Newt Brain Research Articles

Spatio-temporal neural stem cell behavior leads to both perfect and imperfect structural brain regeneration in adult newts.

ABSTRACTAdult newts can regenerate large parts of their brain from adult neural stem cells (NSCs), but how adult NSCs reorganize brain structures during regeneration remains unclear. In development, elaborate brain structures are produced under broadly coordinated regulations of embryonic NSCs in the neural tube, whereas brain regeneration entails exquisite control of the re-establishment of certain brain parts, suggesting that a yet-unknown mechanism directs NSCs upon partial brain excision. Here we report that upon excision of a quarter of the adult newt (Pleurodeles waltl) mesencephalon, active participation of local NSCs around specific brain subregions’ boundaries leads to some imperfect and some perfect brain regeneration along an individual's rostrocaudal axis. Regeneration phenotypes depend on how wound closing occurs using local NSCs, and perfect regeneration replicates development-like processes, but takes more than 1 year. Our findings indicate that newt brain regeneration is supported by modularity of boundary-domain NSCs with self-organizing ability in neighboring fields.This article has an associated First Person interview with the first author of the paper.

Environmental changes in oxygen tension reveal ROS-dependent neurogenesis and regeneration in the adult newt brain.

Organisms need to adapt to the ecological constraints in their habitat. How specific processes reflect such adaptations are difficult to model experimentally. We tested whether environmental shifts in oxygen tension lead to events in the adult newt brain that share features with processes occurring during neuronal regeneration under normoxia. By experimental simulation of varying oxygen concentrations, we show that hypoxia followed by re-oxygenation lead to neuronal death and hallmarks of an injury response, including activation of neural stem cells ultimately leading to neurogenesis. Neural stem cells accumulate reactive oxygen species (ROS) during re-oxygenation and inhibition of ROS biosynthesis counteracts their proliferation as well as neurogenesis. Importantly, regeneration of dopamine neurons under normoxia also depends on ROS-production. These data demonstrate a role for ROS-production in neurogenesis in newts and suggest that this role may have been recruited to the capacity to replace lost neurons in the brain of an adult vertebrate.

Studying newt brain regeneration following subtype specific neuronal ablation.

The realization that neuronal injury does not result in permanent functional or cellular loss in all vertebrates has fascinated regenerative biologists. Neuronal regeneration occurs in a subset of species, including lizards, teleost fish, axolotls, and newts. One tool for studying neuronal regeneration in the adult brain is intraventricular injection of selective neuronal toxins, which leads to loss of subpopulations of neurons. To trace cells involved in the regeneration process, plasmids encoding reporter proteins can be electroporated in vivo into the cells of interest. This protocol describes methods to label the ependymoglial cells of the brain of the red spotted newt Notophthalmus viridescens and follow their response after ablation of dopaminergic neurons.

Progenitor Cell Dynamics in the Newt Telencephalon during Homeostasis and Neuronal Regeneration

SummaryThe adult newt brain has a marked neurogenic potential and is highly regenerative. Ventricular, radial glia-like ependymoglia cells give rise to neurons both during normal homeostasis and after injury, but subpopulations among ependymoglia cells have not been defined. We show here that a substantial portion of GFAP+ ependymoglia cells in the proliferative hot spots of the telencephalon has transit-amplifying characteristics. In contrast, proliferating ependymoglia cells, which are scattered along the ventricular wall, have stem cell features in terms of label retention and insensitivity to AraC treatment. Ablation of neurons remodels the proliferation dynamics and leads to de novo formation of regions displaying features of neurogenic niches, such as the appearance of cells with transit-amplifying features and proliferating neuroblasts. The results have implication both for our understanding of the evolutionary diversification of radial glia cells as well as the processes regulating neurogenesis and regeneration in the adult vertebrate brain.

Brain and pineal 7α-hydroxypregnenolone stimulating locomotor activity: Identification, mode of action and regulation of biosynthesis

Biologically active steroids synthesized in the central and peripheral nervous systems are termed neurosteroids. However, the biosynthetic pathways leading to the formation of neurosteroids are still incompletely elucidated. 7α-Hydroxypregnenolone, a novel bioactive neurosteroid stimulating locomotor activity, has been recently identified in the brain of newts and quail. Subsequently, the mode of action and regulation of biosynthesis of 7α-hydroxypregnenolone have been determined. Moreover, recent studies on birds have demonstrated that the pineal gland, an endocrine organ located close to the brain, is an important site of production of neurosteroids de novo from cholesterol. 7α-Hydroxypregnenolone is a major pineal neurosteroid that stimulates locomotor activity in juvenile chickens, connecting light-induced gene expression with locomotion. This review summarizes the advances in our understanding of the identification, mode of action and regulation of biosynthesis of brain and pineal 7α-hydroxypregnenolone, a potent stimulator of locomotor activity.

ADAR-Related Activation of Adenosine-to-Inosine RNA Editing During Regeneration

Urodele amphibians possess an amazing regenerative capacity that requires the activation of cellular plasticity in differentiated cells and progenitor/stem cells. Many aspects of regeneration in Urodele amphibians recapitulate development, making it unlikely that gene regulatory pathways which are essential for development are mutually exclusive from those necessary for regeneration. One such post-transcriptional gene regulatory pathway, which has been previously shown to be essential for functional metazoan development, is RNA editing. RNA editing catalyses discrete nucleotide changes in RNA transcripts, creating a molecular diversity that could create an enticing connection to the activated cellular plasticity found in newts during regeneration. To assess whether RNA editing occurs during regeneration, we demonstrated that GABRA3 and ADAR2 mRNA transcripts are edited in uninjured and regenerating tissues. Full open-reading frame sequences for ADAR1 and ADAR2, two enzymes responsible for adenosine-to-inosine RNA editing, were cloned from newt brain cDNA and exhibited a strong resemblance to ADAR (adenosine deaminase, RNA-specific) enzymes discovered in mammals. We demonstrated that ADAR1 and ADAR2 mRNA expression levels are differentially expressed during different phases of regeneration in multiple tissues, whereas protein expression levels remain unaltered. In addition, we have characterized a fascinating nucleocytoplasmic shuttling of ADAR1 in a variety of different cell types during regeneration, which could provide a mechanism for controlling RNA editing, without altering translational output of the editing enzyme. The link between RNA editing and regeneration provides further insights into how lower organisms, such as the newt, can activate essential molecular pathways via the discrete alteration of RNA sequences.

Acute Stress Increases the Synthesis of 7α-Hydroxypregnenolone, a New Key Neurosteroid Stimulating Locomotor Activity, through Corticosterone Action in Newts

7α-Hydroxypregnenolone (7α-OH PREG) is a newly identified bioactive neurosteroid stimulating locomotor activity in the brain of newt, a wild animal, which serves as an excellent model to investigate the biosynthesis and biological action of neurosteroids. Here, we show that acute stress increases 7α-OH PREG synthesis in the dorsomedial hypothalamus (DMH) through corticosterone (CORT) action in newts. A 30-min restraint stress increased 7α-OH PREG synthesis in the brain tissue concomitant with the increase in plasma CORT concentrations. A 30-min restraint stress also increased the expression of cytochrome P450(7α) (CYP7B), the steroidogenic enzyme of 7α-OH PREG formation, in the DMH. Decreasing plasma CORT concentrations by hypophysectomy or trilostane administration decreased 7α-OH PREG synthesis in the diencephalon, whereas administration of CORT to these animals increased 7α-OH PREG synthesis. Glucocorticoid receptor was present in DMH neurons expressing CYP7B. Thus, CORT appears to act directly on DMH neurons to increase 7α-OH PREG synthesis. We further investigated the biological action of 7α-OH PREG in the brain under stress. A 30-min restraint stress or central administration of 7α-OH PREG increased serotonin concentrations in the diencephalon. Double immunolabeling further showed colocalization of CYP7B and serotonin in the DMH. These results indicate that acute stress increases the synthesis of 7α-OH PREG via CORT action in the DMH, and 7α-OH PREG activates serotonergic neurons in the DMH that may coordinate behavioral responses to stress. This is the first demonstration of neurosteroid biosynthesis regulated by peripheral steroid hormone and of neurosteroid action in the brain under stress in any vertebrate class.

Identification, localization and expression of LPXRFamide peptides, and melatonin-dependent induction of their precursor mRNA in the newt brain

The existence of RFamide peptides with a C-terminal LPXRFamide (X=L or Q) motif has been identified in the brain of various vertebrate species. However, the presence of LPXRFamide peptides in the urodele brain is not yet known. In this study, we cloned a cDNA encoding the precursor of LPXRFamide peptides from the newt brain by a combination of 3' and 5' rapid amplification of cDNA ends. The deduced LPXRFamide peptide precursor consisted of 233 amino acid residues, encoding four putative LPXRFamide peptides. All the peptide sequences were flanked by a glycine C-terminal amidation signal and basic amino acid on each end as an endoproteolytic site. Mass spectrometric analyses detected a nonapeptide, two decapeptides and an octapeptide produced from the precursor polypeptide in the brain as endogenous ligands. In situ hybridization further revealed the cellular localization of newt LPXRFamide (nLPXRFa) precursor mRNA in the suprachiasmatic nucleus (SCN) in the newt hypothalamus. Immunocytochemistry showed a cluster of cell bodies restricted to the SCN and their terminals in the median eminence. To understand the regulatory mechanism of nLPXRFa peptide expression, we further analyzed the effect of melatonin on the expression of nLPXRFa precursor mRNA. Melatonin administration to newts increased the expression of nLPXRFa precursor mRNA in the diencephalon. These results indicate that the urodele hypothalamus possesses LPXRFamide peptides and the expression of LPXRFamide peptides is regulated by melatonin. The localization of nLPXRFa peptides further suggests that these peptides may be involved in the regulation of pituitary hormone release in newts.

Mode of Action and Functional Significance of 7?-Hydroxypregnenolone Stimulating Locomotor Activity

Previous studies over the past two decades have demonstrated that the brain and other nervous systems possess key steroidogenic enzymes and produces pregnenolone and other various neurosteroids in vertebrates in general. Recently, 7α-hydroxypregnenolone, a novel bioactive neurosteroid, was identified in the brain of newts and quail. Importantly, this novel neurosteroid is produced from pregnenolone through the enzymatic activity of cytochrome P4507α and acts on brain tissue as a neuronal modulator to stimulate locomotor activity in these vertebrates. Subsequently, the mode of action of 7α-hydroxypregnenolone was demonstrated. 7α-Hydroxypregnenolone stimulates locomotor activity through activation of the dopaminergic system. To understand the functional significance of 7α-hydroxypregnenolone in the regulation of locomotor activity, diurnal, and seasonal changes in 7α-hydroxypregnenolone synthesis were further characterized. Melatonin derived from the pineal gland and eyes regulates 7α-hydroxypregnenolone synthesis in the brain, thus inducing diurnal locomotor changes. Prolactin, an adenohypophyseal hormone, regulates 7α-hydroxypregnenolone synthesis in the brain, and also induces seasonal locomotor changes. In addition, 7α-hydroxypregnenolone mediates corticosterone action to modulate locomotor activity under stress. This review summarizes the current knowledge regarding the mode of action and functional significance of 7α-hydroxypregnenolone, a newly identified bioactive neurosteroid stimulating locomotor activity.

MELATONIN REGULATES DIURNAL CHANGES IN LOCOMOTOR ACTIVITY BY REGULATING 7alpha-HYDROXYPREGNENOLONE SYNTHESIS IN NEWTS

Event Abstract Back to Event MELATONIN REGULATES DIURNAL CHANGES IN LOCOMOTOR ACTIVITY BY REGULATING 7alpha-HYDROXYPREGNENOLONE SYNTHESIS IN NEWTS Shogo Haraguchi1, 2, Teppei Koyama1, Kazuyoshi Tsutsui1* and Hubert Vaudry3 1 Waseda University, Department of Biology, Japan 2 Tokyo Gakugei University, Department of Biology, Faculty of Education, Japan 3 University of Rouen, European Institute for Peptide Research, France Steroids can be synthesized de novo in the central and peripheral nervous systems and such steroids are called “neurosteroids”. Seasonally breeding wild animals, such as amphibians, have served as excellent animal models to investigate the biosynthesis and biological actions of neurosteroids. Our previous studies have demonstrated that the brain of amphibians possesses the key steroidogenic enzymes and produces pregnenolone, a precursor of steroid hormones, and other various neurosteroids. We recently found that the brain of seasonally breeding newts actively produces 7alpha-hydroxypregnenolone, a previously undescribed amphibian neurosteroid. Interestingly, this novel neurosteroid acts as a neuronal modulator to stimulate locomotor activity in male newts. Because male newts show marked diurnal changes in locomotor activity, we hypothesized that 7alpha-hydroxypregnenolone may be a key factor for the induction of diurnal changes in locomotor activity in male newts. In this study, we first found diurnal changes in 7alpha-hydroxypregnenolone synthesis in the brain of male newts. The production of 7alpha-hydroxypregnenolone in the male brain increased during the dark phase when locomotor activity of males was high. Thus, diurnal changes in 7alpha-hydroxypregnenolone synthesis in the brain paralleled with locomotor activity in male newts. We then identified melatonin as a key component of the mechanism regulating 7alpha-hydroxypregnenolone synthesis. Decreased synthesis of 7alpha-hydroxypregnenolone occurred in males in vivo after melatonin removal via pinealectomy and orbital enucleation (Px plus Ex). Conversely, increased synthesis of this neurosteroid occurred after melatonin administration to Px plus Ex males. This study demonstrates that melatonin regulates synthesis of 7alpha-hydroxypregnenolone, a key factor for induction of locomotor activity, thus inducing diurnal locomotor changes in male newts Acknowledgements This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan to KT and a Japan Society for the Promotion of Science (JSPS) – Institut National de la Santé de la Recherche Médicale (INSERM) exchange program to KT and HV. Keywords: 7alpha-Hydroxypregnenolone, diurnal changes, locomotor activity, Melatonin, Neurosteroids, newt brain Conference: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology, Ann Arbor, United States, 13 Jul - 16 Jul, 2011. Presentation Type: Poster Topic: Brain and behavior Citation: Haraguchi S, Koyama T, Tsutsui K and Vaudry H (2011). MELATONIN REGULATES DIURNAL CHANGES IN LOCOMOTOR ACTIVITY BY REGULATING 7alpha-HYDROXYPREGNENOLONE SYNTHESIS IN NEWTS. Front. Endocrinol. Conference Abstract: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology. doi: 10.3389/conf.fendo.2011.04.00021 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 06 Jul 2011; Published Online: 09 Aug 2011. * Correspondence: Prof. Kazuyoshi Tsutsui, Waseda University, Department of Biology, Tokyo, 162-8480, Japan, k-tsutsui@waseda.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Shogo Haraguchi Teppei Koyama Kazuyoshi Tsutsui Hubert Vaudry Google Shogo Haraguchi Teppei Koyama Kazuyoshi Tsutsui Hubert Vaudry Google Scholar Shogo Haraguchi Teppei Koyama Kazuyoshi Tsutsui Hubert Vaudry PubMed Shogo Haraguchi Teppei Koyama Kazuyoshi Tsutsui Hubert Vaudry Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Efficient regeneration by activation of neurogenesis in homeostatically quiescent regions of the adult vertebrate brain

In contrast to mammals, salamanders and teleost fishes can efficiently repair the adult brain. It has been hypothesised that constitutively active neurogenic niches are a prerequisite for extensive neuronal regeneration capacity. Here, we show that the highly regenerative salamander, the red spotted newt, displays an unexpectedly similar distribution of active germinal niches with mammals under normal physiological conditions. Proliferation zones in the adult newt brain are restricted to the forebrain, whereas all other regions are essentially quiescent. However, ablation of midbrain dopamine neurons in newts induced ependymoglia cells in the normally quiescent midbrain to proliferate and to undertake full dopamine neuron regeneration. Using oligonucleotide microarrays, we have catalogued a set of differentially expressed genes in these activated ependymoglia cells. This strategy identified hedgehog signalling as a key component of adult dopamine neuron regeneration. These data show that brain regeneration can occur by activation of neurogenesis in quiescent brain regions.

Expression of cytochrome P450 side-chain cleavage enzyme mRNA and production of pregnenolone in the brain of the red-bellied newt Cynops pyrrhogaster

It is becoming clear that the vertebrate brain has the capability of forming steroids de novo, the so-called “neurosteroids”. To understand neurosteroidogenesis in the brain, it is essential to demonstrate the formation of pregnenolone, a main precursor of neurosteroids. In amphibians, the pregnenolone formation from cholesterol is still unclear, although the brain accumulates pregnenolone, pregnenolone sulfate and 7α-hydroxypregnenolone. This study was addressed to obtain basic information about pregnenolone formation in the newt brain. Firstly, we demonstrated that the newt brain produces pregnenolone from cholesterol. Subsequently, cDNA encoding cytochrome P450 side-chain cleavage enzyme (P450scc), a key steroidogenic enzyme catalyzing pregnenolone formation, was isolated from the newt. The sequence analysis showed that the isolated P450scc cDNA contained a putative coding region consisting of 1569bp, which encoded 523 amino acids. The steroid- and heme-binding domains of P450scc were highly shared in amino acids among vertebrates. RT-PCR analysis amplified the authentic fragment corresponding to newt P450scc showed its transcription in the brain. However, the transcription level in the brain was lower than those of the gonad and the kidney including adrenals. The restricted cells in the four major regions of the newt brain, such as the telencephalon, diencephalon, mesencephalon, and rhombencephalon, were demonstrated to express P450scc transcripts by RT-PCR and in situ hybridization. Taken together, these results indicate that the newt brain expresses P450scc mRNA and produces pregnenolone from cholesterol.

Identification of 7α-hydroxypregnenolone, a novel bioactive amphibian neurosteroid stimulating locomotor activity, and its physiological roles in the regulation of locomotion

We now know that steroids can be synthesized de novo by the brain and the peripheral nervous system. Such steroids are called neurosteroids and de novo neurosteroidogenesis from cholesterol is a conserved property of vertebrate brains. Our studies over the past decade have demonstrated that the brain expresses several kinds of steroidogenic enzymes and produces a variety of neurosteroids in sub-mammalian species. However, neurosteroid biosynthetic pathways in amphibians, as well as other vertebrates may still not be fully mapped. We first found that the newt brain actively produces 7α-hydroxypregnenolone, a previously undescribed amphibian neurosteroid. We then demonstrated that 7α-hydroxypregnenolone acts as a novel bioactive neurosteroid to stimulate locomotor activity of newt by means of the dopaminergic system. Subsequently, we analyzed the physiological roles of 7α-hydroxypregnenolone in the regulation of locomotor activity of newt. This paper summarizes the advances made in our understanding of 7α-hydroxypregnenolone, a newly discovered bioactive amphibian neurosteroid stimulating locomotor activity, and its physiological roles in the regulation of locomotion in newt.

7α-Hydroxypregnenolone, a New Key Regulator of Locomotor Activity of Vertebrates: Identification, Mode of Action, and Functional Significance

Steroids synthesized de novo by the central and peripheral nervous systems are called neurosteroids. The formation of neurosteroids from cholesterol in the brain was originally demonstrated in mammals by Baulieu and colleagues. Our studies over the past two decades have also shown that, in birds and amphibians as in mammals, the brain expresses several kinds of steroidogenic enzymes and produces a variety of neurosteroids. Thus, de novo neurosteroidogenesis from cholesterol is a conserved property that occurs throughout vertebrates. However, the biosynthetic pathways of neurosteroids in the brain of vertebrates was considered to be still incompletely elucidated. Recently, 7α-hydroxypregnenolone was identified as a novel bioactive neurosteroid stimulating locomotor activity in the brain of newts and quail through activation of the dopaminergic system. Subsequently, diurnal and seasonal changes in synthesis of 7α-hydroxypregnenolone in the brain were demonstrated. Interestingly, melatonin derived from the pineal gland and eyes regulates 7α-hydroxypregnenolone synthesis in the brain, thus inducing diurnal locomotor changes. Prolactin, an adenohypophyseal hormone, regulates 7α-hydroxypregnenolone synthesis in the brain, and may also induce seasonal locomotor changes. This review highlights the identification, mode of action, and functional significance of 7α-hydroxypregnenolone, a new key regulator of locomotor activity of vertebrates, in terms of diurnal and seasonal changes in 7α-hydroxypregnenolone synthesis, and describes some of their regulatory mechanisms.

Identification, Biosynthesis, and Function of 7α‐Hydroxypregnenolone, a New Key Neurosteroid Controlling Locomotor Activity, in Nonmammalian Vertebrates

It is now clearly established that steroids can be synthesized de novo by the brain and the peripheral nervous systems. Such steroids are called neurosteroids, and de novo neurosteroidogenesis from cholesterol is a conserved property of vertebrate brains. Our studies over the past decade have demonstrated that the brain expresses several kinds of steroidogenic enzymes and produces a variety of neurosteroids in submammalian species. However, the biosynthetic pathway of neurosteroids in nonmammalian vertebrates as well as in mammals may be still incompletely mapped out. We recently found that the brain of newts and quail actively produces 7alpha-hydroxypregnenolone, a novel bioactive neurosteroid, from pregnenolone. Interestingly, 7alpha-hydroxypregnenolone stimulates locomotor activity by means of the dopaminergic system. Subsequently, we demonstrated that melatonin regulates synthesis of 7alpha-hydroxypregnenolone, a key factor for induction of locomotor activity, thus inducing diurnal locomotor changes.

Diurnal Changes in the Synthesis of the Neurosteroid 7α‐Hydroxypregnenolone Stimulating Locomotor Activity in Newts

We recently identified 7alpha-hydroxypregnenolone as a novel amphibian neurosteroid stimulating locomotor activity in newts. Because male newts show marked diurnal changes in locomotor activity, we hypothesized that 7alpha-hydroxypregnenolone may be a key factor for the induction of diurnal changes in locomotor activity in male newts. In this study, we found diurnal changes in 7alpha-hydroxypregnenolone synthesis in the brain of male newts, which paralleled locomotor activity. Interestingly, the production of 7alpha-hydroxypregnenolone in the male newt brain increased during the dark phase when locomotor activity of males was high.

Seasonal Changes in the Synthesis of the Neurosteroid 7α‐Hydroxypregnenolone Stimulating Locomotor Activity in Newts

We recently found that the newt brain actively produces 7alpha-hydroxypregnenolone, a novel amphibian neurosteroid stimulating locomotor activity. It is well known that locomotor activity of male newts increases during the breeding period. To understand the physiological role of 7alpha-hydroxypregnenolone, we investigated seasonal changes in 7alpha-hydroxypregnenolone synthesis in the brain of male newts. Interestingly, 7alpha-hydroxypregnenolone synthesis in the brain showed marked changes during the annual breeding cycle, with a maximal level in the breeding period when locomotor activity of male newts increases. These results suggest that 7alpha-hydroxypregnenolone induces seasonal locomotor changes in male newts.

Expression of two types of arginine vasotocin receptor mRNA in the newt brain

Expression of two types of arginine vasotocin receptor mRNA in the newt brain

Delta and mu opioid receptors from the brain of a urodele amphibian, the rough-skinned newt Taricha granulosa: Cloning, heterologous expression, and pharmacological characterization

Two full-length cDNAs, encoding delta (δ) and mu (μ) opioid receptors, were cloned from the brain of the rough-skinned newt Taricha granulosa, complementing previous work from our laboratory describing the cloning of newt brain kappa (κ) and ORL1 opioid receptors. The newt δ receptor shares 82% amino acid sequence identity with a frog δ receptor and lower (68–70%) identity with orthologous receptors cloned from mammals and zebrafish. The newt μ receptor shares 79% sequence identity with a frog μ receptor, 72% identity with mammalian μ receptors, and 66–69% identity with μ receptors cloned from teleost fishes. Membranes isolated from COS-7 cells transiently expressing the newt δ receptor possessed a single, high-affinity ( K d = 2.4 nM) binding site for the nonselective opioid antagonist [ 3H]naloxone. In competition binding assays, the newt δ receptor displayed highest affinity for Met-enkephalin, relatively low affinity for Leu-enkephalin, β-endorphin, and [ d-penicillamine, d-penicillamine] enkephalin (DPDPE) (a δ-selective agonist in mammals), and very low affinity for μ-, κ-, or ORL1-selective agonists. COS-7 cells expressing the newt μ receptor also possessed a high-affinity ( K d = 0.44 nM) naloxone-binding site that showed highest affinity for β-endorphin, moderate-to-low affinity for Met-enkephalin and Leu-enkephalin and DAMGO (a μ-selective agonist in mammals), and very low affinity for DPDPE and κ- or ORL1-selective agonists. COS-7 cells expressing either receptor type (δ or μ) showed very high affinity ( K d = 0.1–0.3 nM) for the nonselective opioid antagonist diprenorphine. Taricha granulosa expresses the same four subtypes (δ, μ, κ, and ORL1) of opioid receptors found in other vertebrate classes, but ligand selectivity appears less stringent in the newt than has been documented in mammals.

Cloning, heterologous expression and pharmacological characterization of a kappa opioid receptor from the brain of the rough-skinned newt, Taricha granulosa

A full-length cDNA that encodes a kappa (kappa) opioid receptor has been isolated from the brain of a urodele amphibian, the rough-skinned newt Taricha granulosa. The deduced protein contains 385 amino acids and possesses features commonly attributed to G protein-coupled receptors, such as seven putative transmembrane domains. The newt kappa receptor has 75% sequence identity to kappa opioid receptors cloned from mammals, and 66% sequence identity to the kappa opioid receptor reported for the zebrafish, with the greatest divergence in the extracellular N-terminus, the second and third extracellular loops and the intracellular C-terminus. Membranes isolated from COS-7 cells expressing the newt kappa receptor possessed a single, high-affinity (Kd = 1.5 nM) binding site for the kappa-selective agonist U69593. In competition binding assays, the expressed newt kappa receptor displayed high affinity for the kappa-selective agonists GR89696, dynorphin A(1-13), U69593, U50488 and BRL52537, as well as the kappa-selective antagonist nor-binaltorphimine and the non-selective antagonist naloxone. Rank order potencies and affinity constants were similar in competition binding studies that used either whole brain homogenates or membranes isolated from COS-7 cells expressing the newt kappa receptor. The expressed receptor displayed essentially no affinity for the delta-selective agonist DPDPE ([d-penicillamine, d-penicillamine]enkephalin), but showed moderate affinity for the mu-selective agonist DAMGO ([d-Ala-MePhe, Gly-ol]enkephalin) and moderately high affinity for nociceptin (orphanin FQ), the endogenous ligand for the opioid receptor-like (ORL)1 receptor. These findings support the conclusions that a gene for the kappa opioid receptor is expressed in amphibians and that the pharmacology of the newt kappa receptor closely matches mammalian kappa opioid receptors. However, the functional dichotomy between the classic opioid receptors (kappa, delta, mu) and ORL1 appears less strict in amphibians than in mammals.