Olivier Civelli

Orphanin FQ: A neuropeptide that activates an opioidlike G protein-coupled receptor

A heptadecapeptide was identified and purified from porcine brain tissue as a ligand for an orphan heterotrimeric GTP- binding protein (G protein)- coupled receptor (LC132) that is similar in sequence to opioid receptors. This peptide, orphanin FQ, has a primary structure reminiscent of that of opioid peptides. Nanomolar concentrations of orphanin FQ inhibited forskolin-stimulated adenylyl cyclase activity in cells transfected with LC132. This inhibitory activity was not affected by the addition of opioid ligands, nor did the peptide activate opioid receptors. Orphanin FQ bound to its receptor in a saturable manner and with high affinity. When injected intracerebroventricularly into mice, orphanin FQ caused a decrease in locomotor activity but did not induce analgesia in the hot-plate test. However, the peptide produced hyperalgesia in the tail-flick assay. Thus, orphanin FQ may act as a transmitter in the brain by modulating nociceptive and locomotor behavior.

Molecular characterization of the melanin-concentrating-hormone receptor

Orphan G-protein-coupled receptors (GPCRs) are cloned proteins with structural characteristics common to the GPCRs but that bind unidentified ligands. Orphan GPCRs have been used as targets to identify novel transmitter molecules. Here we describe the isolation from brain extracts and the characterization of the natural ligand of a particular orphan GPCR (SLC-1) that is sequentially homologous to the somatostatin receptors,. We show that the natural ligand of this receptor is the neuropeptide melanin-concentrating hormone (MCH). MCH is a cyclic peptide that regulates a variety of functions in the mammalian brain, in particular feeding behaviour,. We demonstrate that nanomolar concentrations of MCH strongly activate SLC-1-related pathways through Gαi and/or Gαq proteins. We have analysed the tissue localization of the MCH receptor and find that it is expressed in several brain regions, in particular those involved in olfactory learning and reinforcement mechanisms, indicating that therapies targeting the MCH receptor should act on the neuronal regulation of food consumption.

Neuropeptide S: a neuropeptide promoting arousal and anxiolytic-like effects.

Arousal and anxiety are behavioral responses that involve complex neurocircuitries and multiple neurochemical components. Here, we report that a neuropeptide, neuropeptide S (NPS), potently modulates wakefulness and could also regulate anxiety. NPS acts by activating its cognate receptor (NPSR) and inducing mobilization of intracellular Ca2+. The NPSR mRNA is widely distributed in the brain, including the amygdala and the midline thalamic nuclei. Central administration of NPS increases locomotor activity in mice and decreases paradoxical (REM) sleep and slow wave sleep in rats. NPS was further shown to produce anxiolytic-like effects in mice exposed to four different stressful paradigms. Interestingly, NPS is expressed in a previously undefined cluster of cells located between the locus coeruleus (LC) and Barringtons nucleus. These results indicate that NPS could be a new modulator of arousal and anxiety. They also show that the LC region encompasses distinct nuclei expressing different arousal-promoting neurotransmitters.

Orphanin FQ is a functional anti-opioid peptide

The heptadecapeptide orphanin FQ has recently been shown to be the endogenous agonist for the orphan opioid-like receptor, LC132. The molecular evidence that LC132 and orphanin FQ are evolutionarily related to other opioid receptors and their ligands suggests that these proteins may also play a role in modulating opiate actions. We now report that orphanin FQ (0.5-10 nmol), injected intracerebroventricularly in mice, does not produce hyperalgesia as suggested previously but rather reverses opioid-mediated (i.e. naloxone-sensitive) stress-induced antinociception in three different algesiometric assays. In addition to its antagonism of endogenous opioid antinociception, orphanin FQ dose-dependently (2.5-25 nmol) reverses systemic morphine antinociception (5 mg/kg, s.c.). Based on these data, we propose that orphanin FQ is a functional anti-opioid peptide.

Localization of orphanin FQ (nociceptin) peptide and messenger RNA in the central nervous system of the rat

Orphanin FQ (OFQ) is the endogenous agonist of the opioid receptor‐like receptor (ORL‐1). It and its precursor, prepro‐OFQ, exhibit structural features suggestive of the opioid peptides. A cDNA encoding the OFQ precursor sequence in the rat recently has been cloned, and the authors recently generated a polyclonal antibody directed against the OFQ peptide. In the present study, the authors used in situ hybridization and immunohistochemistry to examine the distribution of OFQ peptide and mRNA in the central nervous system of the adult rat. OFQ immunoreactivity and prepro‐OFQ mRNA expression correlated virtually in all brain areas studied. In the forebrain, OFQ peptide and mRNA were prominent in the neocortex endopiriform nucleus, claustrum, lateral septum, ventral forebrain, hypothalamus, mammillary bodies, central and medial nuclei of the amygdala, hippocampal formation, paratenial and reticular nuclei of the thalamus, medial habenula, and zona incerta. No OFQ was observed in the pineal or pituitary glands. In the brainstem, OFQ was prominent in the ventral tegmental area, substantia nigra, nucleus of the posterior commissure, central gray, nucleus of Darkschewitsch, peripeduncular nucleus, interpeduncular nucleus, tegmental nuclei, locus coeruleus, raphe complex, lateral parabrachial nucleus, inferior olivary complex, vestibular nuclear complex, prepositus hypoglossus, solitary nucleus, nucleus ambiguous, caudal spinal trigeminal nucleus, and reticular formation. In the spinal cord, OFQ was observed throughout the dorsal and ventral horns. The wide distribution of this peptide provides support for its role in a multitude of functions, including not only nociception but also motor and balance control, special sensory processing, and various autonomic and physiologic processes. J. Comp. Neurol. 406:503–547, 1999.

Opioid receptor‐like (ORL1) receptor distribution in the rat central nervous system: Comparison of ORL1 receptor mRNA expression with 125I‐[14Tyr]‐orphanin FQ binding

The recently discovered neuropeptide orphanin FQ (OFQ), and its opioid receptor‐like (ORL1) receptor, exhibit structural features suggestive of the μ, κ, and δ opioid systems. The anatomic distribution of OFQ immunoreactivity and mRNA expression has been reported recently. In the present analysis, we compare the distribution of orphanin receptor mRNA expression with that of orphanin FQ binding at the ORL1 receptor in the adult rat central nervous system (CNS). By using in vitro receptor autoradiography with 125I‐[14Tyr]‐OFQ as the radioligand, orphanin receptor binding was analyzed throughout the rat CNS. Orphanin binding sites were densest in several cortical regions, the anterior olfactory nucleus, lateral septum, ventral forebrain, several hypothalamic nuclei, hippocampal formation, basolateral and medial amygdala, central gray, pontine nuclei, interpeduncular nucleus, substantia nigra, raphe complex, locus coeruleus, vestibular nuclear complex, and the spinal cord. By using in situ hybridization, cells expressing ORL1 mRNA were most numerous throughout multiple cortical regions, the anterior olfactory nucleus, lateral septum, endopiriform nucleus, ventral forebrain, multiple hypothalamic nuclei, nucleus of the lateral olfactory tract, medial amygdala, hippocampal formation, substantia nigra, ventral tegmental area, central gray, raphe complex, locus coeruleus, multiple brainstem motor nuclei, inferior olive, deep cerebellar nuclei, vestibular nuclear complex, nucleus of the solitary tract, reticular formation, dorsal root ganglia, and spinal cord. The diffuse distribution of ORL1 mRNA and binding supports an extensive role for orphanin FQ in a multitude of CNS functions, including motor and balance control, reinforcement and reward, nociception, the stress response, sexual behavior, aggression, and autonomic control of physiologic processes. J. Comp. Neurol. 412:563–605, 1999.

Expression of the melanin-concentrating hormone (MCH) receptor mRNA in the rat brain.

The melanin‐concentrating hormone (MCH) system is thought to be an important regulator of food intake. Recently the orphan G protein‐coupled receptor SLC‐1 was identified as the MCH receptor (MCHR). Preliminary analyses of MCHR mRNA distribution have supported a role for the MCH system in nutritional homeostasis. We report here a complete anatomical distribution of the MCHR mRNA. We have found high levels of expression of MCHR mRNA in most anatomical areas implicated in control of olfaction, with the exception of the main olfactory bulb. Dense labeling was also detected in the hippocampal formation, subiculum, and basolateral amygdala, all of which are important in learning and memory, and in the shell of the nucleus accumbens, a substrate for motivated behavior and feeding. Within the hypothalamus, MCHR mRNA was moderately expressed in the ventromedial nucleus, arcuate nucleus, and zona incerta, all of which serve key roles in the neuronal circuitry of feeding. In the brainstem, strong expression was observed in the locus coeruleus, which is implicated in arousal, as well as in nuclei that contribute to orofacial function and mastication, including the facial, hypoglossal, motor trigeminal, and dorsal motor vagus nuclei. In most regions there was a good correspondence between MCHR mRNA distribution and that of MCH‐immunoreactive fibers. Taken together, these data suggest that MCH may act at various levels of the brain to integrate various aspects of feeding behavior. However, the extensive MCHR distribution throughout the brain suggests that this receptor may play a role in other functions, most notably reinforcement, arousal, sensorimotor integration, and autonomic control. J. Comp. Neurol. 435:26–40, 2001.

Differential activation of spinal cord dynorphin and enkephalin neurons during hyperalgesia: evidence using cDNA hybridization

A unilateral experimental inflammation of the hindlimb produces hyperalgesia to both mechanical and radiant thermal stimuli that is rapid in onset. During this period, parameters of dynorphin biosynthesis are elevated to a much greater degree than those of the enkephalin system. An increase in the content of the peptide dynorphin A(1-8) occurs in the spinal cord segments that receive sensory input from the affected limb. This is accompanied by a rapid (within 24 h) and pronounced increase in the levels of mRNA coding for the dynorphin protein precursor. Maximum elevations (6- to 8-fold) of preprodynorphin mRNA are observed between days 2 and 5 subsequent to the induction of inflammation. Compared to the increase in mRNA, the increase in dynorphin A(1-8) peptide was appreciably delayed and proportionately less; maximal increases in peptide (3-fold) were seen at day 5 of inflammation. Dorsal spinal cord preproenkephalin mRNA is elevated to a lesser degree (50-80%). However, the increase in preproenkephalin mRNA is apparently not enough to yield a measurable increase in the proenkephalin-derived peptide met5-enkephalin-Arg6-Gly7-Leu8, the levels of which showed no significant change during the 14-day inflammatory period. These data suggest the active participation of opioid neurons, especially those containing dynorphin, at the spinal level, in the modulation of sensory afferent input during peripheral inflammatory pain states.

Distribution of D5 dopamine receptor mRNA in rat brain

The distribution of the messenger RNA encoding the dopamine D5 receptor was determined in the rat brain by in situ hybridization. Using [35S]-labelled riboprobes to either the rat or human D5 receptor, this mRNA was localized to the hippocampus and the parafascicular nucleus of the thalamus. This mRNA could not be visualized in the more traditional brain regions associated with dopaminergic cell bodies or projection fields. This unusual distribution suggests a novel function in the brain for this subtype of the dopamine receptor.

A comparison of D1 receptor binding and mRNA in rat brain using receptor autoradiographic and in situ hybridization techniques

D1, a subtype of the dopamine receptors, is widely distributed in the nervous system and has been shown to be positively coupled to adenylate cyclase. Using a combination of in vitro receptor autoradiographic and in situ hybridization techniques, the present study examines the co-distribution of D1 receptor binding sites and D1 receptor mRNA in adjacent rat brain sections. D1 receptor binding sites were labeled using the selective antagonist [3H](R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzaz epin- 7-ol (SCH23390) (4.6 nM), in the presence of 1 microM ketanserin, while the D1 receptor mRNA was visualized with a 35S-labeled riboprobe corresponding to a region between transmembrane domains III and VI of the rat D1 receptor (base pairs 383-843). Analysis of serial sections suggested a good agreement between D1 receptor binding and mRNA in several brain regions, including the paleocortex, caudate-putamen, nucleus accumbens, amygdala, and suprachiasmatic nucleus. Marked discrepancies between D1 receptor binding and mRNA were observed in other brain regions including the entopeduncular and subthalamic nuclei, substantia nigra (pars reticulata), hippocampus, and cerebellum. While technical considerations may contribute to these results, much of the discordance between the distributions is probably due to the differential localization of D1 receptor mRNA in cell bodies and receptor binding sites on fibers and may provide insights into receptor synthesis, transport, and membrane insertion. In the basal ganglia, for instance, D1 receptors are synthesized in the striatum and are either transported to efferent projections in areas such as the substantia nigra, or remain localized in striatal cells bodies. Ibotenic acid lesions in the striatum are consistent with these conclusions and demonstrate a coordinate loss of D1 receptor binding and mRNA in the caudate-putamen that is accompanied by a degeneration of fibers projecting to substantia nigra and a loss of D1 binding in the pars reticulata. Neurons in the dentate gyrus and in the granular layer of the cerebellum, on the other hand, synthesize D1 receptors and transport them entirely to either their dendritic or axonal fields, respectively, in the molecular layer. This analysis provides a better understanding of dopaminergic receptor systems in the CNS and their anatomical organization.