult patients with alcohol dependence. Its modulating original pharmacological properties by targeting both the positive but also the negative reinforcing effects of alcohol, are responsible for its development in reducing consumption in the alcohol dependence., (Copyright © 2014 L’Encéphale, Paris. Published by Elsevier Masson SAS. All rights reserved.)

Nicotine (NIC) regulates various cellular functions acting on the nicotinic acetylcholine receptor (nAChR). And nAChR consists of ligand-gated cation channels with pentameric structure and composed of α and β subunits. In the central nervous system, α 4 β 2 and α 7 nAChRs are the most abundantly expressed as nAChR subtypes. There are several lines of evidence indicating that systemic administration of NIC elicits the release of endogenous opioids, such as, endorphins, enkephalins and dynorphins, in the brain. NIC exerts numerous acute effects, for example, antinociceptive effects and the activating effects of the hypothalamic-pituitary-adrenal (HPA) axis. In these effects, NIC-induced antinociception, but not HPA axis activation, was inhibited by opioid receptor antagonist, naloxone (NLX), and was also suppressed in morphine tolerated mice, indicating the participation of the endogenous opioid system in NIC-induced antinociception, but not HPA axis activation. Moreover, NIC-induced antinociception was antagonized by both α 4 β 2 and α 7 nAChR antagonists, while NIC-induced HPA axis activation was antagonized by α 4 β 2 nAChR antagonist, but not by α 7 nAChR antagonist. These results suggest that the endogenous opioid system may not be located on the downstream of α 4 β 2 nAChR. On the other hand, NIC has substantial physical dependence liability. NLX elicits NIC withdrawal after repeated NIC administration evaluated by corticosterone increase as a withdrawal sign, and NLX-precipitated NIC withdrawal is inhibited by concomitant administration of other opioid receptor antagonist, naltrexone, indicating the participation of endogenous opioid system in the development of physical dependence on NIC. NLX-precipitated NIC withdrawal was also inhibited by concomitant administration of an α 7 nAChR antagonist, but not an α 4 β 2 nAChR antagonist. Taken together, these findings suggest that the endogenous opioid system may be located on the downstream of α 7 nAChR and participates in the development of physical dependence on NIC.

Background: The discovery of kisspeptin as key central regulator of GnRH secretion has led to a new level of understanding of the neuroendocrine regulation of human reproduction. The related discovery of the kisspeptin-neurokinin B-dynorphin (KNDy) pathway in the last decade has further strengthened our understanding of the modulation of GnRH secretion by endocrine, metabolic and environmental inputs. In this review, we summarize current understanding of the physiological roles of these novel neuropeptides, and discuss the clinical relevance of these discoveries and their potential translational applications., Methods: A systematic literature search was performed using PUBMED for all English language articles up to January 2014. In addition, the reference lists of all relevant original research articles and reviews were examined. This review focuses mainly on published human studies but also draws on relevant animal data., Results: Kisspeptin is a principal regulator of the secretion of gonadotrophins, and through this key role it is critical for the onset of puberty, the regulation of sex steroid-mediated feedback and the control of adult fertility. Although there is some sexual dimorphism, both neuroanatomically and functionally, these functions are apparent in both men and women. Kisspeptin acts upstream of GnRH and, following paracrine stimulatory and inhibitory inputs from neurokinin B and dynorphin (KNDy neuropeptides), signals directly to GnRH neurones to control pulsatile GnRH release. When administered to humans in different isoforms, routes and doses, kisspeptin robustly stimulates LH secretion and LH pulse frequency. Manipulation of the KNDy system is currently the focus of translational research with the possibility of future clinical application to regulate LH pulsatility, increasing gonadal sex steroid secretion in reproductive disorders characterized by decreased LH pulsatility, including hypothalamic amenorrhoea and hypogonadotropic hypogonadism. Conversely there may be scope to reduce the activity of the KNDy system to reduce LH secretion where hypersecretion of LH adds to the phenotype, such as in polycystic ovary syndrome., Conclusions: Kisspeptin is a recently discovered neuromodulator that controls GnRH secretion mediating endocrine and metabolic inputs to the regulation of human reproduction. Manipulation of kisspeptin signalling has the potential for novel therapies in patients with pathologically low or high LH pulsatility., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.)

Menthol and other counterstimuli relieve itch, resulting in an antipruritic state that persists for minutes to hours. However, the neural basis for this effect is unclear, and the underlying neuromodulatory mechanisms are unknown. Previous studies revealed that Bhlhb5(-/-) mice, which lack a specific population of spinal inhibitory interneurons (B5-I neurons), develop pathological itch. Here we characterize B5-I neurons and show that they belong to a neurochemically distinct subset. We provide cause-and-effect evidence that B5-I neurons inhibit itch and show that dynorphin, which is released from B5-I neurons, is a key neuromodulator of pruritus. Finally, we show that B5-I neurons are innervated by menthol-, capsaicin-, and mustard oil-responsive sensory neurons and are required for the inhibition of itch by menthol. These findings provide a cellular basis for the inhibition of itch by chemical counterstimuli and suggest that kappa opioids may be a broadly effective therapy for pathological itch., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Neuropeptides play an important role in modulating seizures and epilepsy. Unlike neurotransmitters which operate on a millisecond time-scale, neuropeptides have longer half lives; this leads to modulation of neuronal and network activity over prolonged periods, so contributing to setting the seizure threshold. Most neuropeptides are stored in large dense vesicles and co-localize with inhibitory interneurons. They are released upon high frequency stimulation making them attractive targets for modulation of seizures, during which high frequency discharges occur. Numerous neuropeptides have been implicated in epilepsy; one, ACTH, is already used in clinical practice to suppress seizures. Here, we concentrate on neuropeptides that have a direct effect on seizures, and for which therapeutic interventions are being developed. We have thus reviewed the abundant reports that support a role for neuropeptide Y (NPY), galanin, ghrelin, somatostatin and dynorphin in suppressing seizures and epileptogenesis, and for tachykinins having pro-epileptic effects. Most in vitro and in vivo studies are performed in hippocampal tissue in which receptor expression is usually high, making translation to other brain areas less clear. We highlight recent therapeutic strategies to treat epilepsy with neuropeptides, which are based on viral vector technology, and outline how such interventions need to be refined in order to address human disease., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Although Berberine (BER) is popular in treating gastrointestinal (GI) disorders, its mechanisms are not clear yet. In order to investigate the effects and possible mechanism of BER on GI motility in rodents, we first explored GI motility by recording the myoelectrical activity of jejunum and colon in rats, and upper GI transit with a charcoal marker in mice. Then, the plasma levels of gastrin, motilin, somatostatin and glucagon-like-peptide-1 (Glp-1) were measured by ELISA or radioimmunoassay (RIA). Furthermore, endogenous opioid-peptides (β-endorphin, dynorphin-A, met-enkephalin) were detected by RIA after treatment with BER. Our results showed that BER concentration-dependently inhibited myoelectrical activity and GI transit, which can be antagonized by opioid-receptor antagonists to different extents. The elevated somatostatin and Glp-1, and decreased gastrin and motilin in plasma, which were caused by BER application, also could be antagonized by the opioid-receptor antagonists. Additionally, plasma level of β-endorphin, but not dynorphin-A and met-enkephalin, was increased by applying BER. Taken together, these studies show that BER plays inhibiting roles on GI motility and up-regulating roles on somatostatin, Glp-1 and down-regulating roles on gastrin, motilin. The pharmacological mechanisms of BER on GI motility and plasma levels of GI hormones were discovered to be closely related to endogenous opioid system., (Copyright © 2013 John Wiley & Sons, Ltd.)

Despite affecting millions of individuals, the etiology of hot flushes remains unknown. Here we review the physiology of hot flushes, CNS pathways regulating heat-dissipation effectors, and effects of estrogen on thermoregulation in animal models. Based on the marked changes in hypothalamic kisspeptin, neurokinin B and dynorphin (KNDy) neurons in postmenopausal women, we hypothesize that KNDy neurons play a role in the mechanism of flushes. In the rat, KNDy neurons project to preoptic thermoregulatory areas that express the neurokinin 3 receptor (NK3R), the primary receptor for NKB. Furthermore, activation of NK₃R in the median preoptic nucleus, part of the heat-defense pathway, reduces body temperature. Finally, ablation of KNDy neurons reduces cutaneous vasodilatation and partially blocks the effects of estrogen on thermoregulation. These data suggest that arcuate KNDy neurons relay estrogen signals to preoptic structures regulating heat-dissipation effectors, supporting the hypothesis that KNDy neurons participate in the generation of flushes., (Published by Elsevier Inc.)

The brain stress-response system is critically involved in the addiction process, stimulating drug consumption and the relapse to drug taking in abstinent addicts. At the same time, its functioning is affected by chronic drug exposure. Here, we have investigated the role of the endogenous opioid peptide dynorphin as a modulator of effects of long-term ethanol consumption on the brain stress-response system. Using the two-bottle choice paradigm, we demonstrate an enhanced ethanol preference in male dynorphin knockout mice. Exposure to mild foot shock increased ethanol consumption in wild-type control littermates, but not in dynorphin-deficient animals. Blood adrenocorticotropic hormone levels determined 5 minutes after the shock were not affected by the genotype. We also determined the neuronal reactivity after foot shock exposure using c-Fos immunoreactivity in limbic structures. This was strongly influenced by both genotype and chronic ethanol consumption. Long-term alcohol exposure elevated the foot shock-induced c-Fos expression in the basolateral amygdala in wild-type animals, but had the opposite effect in dynorphin-deficient mice. An altered c-Fos reactivity was also found in the periventricular nucleus, the thalamus and the hippocampus of dynorphin knockouts. Together these data suggest that dynorphin plays an important role in the modulation of the brain stress-response systems after chronic ethanol exposure., (© 2012 The Authors, Addiction Biology © 2012 Society for the Study of Addiction.)

Psychostimulants such as amphetamine and cocaine are believed to produce dependence by causing rapid, supraphysiological elevations in synaptic dopamine (DA) within the nucleus accumbens (NAc) (Volkow et al. 2009, Neuropharmacology 56: 3-8). These changes in forebrain DA transmission are similar to those evoked by natural reinforcers (Louilot et al. 1991, Brain Res 553: 313-317; Roitman et al. 2004, J Neurosci 24: 1265-1271), but are of greater magnitude and longer duration. Repeated drug exposure causes compensatory neuroadaptations in neurons of the NAc, some of which may modulate excess DA in a homeostatic fashion. One such adaptation is the activation of the transcription factor CREB (cAMP response element-binding protein) within neurons of the NAc. Although elevated levels of transcriptionally active CREB appear to attenuate DA transmission by increasing expression of the endogenous κ opioid receptor (KOR) ligand dynorphin, increased dynorphin transmission may ultimately have undesirable effects that contribute to drug withdrawal states as well as comorbid psychiatric illnesses such as depression. This state may prompt a return to drug use to mitigate the adverse effects of withdrawal. This article summarizes our current understanding of how CREB and dynorphin contribute to the dysregulation of motivation and describes novel therapeutic strategies that derive from preclinical research in this area.

Activation of the dynorphin/κ-opioid receptor (KOR) system by repeated stress exposure or agonist treatment produces place aversion, social avoidance, and reinstatement of extinguished cocaine place preference behaviors by stimulation of p38α MAPK, which subsequently causes the translocation of the serotonin transporter (SERT, SLC6A4) to the synaptic terminals of serotonergic neurons. In the present study we extend those findings by showing that stress-induced potentiation of cocaine conditioned place preference occurred by a similar mechanism. In addition, SERT knock-out mice did not show KOR-mediated aversion, and selective reexpression of SERT by lentiviral injection into the dorsal raphe restored the prodepressive effects of KOR activation. Kinetic analysis of several neurotransporters demonstrated that repeated swim stress exposure selectively increased the V(max) but not K(m) of SERT without affecting dopamine transport or the high-capacity, low-affinity transporters. Although the serotonergic neurons in the dorsal raphe project throughout the forebrain, a significant stress-induced increase in cell-surface SERT expression was only evident in the ventral striatum, and not in the dorsal striatum, hippocampus, prefrontal cortex, amygdala, or dorsal raphe. Stereotaxic microinjections of the long-lasting KOR antagonist norbinaltorphimine demonstrated that local KOR activation in the nucleus accumbens, but not dorsal raphe, mediated this stress-induced increase in ventral striatal surface SERT expression. Together, these results support the hypothesis that stress-induced activation of the dynorphin/KOR system produces a transient increase in serotonin transport locally in the ventral striatum that may underlie some of the adverse consequences of stress exposure, including the potentiation of the rewarding effects of cocaine.

Chronic opiate usage, whether prescribed or illicit, has been associated with changes in bone mass and is a recognized risk factor for the development of osteoporosis; however, the mechanism behind this effect is unknown. Here we show that lack of dynorphin, an endogenous opioid, in mice (Dyn-/-), resulted in a significantly elevated cancellous bone volume associated with greater mineral apposition rate and increased resorption indices. A similar anabolic phenotype was evident in bone of mice lacking dynorphin's cognate receptor, the kappa opioid receptor. Lack of opioid receptor expression in primary osteoblastic cultures and no change in bone cell function after dynorphin agonist treatment in vitro indicates an indirect mode of action. Consistent with a hypothalamic action, central dynorphin signaling induces extracellular signal-regulated kinase (ERK) phosphorylation and c-fos activation of neurons in the arcuate nucleus of the hypothalamus (Arc). Importantly, this signaling also leads to an increase in Arc NPY mRNA expression, a change known to decrease bone formation. Further implicating NPY in the skeletal effects of dynorphin, Dyn-/-/NPY-/- double mutant mice showed comparable increases in bone formation to single mutant mice, suggesting that dynorphin acts upstream of NPY signaling to control bone formation. Thus the dynorphin system, acting via NPY, may represent a pathway by which higher processes including stress, reward/addiction and depression influence skeletal metabolism. Moreover, understanding of these unique interactions may enable modulation of the adverse effects of exogenous opioid treatment without directly affecting analgesic responses., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

Addictions to cocaine or heroin/prescription opioids [short-acting μ-opioid receptor (MOPr) agonists] involve relapsing cycles, with experimentation/escalating use, withdrawal/abstinence, and relapse/re-escalation. κ-Opioid receptors (KOPr; encoded by OPRK1), and their endogenous agonists, the dynorphins (encoded by PDYN), have counter-modulatory effects on reward caused by cocaine or MOPr agonist exposure, and exhibit plasticity in addictive-like states. KOPr/dynorphin activation is implicated in depression/anxiety, often comorbid with addictions. In this opinion article we propose that particular stages of the addiction cycle are differentially affected by KOPr/dynorphin systems. Vulnerability and resilience can be due to pre-existing (e.g., genetic) factors, or epigenetic modifications of the OPRK1 or PDYN genes during the addiction cycle. Pharmacotherapeutic approaches limiting changes in KOPr/dynorphin tone, especially with KOPr partial agonists, may hold potential for the treatment of specific drug addictions and psychiatric comorbidity., (Copyright © 2012. Published by Elsevier Ltd.)

Background: The striato-nigral projecting pathway contains the highest concentrations of dynorphin in the brain. The functional role of this opioid peptide in the regulation of mesencephalic dopaminergic (DAergic) neurons is not clear. We reported previously that exogenous dynorphin exerts potent neuroprotective effects against inflammation-induced dopaminergic neurodegeneration in vitro. The present study was performed to investigate whether endogenous dynorphin has neuroprotective roles in vivo., Methods: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine (MA), two commonly used neurotoxins in rodent models of Parkinson's disease, were administered to wild-type (Dyn⁺/⁺) and prodynorphin-deficient mice (Dyn⁻/⁻). We examined dopaminergic neurotoxicity by using an automated video tracking system, HPLC, immunocytochemistry, and reverse transcription and polymerase chain reaction (RT-PCR)., Results: Treatment with MPTP resulted in behavioral impairments in both strains. However, these impairments were more pronounced in Dyn-l- than in Dyn⁺/⁺. Dyn⁻/⁻ showed more severe MPTP-induced dopaminergic neuronal loss in the substantia nigra and striatum than Dyn⁺/⁺. Similarly, the levels of dopamine and its metabolites in the striatum were depleted to a greater extent in Dyn⁻/⁻ than in Dyn⁺/⁺. Additional mechanistic studies revealed that MPTP treatment caused a higher degree of microglial activation and M1 phenotype differentiation in Dyn⁻/⁻ than in Dyn⁺/⁺. Consistent with these observations, prodynorphin deficiency also exacerbated neurotoxic effects induced by MA, although this effect was less pronounced than that of MPTP., Conclusions: The in vivo results presented here extend our previous in vitro findings and further indicate that endogenous dynorphin plays a critical role in protecting dopaminergic neurons through its anti-inflammatory effects.

This review represents the focus of a symposium that was presented at the "Alcoholism and Stress: A Framework for Future Treatment Strategies" conference in Volterra, Italy on May 3-6, 2011 and organized/chaired by Dr. Brendan M. Walker. The primary goal of the symposium was to evaluate and disseminate contemporary findings regarding the emerging role of kappa-opioid receptors (KORs) and their endogenous ligands dynorphins (DYNs) in the regulation of escalated alcohol consumption, negative affect and cognitive dysfunction associated with alcohol dependence, as well as DYN/KOR mediation of the effects of chronic stress on alcohol reward and seeking behaviors. Dr. Glenn Valdez described a role for KORs in the anxiogenic effects of alcohol withdrawal. Dr. Jay McLaughlin focused on the role of KORs in repeated stress-induced potentiation of alcohol reward and increased alcohol consumption. Dr. Brendan Walker presented data characterizing the effects of KOR antagonism within the extended amygdala on withdrawal-induced escalation of alcohol self-administration in dependent animals. Dr. Georgy Bakalkin concluded with data indicative of altered DYNs and KORs in the prefrontal cortex of alcohol dependent humans that could underlie diminished cognitive performance. Collectively, the data presented within this symposium identified the multifaceted contribution of KORs to the characteristics of acute and chronic alcohol-induced behavioral dysregulation and provided a foundation for the development of pharmacotherapeutic strategies to treat certain aspects of alcohol use disorders., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The role of kappa-opioid receptors (KOR) in the regulation of alcohol-related behaviors is not completely understood. For example, alcohol consumption has been reported to increase following treatment with KOR antagonists in rats, but was decreased in mice with genetic deletion of KOR. Recent studies have further suggested that KOR antagonists may selectively decrease alcohol self-administration in rats following a history of dependence. We assessed the effects of the KOR antagonist JDTic on alcohol self-administration, reinstatement of alcohol seeking induced by alcohol-associated cues or stress, and acute alcohol withdrawal-induced anxiety ('hangover anxiety'). JDTic dose-dependently reversed hangover anxiety when given 48 hours prior to testing, a time interval corresponding to the previously demonstrated anxiolytic efficacy of this drug. In contrast, JDTic decreased alcohol self-administration and cue-induced reinstatement of alcohol seeking when administered 2 hours prior to testing, but not at longer pre-treatment times. For comparison, we determined that the prototypical KOR antagonist nor-binaltorphimine can suppress self-administration of alcohol at 2 hours pre-treatment time, mimicking our observations with JDTic. The effects of JDTic were behaviorally specific, as it had no effect on stress-induced reinstatement of alcohol seeking, self-administration of sucrose, or locomotor activity. Further, we demonstrate that at a 2 hours pre-treatment time JDTic antagonized the antinociceptive effects of the KOR agonist U50,488H but had no effect on morphine-induced behaviors. Our results provide additional evidence for the involvement of KOR in regulation of alcohol-related behaviors and provide support for KOR antagonists, including JDTic, to be evaluated as medications for alcoholism., (Published 2012. This article is a U.S. Government work and is in the public domain in the USA.)

Apelin receptor (APJ) and kappa opioid receptor (KOR) are members of the family A of G protein-coupled receptors (GPCRs). These two receptors are involved in the central nervous system regulation of the cardiovascular system. Here, we explore the possibility of heterodimerization between APJ and KOR and investigate their novel signal transduction characteristics. Co-immunoprecipitation (Co-IP), co-localization and bioluminescence resonance energy transfer (BRET) assays confirmed the heterodimerization of APJ and KOR. In APJ and KOR stably transfected HEK293 cells, treatment with APJ ligand apelin-13 or KOR ligand dynorphinA (1-13) resulted in higher phosphorylation levels of extracellular-regulated kinases 1/2 (ERK1/2) compared to HEK293 cells transfected with either APJ or KOR alone. The siRNA knockdown of either APJ or KOR receptor in human umbilical vein endothelial cells (HUVECs) resulted in significant reduction of the apelin-13 induced ERK activation. Additionally both forskolin (FSK)-induced cAMP levels and cAMP response element reporter activities were significantly reduced, whereas the serum response element luciferase (SRE-luc) reporter activity was significantly upregulated. Moreover, the ERK phosphorylation and SRE-luc activity were abrogated by the protein kinase C (PKC) inhibitor. These results demonstrate for the first time that human APJ forms a heterodimer with KOR and leads to increased PKC and decreased protein kinase A activity leading to a significant increase in cell proliferation, which may translate to the regulation of diverse biological actions and offers the potential for the development of more selective and tissue specific drug therapies., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The dynorphin/κ-opioid receptor system has been implicated in the pathogenesis and pathophysiology of several psychiatric disorders. In the present review, we present evidence indicating a key role for this system in modulating neurotransmission in brain circuits that subserve mood, motivation, and cognitive function. We overview the pharmacology, signaling, post-translational, post-transcriptional, transcriptional, epigenetic and cis regulation of the dynorphin/κ-opioid receptor system, and critically review functional neuroanatomical, neurochemical, and pharmacological evidence, suggesting that alterations in this system may contribute to affective disorders, drug addiction, and schizophrenia. We also overview the dynorphin/κ-opioid receptor system in the genetics of psychiatric disorders and discuss implications of the reviewed material for therapeutics development.

Background/aims: Multiple opioid receptor (OR) types and endogenous opioid peptides exist in the spinal dorsal horn and there may be interactions among these receptor types that involve opioid peptides. In a previous study we observed that antinociceptive effects of the selective κ-opioid receptor (κOR) agonist, U50,488H, was attenuated in μ-opioid receptor (μOR) knockout mice as compared to wild-type mice when administered spinally. This suggests that an interaction between κORs and μORs exits in the spinal cord. The present study was aimed at investigating whether endogenous opioid peptides were involved in such interaction., Methods: We examined whether the presence of antibodies to endogenous opioid peptides, endomorphin-2, met-enkephalin and dynorphin A affected the antinociceptive effects of spinal U50,488H in rats. The tail-flick test was used to assess pain thresholds., Results: The increase in tail-flick latency after spinal U50,488H was attenuated when the rats were pretreated intrathecally with antiserum against endomorphin-2. Pretreatments with antisera against met-enkephalin and dynorphin A had no effect on U50,488H antinociception. The antisera alone did not affect pain threshold., Conclusion: The results suggest that endomorphin-2, an endogenous opioid peptide highly selective to the μOR, plays a role in antinociception induced by κOR activation in the spinal cord., (Copyright © 2012 S. Karger AG, Basel.)

Neurokinin B (NKB) and its receptor (neurokinin-3 receptor) are coexpressed with kisspeptin and dynorphin A (Dyn) within neurons of the hypothalamic arcuate nucleus, the suggested site of the GnRH pulse generator. It is thought that these neuropeptides interact to regulate gonadotropin secretion. Using the ovariectomized (OVX) and OVX 17β-estradiol-replaced rat models, we have carried out a series of in vivo neuropharmacological and electrophysiological experiments to elucidate the hierarchy between the kisspeptin, NKB, and Dyn signaling systems. Rats were implanted with intracerebroventricular cannulae and cardiac catheters for frequent (every 5 min) automated serial blood sampling. Freely moving rats were bled for 6 h, with intracerebroventricular injections taking place after a 2-h control bleeding period. A further group of OVX rats was implanted with intra-arcuate electrodes for the recording of multiunit activity volleys, which coincide invariably with LH pulses. Intracerebroventricular administration of the selective neurokinin-3 receptor agonist, senktide (100-600 pmol), caused a dose-dependent suppression of LH pulses and multiunit activity volleys. The effects of senktide did not differ between OVX and 17β-estradiol-replaced OVX animals. Pretreatment with a selective Dyn receptor (κ opioid receptor) antagonist, norbinaltorphimine (6.8 nmol), blocked the senktide-induced inhibition of pulsatile LH secretion. Intracerebroventricular injection of senktide did not affect the rise in LH concentrations after administration of kisspeptin (1 nmol), and neither did kisspeptin preclude the senktide-induced suppression of LH pulses. These data show that NKB suppresses the frequency of the GnRH pulse generator in a Dyn/κ opioid receptor-dependent fashion.

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.

Background: The kappa opioid receptor (KOR) system contributes to the prodepressive and aversive consequences of stress and is implicated in the facilitation of conditioned fear and anxiety in rodents. Here, we sought to identify neural circuits that mediate KOR system effects on fear and anxiety in rats., Methods: We assessed whether fear conditioning induces plasticity in KOR or dynorphin (the endogenous KOR ligand) messenger RNA (mRNA) expression in the basolateral (BLA) and central (CeA) nuclei of the amygdala, hippocampus, or striatum. We then assessed whether microinfusions of the KOR antagonist JDTic (0-10 μg/side) into the BLA or CeA affect the expression of conditioned fear or anxiety. Finally, we examined whether fear extinction induces plasticity in KOR mRNA expression that relates to the quality of fear extinction., Results: Fear conditioning upregulated KOR mRNA in the BLA by 65% and downregulated it in the striatum by 22%, without affecting KOR levels in the CeA or hippocampus, or dynorphin levels in any region. KOR antagonism in either the BLA or CeA decreased conditioned fear in the fear-potentiated startle paradigm, whereas KOR antagonism in the BLA, but not the CeA, produced anxiolytic-like effects in the elevated plus maze. Effective fear extinction was associated with a 67% reduction in KOR mRNA in the BLA., Conclusions: These findings suggest that fear conditioning and extinction dynamically regulate KOR expression in the BLA and provide evidence that the BLA and CeA are important neural substrates mediating the anxiolytic-like effects of KOR antagonists in models of fear and anxiety., (Copyright © 2011 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Orexin (also known as hypocretin) neurons play a key role in regulating sleep-wake behavior, but the links between orexin neuron electrophysiology and function have not been explored. Orexin neurons are wake-active, and spiking activity in orexin neurons may anticipate transitions to wakefulness by several seconds. However, it is suggested that while the orexin system is necessary to maintain sustained wake bouts, orexin has little effect on brief wake bouts. In vitro experiments investigating the actions of orexin and dynorphin, a colocalized neuropeptide, on orexin neurons indicate that the dynamics of desensitization to dynorphin may represent a mechanism for modulating local network activity and resolving the apparent discrepancy between the onset of firing in orexin neurons and the onset of functional orexin effects. To investigate the role of dynorphin on orexin neuron activity, a Hodgkin-Huxley-type model orexin neuron was developed in which baseline electrophysiology, orexin/dynorphin action, and dynorphin desensitization were closely tied to experimental data. In this model framework, model orexin neuron responses to orexin/dynorphin action were calibrated by simulating the physiologic effects of static orexin and dynorphin bath application on orexin neurons. Then behavior in a small network of model orexin neurons was simulated with pure orexin, pure dynorphin, or combined orexin and dynorphin coupling based on the mechanisms established in the static case. It was found that the dynamics of desensitization to dynorphin can mediate a clear shift from a network in which firing is suppressed by dynorphin-mediated inhibition to a network of neurons with high firing rates sustained by orexin-mediated excitation. The findings suggest that dynamic interactions between orexin and dynorphin at transitions from sleep to wake may delay onset of functional orexin effects., (© 2011 Sage Publications)

Opioids inhibit release of primary afferent transmitters but it is unclear whether the converse occurs. To test the hypothesis that primary afferent transmitters influence opioid-ergic tone, we studied the functional and anatomical relationships between pituitary adenylyl cyclase-activating polypeptide (PACAP) and dynorphin 1-17 (Dyn) in spinal cord. We found that activation of the PACAP-specific receptor PAC(1) (PAC(1)R) inhibited, whereas PAC(1)R blockade augmented, spinal release of Dyn. It is noteworthy that in the formalin-induced pain model PAC(1)R blockade (via PACAP6-38) also resulted in antinociception that was abolished by spinal κ-opioid receptor blockade. These findings indicate that Dyn release is tonically inhibited by PACAP and that blocking this inhibition, which increases the spinal release of Dyn, results in antinociception. Consistent with this conclusion, we found in the spinal dorsal horn that Dyn-immunoreactive neurons 1) expressed PAC(1)R and 2) were apposed by PACAP terminals. Present results, in combination with the previous demonstration that the release of spinal Dyn is tonically inhibited by opioid- and nociceptin/orphanin FQ-coupled pathways (J Pharmacol Exp Ther 298:1213-1220, 2001), indicate that spinal Dyn-ergic neurons integrate multiple inhibitory inputs, the interruption of any one of which (i.e., disinhibition) is sufficient to enhance spinal Dyn release and generate antinociception. Gaining a better understanding of the role of primary afferent neurotransmitters in negatively modulating the spinal release of Dyn and the physiological use of disinhibition to increase spinal Dyn activity could suggest novel clinically useful approaches for harnessing endogenous Dyn for pain control.

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., (Copyright © 2011 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Stress is known to elicit pain relief, a phenomenon referred to as stress-induced analgesia. Based on stress parameters, opioid and non-opioid intrinsic pain inhibitory systems can be activated. In the present study, we assessed whether changing the duration of stress would affect the involvement of endogenous opioids in antinociception elicited by swim in warm water (32 °C), known to be opioid-mediated. Using mice lacking beta-endorphin, enkephalins or dynorphins and their respective wild-type littermates, we assessed the role of each opioid peptide in antinociception induced by a short (3 min) vs. long (15 min) swim. Mice were tested for baseline hot plate latency, exposed to swim (3 or 15 min) in warm water (32 °C) and then tested for antinociception at 5, 15 and 30 min. Our results revealed that both swim paradigms induced significant antinociception in wild-type mice. However, the short swim failed to induce antinociception in beta-endorphin-deficient mice, illustrating that beta-endorphin is important in this form of stress-induced antinociception. On the other hand, antinociception elicited by the long swim was only slightly reduced in beta-endorphin-deficient mice despite pretreatment with naloxone, a non-selective opioid receptor antagonist, significantly attenuated the antinociception elicited by the long swim. Nevertheless, a delayed hyperalgesic response developed in mice lacking beta-endorphin following exposure to either swim paradigm. On the other hand, mice lacking enkephalins or dynorphins and their respective wild-type littermates expressed a comparable antinociceptive response and did not exhibit the delayed hyperalgesic response. Together, our results suggest that the endogenous opioid peptide beta-endorphin not only mediates antinociception induced by the short swim but also prevents the delayed hyperalgesic response elicited by either swim paradigm., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Altered dynorphin opioid peptide systems contribute to increased ethanol self-administration during withdrawal following chronic alcohol exposure. We previously identified that the κ-opioid receptor antagonist nor-binaltorphimine (nor-BNI) selectively reduced ethanol self-administration in dependent animals. The purpose of this study was twofold: (1) determine whether peripherally administered nor-BNI could reduce dependence-induced ethanol self-administration and (2) confirm the selective κ-opioid effects of nor-BNI by administering it 24 hours prior to ethanol self-administration sessions occurring during acute withdrawal. Nor-BNI decreased ethanol self-administration in ethanol-dependent animals, with no effect in nondependent animals. Thus, the κ-opioid/dynorphin system is a viable pharmacotherapeutic target for the treatment of alcoholism.

Loss-of-function mutations in the genes encoding either neurokinin B (NKB) or its receptor, NK3 (NK3R), result in hypogonadotropic hypogonadism, characterized by an absence of pubertal development and low circulating levels of LH and gonadal steroids. These studies implicate NKB and NK3R as essential elements of the human reproductive axis. Studies over the last two decades provide evidence that a group of neurons in the hypothalamic infundibular/arcuate nucleus form an important component of this regulatory circuit. These neurons are steroid-responsive and coexpress NKB, kisspeptin, dynorphin, NK3R, and estrogen receptor α (ERα) in a variety of mammalian species. Compelling evidence in the human indicates these neurons function in the hypothalamic circuitry regulating estrogen negative feedback on gonadotropin-releasing hormone (GnRH) secretion. Moreover, in the rat, they form a bilateral, interconnected network that projects to NK3R-expressing GnRH terminals in the median eminence. This network provides an anatomical framework to explain how coordination among NKB/kisspeptin/dynorphin/NK3R/ERα neurons could mediate feedback information from the gonads to modulate pulsatile GnRH secretion. There is substantial (but indirect) evidence that this network may be part of the neural circuitry known as the "GnRH pulse generator," with NK3R signaling as an important component. This theory provides a compelling explanation for the occurrence of hypogonadotropic hypogonadism in patients with inactivating mutations in the TAC3 or TACR3 genes. Future studies will be needed to determine whether NKB signaling plays a permissive role in the onset of puberty or is part of the driving force initiating the maturation of reproductive function., (Published by Elsevier B.V.)