Endogenous Opioid Signaling in the Mouse Retina Modulates Pupillary Light Reflex.

Opioid peptides and their receptors are expressed in the mammalian retina; however, little is known about how they might affect visual processing. The melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs), which mediate important non-image-forming visual processes such as the pupillary light reflex (PLR), express ß-endorphin-preferring, µ-opioid receptors (MORs). The objective of the present study was to elucidate if opioids, endogenous or exogenous, modulate pupillary light reflex (PLR) via MORs expressed by ipRGCs. MOR-selective agonist [D-Ala2, MePhe4, Gly-ol5]-enkephalin (DAMGO) or antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) was administered via intravitreal injection. PLR was recorded in response to light stimuli of various intensities. DAMGO eliminated PLR evoked by light with intensities below melanopsin activation threshold but not that evoked by bright blue irradiance that activated melanopsin signaling, although in the latter case, DAMGO markedly slowed pupil constriction. CTAP or genetic ablation of MORs in ipRGCs slightly enhanced dim-light-evoked PLR but not that evoked by a bright blue stimulus. Our results suggest that endogenous opioid signaling in the retina contributes to the regulation of PLR. The slowing of bright light-evoked PLR by DAMGO is consistent with the observation that systemically applied opioids accumulate in the vitreous and that patients receiving chronic opioid treatment have slow PLR.

Altered mRNA Levels of Stress-Related Peptides in Mouse Hippocampus and Caudate-Putamen in Withdrawal after Long-Term Intermittent Exposure to Tobacco Smoke or Electronic Cigarette Vapour.

Nicotine addiction is a severe public health problem. The aim of this study was to investigate the alterations in key neurotransmissions after 60 days of withdrawal from seven weeks of intermittent cigarette smoke, e-cigarette vapours, or an e-cigarette vehicle. In the nicotine withdrawal groups, increased depressive and anxiety/obsessive-compulsive-like behaviours were demonstrated in the tail suspension, sucrose preference and marble burying tests. Cognitive impairments were detected in the spatial object recognition test. A significant increase in Corticotropin-releasing factor (Crf) and Crf1 mRNA levels was observed, specifically after cigarette withdrawal in the caudate-putamen nucleus (CPu). The nociceptin precursor levels were reduced by cigarette (80%) and e-cigarette (50%) withdrawal in the CPu. The delta opioid receptor showed a significant reduction in the hippocampus driven by the exposure to an e-cigarette solubilisation vehicle, while the mRNA levels doubled in the CPu of mice that had been exposed to e-cigarettes. Withdrawal after exposure to e-cigarette vapour induced a 35% Bdnf mRNA decrease in the hippocampus, whereas Bdnf was augmented by 118% by cigarette withdrawal in the CPu. This study shows that long-term withdrawal-induced affective and cognitive symptoms associated to lasting molecular alterations in peptidergic signalling may determine the impaired neuroplasticity in the hippocampal and striatal circuitry.

Five Decades of Research on Opioid Peptides: Current Knowledge and Unanswered Questions.

In the mid-1970s, an intense race to identify endogenous substances that activated the same receptors as opiates resulted in the identification of the first endogenous opioid peptides. Since then, >20 peptides with opioid receptor activity have been discovered, all of which are generated from three precursors, proenkephalin, prodynorphin, and proopiomelanocortin, by sequential proteolytic processing by prohormone convertases and carboxypeptidase E. Each of these peptides binds to all three of the opioid receptor types (µ, δ, or κ), albeit with differing affinities. Peptides derived from proenkephalin and prodynorphin are broadly distributed in the brain, and mRNA encoding all three precursors are highly expressed in some peripheral tissues. Various approaches have been used to explore the functions of the opioid peptides in specific behaviors and brain circuits. These methods include directly administering the peptides ex vivo (i.e., to excised tissue) or in vivo (in animals), using antagonists of opioid receptors to infer endogenous peptide activity, and genetic knockout of opioid peptide precursors. Collectively, these studies add to our current understanding of the function of endogenous opioids, especially when similar results are found using different approaches. We briefly review the history of identification of opioid peptides, highlight the major findings, address several myths that are widely accepted but not supported by recent data, and discuss unanswered questions and future directions for research. SIGNIFICANCE STATEMENT: Activation of the opioid receptors by opiates and synthetic drugs leads to central and peripheral biological effects, including analgesia and respiratory depression, but these may not be the primary functions of the endogenous opioid peptides. Instead, the opioid peptides play complex and overlapping roles in a variety of systems, including reward pathways, and an important direction for research is the delineation of the role of individual peptides.

A Peptidomic Approach to Characterize Peptides Involved in Cerebellar Cortex Development Leads to the Identification of the Neurotrophic Effects of Nociceptin.

The cerebellum is a brain structure involved in motor and cognitive functions. The development of the cerebellar cortex (the external part of the cerebellum) is under the control of numerous factors. Among these factors, neuropeptides including PACAP or somatostatin modulate the survival, migration and/or differentiation of cerebellar granule cells. Interestingly, such peptides contributing to cerebellar ontogenesis usually exhibit a specific transient expression profile with a low abundance at birth, a high expression level during the developmental processes, which take place within the first two postnatal weeks in rodents, and a gradual decline toward adulthood. Thus, to identify new peptides transiently expressed in the cerebellum during development, rat cerebella were sampled from birth to adulthood, and analyzed by a semi-quantitative peptidomic approach. A total of 33 peptides were found to be expressed in the cerebellum. Among these 33 peptides, 8 had a clear differential expression pattern during development, 4 of them i.e. cerebellin 2, nociceptin, somatostatin and VGF [353-372], exhibiting a high expression level during the first two postnatal weeks followed by a significative decrease at adulthood. A focus by a genomic approach on nociceptin, confirmed that its precursor mRNA is transiently expressed during the first week of life in granule neurons within the internal granule cell layer of the cerebellum, and showed that the nociceptin receptor is also actively expressed between P8 and P16 by the same neurons. Finally, functional studies revealed a new role for nociceptin, acting as a neurotrophic peptide able to promote the survival and differentiation of developing cerebellar granule neurons.

ERK and p38 contribute to the regulation of nociceptin and the nociceptin receptor in human peripheral blood leukocytes.

Little is known about the mechanisms involved in the regulation of nociceptin and its receptor (nociceptin opioid peptide receptor, NOP) in response to inflammation and pain in humans. In this study, specific signaling pathways contributing to the regulation of nociceptin and NOP in human peripheral blood leukocytes were investigated. After approval by the ethics committee, peripheral blood obtained from healthy donors was cultured with or without phorbol-12-myristate-13-acetate (PMA). Prepronociceptin (ppNOC) and NOP mRNA were analyzed by real-time quantitative polymerase chain reaction, and nociceptin concentrations in culture supernatants by fluorescent enzyme immunoassay. Nociceptin and NOP protein levels in blood leukocyte subsets were determined using flow cytometry. To examine the contribution of signaling pathways to ppNOC and NOP regulation, blood was pre-treated with kinase inhibitors specific for ERK, JNK, p38, and NFκB pathways prior to culturing with or without PMA. PMA dose-dependently upregulated ppNOC mRNA but downregulated NOP mRNA in human peripheral blood leukocytes. PMA 10 ng/ml increased ppNOC after 6 h and suppressed NOP after 3 h compared to controls (both P <0.005). Nociceptin concentrations were increased in supernatants of PMA-induced blood samples after 24 h ( P <0.005), whereas expression of cell-membrane NOP was decreased by PMA in blood leukocyte subsets (all P <0.05). Blockade of ERK or p38 pathways partially prevented PMA effects on ppNOC and NOP mRNA (all P <0.05). The combination of ERK and p38 inhibitors completely reversed the effects of PMA ( P <0.05). ERK and p38 are two major signaling pathways regulating nociceptin and its receptor in human peripheral blood leukocytes under inflammatory conditions.

Intracrine Endorphinergic Systems in Modulation of Myocardial Differentiation.

A wide variety of peptides not only interact with the cell surface, but govern complex signaling from inside the cell. This has been referred to as an "intracrine" action, and the orchestrating molecules as "intracrines". Here, we review the intracrine action of dynorphin B, a bioactive end-product of the prodynorphin gene, on nuclear opioid receptors and nuclear protein kinase C signaling to stimulate the transcription of a gene program of cardiogenesis. The ability of intracrine dynorphin B to prime the transcription of its own coding gene in isolated nuclei is discussed as a feed-forward loop of gene expression amplification and synchronization. We describe the role of hyaluronan mixed esters of butyric and retinoic acids as synthetic intracrines, controlling prodynorphin gene expression, cardiogenesis, and cardiac repair. We also discuss the increase in prodynorphin gene transcription and intracellular dynorphin B afforded by electromagnetic fields in stem cells, as a mechanism of cardiogenic signaling and enhancement in the yield of stem cell-derived cardiomyocytes. We underline the possibility of using the diffusive features of physical energies to modulate intracrinergic systems without the needs of viral vector-mediated gene transfer technologies, and prompt the exploration of this hypothesis in the near future.

Opioid gene expression changes and post-translational histone modifications at promoter regions in the rat nucleus accumbens after acute and repeated 3,4-methylenedioxy-methamphetamine (MDMA) exposure.

The recreational drug of abuse 3,4-methylenedioxymethamphetamine (MDMA) has been shown to produce neurotoxic damage and long-lasting changes in several brain areas. In addition to the involvement of serotoninergic and dopaminergic systems, little information exists about the contribution of nociceptin/orphaninFQ (N/OFQ)-NOP and dynorphin (DYN)-KOP systems in neuronal adaptations evoked by MDMA. Here we investigated the behavioral and molecular effects induced by acute (8mg/kg) or repeated (8mg/kg twice daily for seven days) MDMA exposure. MDMA exposure affected body weight gain and induced hyperlocomotion; this latter effect progressively decreased after repeated administration. Gene expression analysis indicated a down-regulation of the N/OFQ system and an up-regulation of the DYN system in the nucleus accumbens (NAc), highlighting an opposite systems regulation in response to MDMA exposure. Since histone modifications have been strongly associated to the addiction-related maladaptive changes, we examined two permissive (acH3K9 and me3H3K4) and two repressive transcription marks (me3H3K27 and me2H3K9) at the pertinent opioid gene promoter regions. Chromatin immunoprecipitation assays revealed that acute MDMA increased me3H3K4 at the pN/OFQ, pDYN and NOP promoters. Following acute and repeated treatment a significant decrease of acH3K9 at the pN/OFQ promoter was observed, which correlated with gene expression results. Acute treatment caused an acH3K9 increase and a me2H3K9 decrease at the pDYN promoter which matched its mRNA up-regulation. Our data indicate that the activation of the DYNergic stress system together with the inactivation of the N/OFQergic anti-stress system contribute to the neuroadaptive actions of MDMA and offer novel epigenetic information associated with MDMA abuse.

Epigenetic regulation of nociceptin/orphanin FQ and corticotropin-releasing factor system genes in frustration stress-induced binge-like palatable food consumption.

Evidence suggests that binge eating may be caused by a unique interaction between dieting and stress. We developed a binge-eating model in which female rats with a history of intermittent food restriction show binge-like palatable food consumption after a 15-minute exposure to the sight of the palatable food (frustration stress). The aim of the present study was to investigate the regulation of the stress neurohormone corticotropin-releasing factor (CRF) system and of the nociceptin/orphanin FQ (N/OFQ) system genes in selective rat brain regions, using our animal model. Food restriction by itself seems to be responsible in the hypothalamus for the downregulation on messenger RNA levels of CRF-1 receptor, N/OFQ and its receptor (NOP). For the latter, this alteration might be due to selective histone modification changes. Instead, CRF gene appears to be upregulated in the hypothalamus as well as in the ventral tegmental area only when rats are food restricted and exposed to frustration stress, and, of relevance, these changes appear to be due to a reduction in DNA methylation at gene promoters. Moreover, also CRF-1 receptor gene resulted to be differentially regulated in these two brain regions. Epigenetic changes may be viewed as adaptive mechanisms to environmental perturbations concurring to facilitate food consumption in adverse conditions, that is, in this study, under food restriction and stressful conditions. Our data on N/OFQ and CRF signaling provide insight on the use of this binge-eating model for the study of epigenetic modifications in controlled genetic and environmental backgrounds.

Viral vectors encoding endomorphins and serine histogranin attenuate neuropathic pain symptoms after spinal cord injury in rats.

BACKGROUND: The treatment of spinal cord injury (SCI)-induced neuropathic pain presents a challenging healthcare problem. The lack of available robust pharmacological treatments underscores the need for novel therapeutic methods and approaches. Due to the complex character of neuropathic pain following SCI, therapies targeting multiple mechanisms may be a better choice for obtaining sufficient long-term pain relief. Previous studies in our lab showed analgesic effects using combinations of an NMDA antagonist peptide [Ser1]histogranin (SHG), and the mu-opioid peptides endomorphins (EMs), in several pain models. As an alternative to drug therapy, this study evaluated the analgesic potential of these peptides when delivered via gene therapy. RESULTS: Lentiviruses encoding SHG and EM-1 and EM-2 were intraspinally injected, either singly or in combination, into rats with clip compression SCI 2 weeks following injury. Treated animals showed significant reduction in mechanical and thermal hypersensitivity, compared to control groups injected with GFP vector only. The antinociceptive effects of individually injected components were modest, but the combination of EMs and SHG produced robust and sustained antinociception. The onset of the analgesic effects was observed between 1-5 weeks post-injection and sustained without decrement for at least 7 weeks. No adverse effects on locomotor function were observed. The involvement of SHG and EMs in the observed antinociception was confirmed by pharmacologic inhibition using intrathecal injection of either the opioid antagonist naloxone or an anti-SHG antibody. Immunohistochemical analysis showed the presence of SHG and EMs in the spinal cord of treated animals, and immunodot-blot analysis of CSF confirmed the presence of these peptides in injected animals. In a separate group of rats, delayed injection of viral vectors was performed in order to mimic a more likely clinical scenario. Comparable and sustained antinociceptive effects were observed in these animals using the SHG-EMs combination vectors compared to the group with early intervention. CONCLUSIONS: Findings from this study support the potential for direct gene therapy to provide a robust and sustained alleviation of chronic neuropathic pain following SCI. The combination strategy utilizing potent mu-opioid peptides with a naturally-derived NMDA antagonist may produce additive or synergistic analgesic effects without the tolerance development for long-term management of persistent pain.

Possible involvement of endogenous opioid system located downstream of α7 nicotinic acetylcholine receptor in mice with physical dependence on nicotine.

We previously reported that nicotine (NIC)-induced analgesia was elicited in part by activation of the endogenous opioid system. Moreover, it is well known that NIC has physical-dependence liability, but its mechanism is unclear. Therefore, we examined whether physical dependence on NIC was mediated by activation of the endogenous opioid system in ICR mice. We evaluated increased serum corticosterone (SCS) as an indicator of NIC withdrawal, as it is a quantitative indicator of naloxone (opioid receptor antagonist, NLX)-precipitated morphine withdrawal in mice. In this study, NLX precipitated an SCS increase in mice receiving repeated NIC, by a dose-dependent mechanism, and correlated with the dose and number of days of repeated NIC administration. When an opioid receptor antagonist (naltrexone) was concomitantly administered with repeated NIC, the NLX-precipitated SCS increase was not elicited. Concomitant administration of the α7 nicotinic acetylcholine receptor (nAChR) antagonist (methyllycaconitine) with repeated NIC, but not the α4ß2 nAChR antagonist (dihydro-ß-erythroidine), did not elicit an SCS increase by NLX. Thus, a physical dependence on NIC was in part mediated by the activation of the endogenous opioid system, located downstream of α7 nAChR.

Association between the A107V substitution in the δ-opioid receptors and ethanol drinking in mice selected for high and low analgesia.

Experimental evidence suggests that endogenous opioids play an important role in the development of ethanol addiction. In this study, we employed two mouse lines divergently bred for opioid-mediated stress-induced analgesia. In comparison with HA (high analgesia line) mice, LA (low analgesia line) mice, having lower opioid receptor system activity, manifest enhanced basal as well as stress-induced ethanol drinking. Here, we found that recently discovered C320T transition in exon 2 of the δ-opioid receptor gene (EU446125.1), which results in an A107V substitution (ACA23171.1), leads to higher ethanol preference in CT mice compared with CC homozygotes. This genetic association is particularly evident under chronic mild stress (CMS) conditions. The interaction between stress and ethanol intake was significantly stronger in HA than in LA mice. Ethanol almost completely attenuated the pro-depressive effect of CMS (assessed with the tail suspension test) in both the CC and CT genotypes in the HA line. In the LA mice, a lack of response to ethanol was observed in the CC genotype, whereas ethanol consumption strengthened depressive-like behaviours in CT individuals. Our results suggest that constitutively active A107V substitution in δ-opioid receptors may be involved in stress-enhanced vulnerability to ethanol abuse and in the risk of ethanol dependence.

Nociceptin/orphanin FQ receptor agonists attenuate L-DOPA-induced dyskinesias.

In the present study we investigated whether the neuropeptide nociceptin/orphanin FQ (N/OFQ), previously implicated in the pathogenesis of Parkinson's disease, also affects L-DOPA-induced dyskinesia. In striatal slices of naive rodents, N/OFQ (0.1-1 µm) prevented the increase of ERK phosphorylation and the loss of depotentiation of synaptic plasticity induced by the D1 receptor agonist SKF38393 in spiny neurons. In vivo, exogenous N/OFQ (0.03-1 nmol, i.c.v.) or a synthetic N/OFQ receptor agonist given systemically (0.01-1 mg/Kg) attenuated dyskinesias expression in 6-hydroxydopamine hemilesioned rats primed with L-DOPA, without causing primary hypolocomotive effects. Conversely, N/OFQ receptor antagonists worsened dyskinesia expression. In vivo microdialysis revealed that N/OFQ prevented dyskinesias simultaneously with its neurochemical correlates such as the surge of nigral GABA and glutamate, and the reduction of thalamic GABA. Regional microinjections revealed that N/OFQ attenuated dyskinesias more potently and effectively when microinjected in striatum than substantia nigra (SN) reticulata, whereas N/OFQ receptor antagonists were ineffective in striatum but worsened dyskinesias when given in SN. Quantitative autoradiography showed an increase in N/OFQ receptor binding in striatum and a reduction in SN of both unprimed and dyskinetic 6-hydroxydopamine rats, consistent with opposite adaptive changes of N/OFQ transmission. Finally, the N/OFQ receptor synthetic agonist also reduced dyskinesia expression in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated dyskinetic macaques without affecting the global parkinsonian score. We conclude that N/OFQ receptor agonists may represent a novel strategy to counteract L-DOPA-induced dyskinesias. Their action is possibly mediated by upregulated striatal N/OFQ receptors opposing the D1 receptor-mediated overactivation of the striatonigral direct pathway.

Dynorphin--still an extraordinarily potent opioid peptide.

This issue of Molecular Pharmacology is dedicated to Dr. Avram Goldstein, the journal's founding editor and one of the leaders in the development of modern pharmacology. This article focuses on his contributions to the discovery of the dynorphins and evidence that members of this family of opioid peptides are endogenous agonists for the kappa opioid receptor. In his original publication describing the purification and sequencing of dynorphin A, Avram described this peptide as "extraordinarily potent" ("dyn" from the Greek, dynamis = power and "orphin" for endogenous morphine peptide). The name originally referred to its high affinity and great potency in the bioassay that was used to follow its activity during purification, but the name has come to have a second meaning: studies of its physiologic function in brain continue to provide powerful insights to the molecular mechanisms controlling mood disorders and drug addiction. During the 30 years since its discovery, we have learned that the dynorphin peptides are released in brain during stress exposure. After they are released, they activate kappa opioid receptors distributed throughout the brain and spinal cord, where they trigger cellular responses resulting in different stress responses: analgesia, dysphoria-like behaviors, anxiety-like responses, and increased addiction behaviors in experimental animals. Avram predicted that a detailed molecular analysis of opiate drug actions would someday lead to better treatments for drug addiction, and he would be gratified to know that subsequent studies enabled by his discovery of the dynorphins resulted in insights that hold great promise for new treatments for addiction and depressive disorders.

Serine 363 is required for nociceptin/orphanin FQ opioid receptor (NOPR) desensitization, internalization, and arrestin signaling.

We determined the role of carboxyl-terminal regulation of NOPR (nociceptin, orphanin FQ receptor) signaling and function. We mutated C-terminal serine and threonine residues and examined their role in NOPR trafficking, homologous desensitization, and arrestin-dependent MAPK signaling. The NOPR agonist, nociceptin, caused robust NOPR-YFP receptor internalization, peaking at 30 min. Mutation of serine 337, 346, and 351, had no effect on NOPR internalization. However, mutation of C-terminal threonine 362, serine 363, and threonine 365 blocked nociceptin-induced internalization of NOPR. Furthermore, point mutation of only Ser-363 was sufficient to block NOPR internalization. Homologous desensitization of NOPR-mediated calcium channel blockade and inhibition of cAMP were also shown to require Ser-363. Additionally, NOPR internalization was absent when GRK3, and Arrestin3 were knocked down using siRNA, but not when GRK2 and Arrestin2 were knocked down. We also found that nociceptin-induced NOPR-mediated JNK but not ERK signaling requires Ser-363, GRK3, and Arrestin3. Dominant-positive Arrestin3 but not Arrestin2 was sufficient to rescue NOPR-S363A internalization and JNK signaling. These findings suggest that NOPR function may be regulated by GRK3 phosphorylation of Ser-363 and Arrestin3 and further demonstrates the complex nature of G-protein-dependent and -independent signaling in opioid receptors.

Different alcohol exposures induce selective alterations on the expression of dynorphin and nociceptin systems related genes in rat brain.

Molecular mechanisms of adaptive transformations caused by alcohol exposure on opioid dynorphin and nociceptin systems have been investigated in the rat brain. Alcohol was intragastrically administered to rats to resemble human drinking with several hours of exposure: water or alcohol (20% in water) at a dose of 1.5 g/kg three times daily for 1 or 5 days. The development of tolerance and dependence were recorded daily. Brains were dissected 30 minutes (1- and 5-day groups) or 1, 3 or 7 days after the last administration for the three other 5-day groups (groups under withdrawal). Specific alterations in opioid genes expression were ascertained. In the amygdala, an up-regulation of prodynorphin and pronociceptin was observed in the 1-day group; moreover, pronociceptin and the kappa opioid receptor mRNAs in the 5-day group and both peptide precursors in the 1-day withdrawal group were also up-regulated. In the prefrontal cortex, an increase in prodynorhin expression in the 1-day group was detected. These data indicate a relevant role of the dynorphinergic system in the negative hedonic states associated with multiple alcohol exposure. The pattern of alterations observed for the nociceptin system appears to be consistent with its role of functional antagonism towards the actions of ethanol associated with other opioid peptides. Our findings could help to the understanding of how alcohol differentially affects the opioid systems in the brain and also suggest the dynorphin and nociceptin systems as possible targets for the treatment and/or prevention of alcohol dependence.

The endogenous opioid system in human alcoholics: molecular adaptations in brain areas involved in cognitive control of addiction.

The endogenous opioid system (EOS) plays a critical role in addictive processes. Molecular dysregulations in this system may be specific for different stages of addiction cycle and neurocircuitries involved and therefore may differentially contribute to the initiation and maintenance of addiction. Here we evaluated whether the EOS is altered in brain areas involved in cognitive control of addiction including the dorsolateral prefrontal cortex (dl-PFC), orbitofrontal cortex (OFC) and hippocampus in human alcohol-dependent subjects. Levels of EOS mRNAs were measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and levels of dynorphins by radioimmunoassay (RIA) in post-mortem specimens obtained from 14 alcoholics and 14 controls. Prodynorphin mRNA and dynorphins in dl-PFC, κ-opioid receptor mRNA in OFC and dynorphins in hippocampus were up-regulated in alcoholics. No significant changes in expression of proenkephalin, and µ- and δ-opioid receptors were evident; pro-opiomelanocortin mRNA levels were below the detection limit. Activation of the κ-opioid receptor by up-regulated dynorphins in alcoholics may underlie in part neurocognitive dysfunctions relevant for addiction and disrupted inhibitory control.

Nociceptin is upregulated by FSH signaling in Sertoli cells in murine testes.

In postnatal testes, follicle-stimulating hormone (FSH) acts on somatic Sertoli cells to activate gene expression directly via an intracellular signaling pathway composed of cAMP, cAMP-dependent protein kinase (PKA), and cAMP-response element-binding protein (CREB), and promotes germ cell development indirectly. Yet, the paracrine factors mediating the FSH effects to germ cells remained elusive. Here we show that nociceptin, known as a neuropeptide, is upregulated by FSH through cAMP/PKA/CREB pathway in Sertoli cells in murine testes. Chromatin immunoprecipitation from Sertoli cells shows that CREB phosphorylated at Ser133 associates with prepronociceptin gene encoding nociceptin. Analyses with Sertoli cells and testes demonstrates that both prepronociceptin mRNA and the nociceptin peptide are induced after FSH signaling is activated. In addition, the nociceptin peptide is induced in testes after 9days post partum following FSH surge. Thus, our findings may identify nociceptin as a novel paracrine mediator of the FSH effects in the regulation of spermatogenesis.

Hypocretin/orexin and nociceptin/orphanin FQ coordinately regulate analgesia in a mouse model of stress-induced analgesia.

Stress-induced analgesia (SIA) is a key component of the defensive behavioral "fight-or-flight" response. Although the neural substrates of SIA are incompletely understood, previous studies have implicated the hypocretin/orexin (Hcrt) and nociceptin/orphanin FQ (N/OFQ) peptidergic systems in the regulation of SIA. Using immunohistochemistry in brain tissue from wild-type mice, we identified N/OFQ-containing fibers forming synaptic contacts with Hcrt neurons at both the light and electron microscopic levels. Patch clamp recordings in GFP-tagged mouse Hcrt neurons revealed that N/OFQ hyperpolarized, decreased input resistance, and blocked the firing of action potentials in Hcrt neurons. N/OFQ postsynaptic effects were consistent with opening of a G protein-regulated inwardly rectifying K+ (GIRK) channel. N/OFQ also modulated presynaptic release of GABA and glutamate onto Hcrt neurons in mouse hypothalamic slices. Orexin/ataxin-3 mice, in which the Hcrt neurons degenerate, did not exhibit SIA, although analgesia was induced by i.c.v. administration of Hcrt-1. N/OFQ blocked SIA in wild-type mice, while coadministration of Hcrt-1 overcame N/OFQ inhibition of SIA. These results establish what is, to our knowledge, a novel interaction between the N/OFQ and Hcrt systems in which the corticotropin-releasing factor and N/OFQ systems coordinately modulate the Hcrt neurons to regulate SIA.

Pharmacological regulation of gene expression.

Pharmacological regulation of gene expression was one of the top professional interests of Dr. Costa. He promoted the idea that drugs can improve the endogenous mechanisms of synaptic plasticity by modulating gene expression. In this article I reflect upon Dr. Costa's leadership in projects undertaken at FGIN that were aimed at elucidating how neurotransmitter receptor activation could affect brain function by modulating genes and their products. I will be presenting examples of how pharmacological tools can change gene expression. These include the ability of drugs of abuse to alter the synthesis of opioid peptides or an endogenous ligand for GABAA receptor. I will conclude with a brief summary of intriguing discoveries about the regulation of nerve growth factor (NGF) and its receptors by beta-receptor agonists, adrenal steroids and cytokines.