Endocrine-disrupting effects of nonylphenol in the newt, Triturus carnifex (Amphibia, Urodela)

The aim of our study was to verify whether environmental concentrations of nonylphenol influenced the adrenal gland of Triturus carnifex. Newts were exposed to 19 μg/L nominal concentration of nonylphenol throughout the periods of December-January and March-April, corresponding to different stages of the chromaffin cell functional cycle. The morphological features of the steroidogenic and chromaffin tissues, and the serum levels of ACTH, aldosterone, corticosterone, norepinephrine and epinephrine were evaluated. Nonylphenol did not influence ACTH serum levels. During the two periods examined, the steroidogenic tissue had the same reaction: the quantity of cytoplasmic lipids, and the corticosteroid serum levels, decreased, suggesting the inhibition of synthesis and release of corticosteroids. During the two periods examined, the chromaffin tissue reacted differently to nonylphenol. During December-January, the numeric ratio of norepinephrine granules to epinephrine granules, and the epinephrine serum levels, increased, suggesting the stimulation of epinephrine release. During March-April, the numeric ratio of norepinephrine granules to epinephrine granules did not change, and the norepinephrine serum levels decreased, suggesting the inhibition of norepinephrine release. Our results show that nonylphenol influences the activity of the newt adrenal gland; considering the physiological role of this gland, our results suggest that nonylphenol may contribute to amphibian decline.     

High aquatic niche overlap in the newts Triturus cristatus and T. marmoratus (Amphibia,Urodela)

We studied spatial niche metrics of large-bodied newts (Triturus cristatus and T. marmoratus) in three breeding ponds in western France. Adults and larvae were sampled with underwater funnel traps. Larvae were identified to the species with diagnostic microsatellite DNA markers. The distribution of adult T. cristatus and T. marmoratus across pond regions differed in one out of six cases, no differences were observed between larvae (two ponds studied). Niche overlap and niche breadth indices across resource states defined as pond regions or individual traps were high (Schoener's C: pond regions 0.60–0.98, traps 0.35–0.71; Levins' B: pond regions 0.71–0.98, traps 0.35–0.76). Adults of large-bodied newts significantly differed in resource use from small-bodied newts (T. helveticus). The results are discussed in view of the occurrence of interspecific breeding attempts, and the unpredictable ecological characteristics of newt breeding ponds.     

Organization of histamine-containing neurons in the brain of the crested newt,Triturus carnifex

The distribution of immunoreactivity for histamine was studied in the brain of the urodele Triturus carnifex using the indirect immunofluorescence method. Histamine-immunoreactive cell bodies were localized in the caudal hypothalamus within the dorsolateral walls of the infundibular recesses. These immunoreactive cell bodies were pear-shaped, bipolar and frequently of the cerebrospinal-fluid-contacting type. Histaminergic nerve fibers were detected in almost all parts of the brain. Dense innervation was seen in the telencephalic medial pallium and ventral striatum, the neuropil of the preoptic area, the septum, the paraventricular organ, the posterior commissure, the caudal hypothalamus, the ventral and lateral mesencephalic tegmentum. Medium density innervation was observed in the lateral mesencephalic tegmentum and optic tectum. Poor innervation was present in the telencephalic dorsal pallium and in the central gray of the medulla oblongata. Few fibers occurred in the olfactory bulbs and in the telencephalic lateral pallium. Double immunofluorescence staining, using an antibody against tyrosine hydroxylase, showed that histamine-immunostained somata and those containing tyrosine-hydroxylase-like immunoreactivity were co-distributed in the tuberal hypothalamus. No co-occurrence of histamine-like and tyrosine hydroxylase-like immunostaining was seen in the same neuron. The pattern of histamine-immunoreactive neurons in the newt was similar to that described in other vertebrates. Our observations, carried out on the apparently simplified brain of the newt confirm that the basic histaminergic system is well conserved throughout vertebrates.     

Distribution of catecholaminergic and serotoninergic systems in forebrain and midbrain of the newt,Triturus alpestris (Urodela)

Summary Mapping of monoaminergic systems in the brain of the newt Triturus alpestris was achieved with antisera against (1) thyrosine hydroxylase (TH), (2) formaldehyde-conjugated dopamine (DA), and (3) formaldehyde-conjugated serotonin (5-HT). In the telencephalon, the striatum was densely innervated by a large number of 5-HT-, DA-and TH-immunoreactive (IR) fibers; IR fibers were more scattered in the amygdala, the medial and lateral forebrain bundles, and the anterior commissure. In the anterior and medial diencephalon, TH-IR perikarya contacting the cerebrospinal fluid (CSF-C perikarya) were located in the preoptic recess organ (PRO), the organum vasculosum laminae terminalis and the suprachiasmatic nucleus. Numerous TH-IR perikarya, not contacting the CSF, were present in the posterior preoptic nucleus and the ventral thalamus. At this level, DA-IR CSF-C neurons were only located in the PRO. In the posterior diencephalon, large populations of 5-HT-IR and DA-IR CSF-C perikarya were found in the paraventricular organ (PVO) and the nucleus infundibularis dorsalis (NID); the dorsal part of the NID additionally presented TH-IR CSF-C perikarya. Most regions of the diencephalon showed an intense monoaminergic innervation. In addition, numerous TH-IR, DA-IR and 5-HT-IR fibers, orginating from the anterior and posterior hypothalamic nuclei, extended ventrally and reached the median eminence and the pars intermedia of the pituitary gland. In the midbrain, TH-IR perikarya were located dorsally in the pretectal area. Ventrally, a large group of TH-IR cell bodies and some weakly stained DA-IR and 5-HT-IR neurons were observed in the posterior tuberculum. No dopaminergic system equivalent to the substantia nigra was revealed. The possible significance of the differences in the distribution of TH-IR and DA-IR neurons is discussed, with special reference to the CSF-C neurons.Abbreviations AM amygdala - CAnt commissura anterior - CH commissura hippocampi - CP commissura posterior - Ctm commissura tecti mesencephali - DH dorsal hypothalamus - DTh dorsal thalamus - FLM fasciculus longitudinalis medialis - Fsol fasciculus solitarius - H habenula - LFB lateral forebrain bundle - ME median eminence - MFB medial forebrain bundle - NID nucleus infundibularis dorsalis - nIP neuropil of nucleus interpeduncularis - NPOP nucleus preopticus posterior - NS nucleus septi - OVLT organum vasculosum laminae terminalis - PD pars distalis - Pdo dorsal pallium - PHi primordium hippocampi - PI pars intermedia - Pl lateral pallium - PN pars nervosa - PRO preoptic recess organ - Ptec pretectal area - PVO paraventricular organ - Ra nucleus raphe - Rm nucleus reticularis medius - SCO subcommisural organ - ST striatum; strm stria medullaris thalami - strt stria terminalis thalami - TM tegmentum mesencephali - TO tectum opticum - TP tuberculum posterius - trch tractus cortico-habenularis - trmp tractus mamillopeduncularis - VH ventral hypothalamus - Vm nucleus motorius nervi trigemini - VTh ventral thalamus - II optic nerve     

The organization of serotonin-immunoreactive neuronal systems in the brain of the crested newt,Triturus cristatus carnifex Laur.

Summary The distribution of serotonin (5-HT) immunoreactive structures has been investigated in the brain of the crested newt by means of indirect immunofluorescence, and unlabeled antibody peroxidase-antiperoxidase-complex (PAP) or biotin-avidin-system (BAS) techniques. In the newt, the bulk of the serotoninergic system extends from the raphe region of the medulla oblongata, through the isthmus, toward the mesencephalic tegmentum, and is characterized by pyriform neurons mainly located in a subependymal position, close to the midline. Also in the caudal hypothalamus, in addition to some 5-HT-positive adenohypophysial cells, many immunoreactive CSF-contacting neurons are found lining the paraventricular organ and the nucleus infundibularis dorsalis. A rich serotoninergic innervation was observed in the preoptic area and in the habenular complex. Concerning the telencephalon, immunopositive nerve fibers are encountered in the dorsal pallium, primordium hippocampi, striatum and olfactory bulbs. The general organization of serotoninergic systems in the newt brain exhibit close similarities to that described in higher vertebrates.     

Additional acetylcholine (ACh) binding site at alpha4/alpha4 interface of (alpha4beta2)2alpha4 nicotinic receptor influences agonist sensitivity

Nicotinic acetylcholine receptor (nAChR) α4 and β2 subunits assemble in two alternate stoichiometries to produce (α4β2)(2)α4 and (α4β2)(2)β2, which display different agonist sensitivities. Functionally relevant agonist binding sites are thought to be located at α4(+)/β2(-) subunit interfaces, but because these interfaces are present in both receptor isoforms, it is unlikely that they account for differences in agonist sensitivities. In contrast, incorporation of either α4 or β2 as auxiliary subunits produces isoform-specific α4(+)/α4(-) or β2(+)/β2(-) interfaces. Using fully concatenated (α4β2)(2)α4 nAChRs in conjunction with structural modeling, chimeric receptors, and functional mutagenesis, we have identified an additional site at the α4(+)/α4(-) interface that accounts for isoform-specific agonist sensitivity of the (α4β2)(2)α4 nAChR. The additional site resides in a region that also contains a potentiating Zn(2+) site but is engaged by agonists to contribute to receptor activation. By engineering α4 subunits to provide a free cysteine in loop C at the α4(+)α4(-) interface, we demonstrated that the acetylcholine responses of the mutated receptors are attenuated or enhanced, respectively, following treatment with the sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate or aminoethyl methanethiosulfonate. The findings suggest that agonist occupation of the site at the α4(+)/(α4(-) interface leads to channel gating through a coupling mechanism involving loop C. Overall, we propose that the additional agonist site at the α4(+)/α4(-) interface, when occupied by agonist, contributes to receptor activation and that this additional contribution underlies the agonist sensitivity signature of (α4β2)(2)α4 nAChRs.     

Two neuronal nicotinic acetylcholine receptors, alpha4beta4 and alpha7, show differential agonist binding modes

Nicotinic acetylcholine receptors (nAChRs) are pentameric, neurotransmitter-gated ion channels responsible for rapid excitatory neurotransmission in the central and peripheral nervous systems, resulting in skeletal muscle tone and various cognitive effects in the brain. These complex proteins are activated by the endogenous neurotransmitter ACh as well as by nicotine and structurally related agonists. Activation and modulation of nAChRs has been implicated in the pathology of multiple neurological disorders, and as such, these proteins are established therapeutic targets. Here we use unnatural amino acid mutagenesis to examine the ligand binding mechanisms of two homologous neuronal nAChRs: the α4β4 and α7 receptors. Despite sequence identity among the residues that form the core of the agonist-binding site, we find that the α4β4 and α7 nAChRs employ different agonist-receptor binding interactions in this region. The α4β4 receptor utilizes a strong cation-π interaction to a conserved tryptophan (TrpB) of the receptor for both ACh and nicotine, and nicotine participates in a strong hydrogen bond with a backbone carbonyl contributed by TrpB. Interestingly, we find that the α7 receptor also employs a cation-π interaction for ligand recognition, but the site has moved to a different aromatic amino acid of the agonist-binding site depending on the agonist. ACh participates in a cation-π interaction with TyrA, whereas epibatidine participates in a cation-π interaction with TyrC2.     

Regulation of alpha3 nicotinic acetylcholine receptor subunit mRNA levels by nerve growth factor and cyclic AMP in PC12 cells

To investigate the effects of nerve growth factor (NGF) and cyclic AMP (cAMP) on the level of the nicotinic acetylcholine receptor subunit alpha3 mRNA, we used PC12h cells, PC12 cells expressing dominant-negative Ras protein, and the parental PC12 cells. PC12h cells have NGF-responsive tyrosine hydroxylase activity. Expression of dominant-negative Ras protein prevents the signaling through the Ras-mitogen-activated protein kinase cascade. The morphological changes of the parental PC12 cells in response to NGF and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPTcAMP), a cell-penetrating cAMP analogue, were similar to those of PC12h cells. NGF up-regulated the alpha3 mRNA level in PC12h cells and down-regulated the alpha3 mRNA level in the parental PC12 cells. Expression of dominant-negative Ras protein and an inhibitor of mitogen-activated protein kinase kinase inhibited the effects of NGF on alpha3 mRNA level. CPTcAMP down-regulated the alpha3 mRNA level in all three PC12 cell lines. An inhibitor of protein kinase A inhibited the CPTcAMP-induced down-regulation of alpha3 mRNA. The alpha3 mRNA down-regulation required prolonged treatment with CPTcAMP even after cAMP response element binding protein phosphorylation was decreased. Membrane depolarization with high K+ had no effect on the alpha3 mRNA level in PC12h cells. Based on these results, we propose that at least two unknown effectors regulate alpha3 mRNA levels in PC12 cells.     

Contributions from Caenorhabditis elegans functional genetics to antiparasitic drug target identification and validation: nicotinic acetylcholine receptors, a case study

Following the complete sequencing of the genome of the free-living nematode, Caenorhabditis elegans, in 1998, rapid advances have been made in assigning functions to many genes. Forward and reverse genetics have been used to identify novel components of synaptic transmission as well as determine the key components of antiparasitic drug targets. The nicotinic acetylcholine receptors (nAChRs) are prototypical ligand-gated ion channels. The functions of these transmembrane proteins and the roles of the different members of their extensive subunit families are increasingly well characterised. The simple nervous system of C. elegans possesses one of the largest nicotinic acetylcholine receptor gene families known for any organism and a combination of genetic, microarray, physiological and reporter gene expression studies have added greatly to our understanding of the components of nematode muscle and neuronal nAChR subtypes. Chemistry-to-gene screens have identified five subunits that are components of nAChRs sensitive to the antiparasitic drug, levamisole. A novel, validated target acting downstream of the levamisole-sensitive nAChR has also been identified in such screens. Physiology and molecular biology studies on nAChRs of parasitic nematodes have also identified levamisole-sensitive and insensitive subtypes and further subdivisions are under investigation.     

The nicotinic acetylcholine receptor gene family of the nematode Caenorhabditis elegans: an update on nomenclature

The simple nematode, Caenorhabditis elegans, possesses the most extensive known gene family of nicotinic acetylcholine receptor (nAChR)-like subunits. Whilst all show greatest similarity with nAChR subunits of both invertebrates and vertebrates, phylogenetic analysis suggests that just over half of these (32) may represent other members of the cys-loop ligand-gated ion channel superfamily. We have introduced a novel nomenclature system for these “Orphan” subunits, designating them as lgc genes (ligand-gated ion channels of the cys-loop superfamily), which can also be applied in future to unnamed and uncharacterised members of the cys-loop ligand-gated ion channel superfamily. We present here the resulting updated version of the C. elegans nAChR gene family and related ligand-gated ion channel genes.     

Evolution of highly repeated DNA within the genusTriturus (Amphibia,Urodela)

Highly repeated DNA is a main feature of urodele amphibian genomes. InTriturus this class of DNA consists of several sequence families differently arranged at both the molecular and the chromosomal level, showing varying degrees of conservation across species. Present data on highly repeated DNA inTriturus are here summarized and discussed with regard to the evolution and possible functional role of these sequences.     

Search for alpha4 and alpha7 nicotinic acetylcholine receptor markers in a pedigree of benign familial infantile convulsions (BFIC)

In this study we investigate the possible involvement of the recently reported locus for benign familial infantile convulsions (BFIC) in human chromosome 19 and that of the neuronal acetylcholine receptor 4 (CHRNA4) and 7 (CHRNA7) subunits in a family with at least twelve clinically diagnosed cases of BFIC. Six polymorphic microsatellite markers covering the BFIC locus on chromosomal region 19q, one marker for CHRNA4 (chromosome 20) and two for CHRNA7 (chromosome 15) were used for the screening. The two-point lod score analysis showed no evidence of BFIC phenotype on chromosome 19. Similarly, when markers for chromosome 20 (CHRNA4 intron1, Amplimer: CHRNA4. PCR.1) and chromosome 15 (D15S165 and D15S1010) were used, score analysis showed no indication of linkage. The most likely interpretation of these results is that BFIC is a genetically heterogeneous form of epilepsy.     

The distribution of GABA-like-immunoreactive neurons in the brain of the newt,Triturus cristatus carnifex,and the green frog,Rana esculenta

Summary The distribution of -aminobutyric acid (GABA) immunoreactivity was studied in the brain of two amphibian species (Triturus cristatus carnifex, Urodela; Rana esculenta, Anura) by employing a specific GABA antiserum. A noteworthy immunoreactive neuronal system was found in the telencephalic dorsal and medial pallium (primordium pallii dorsalis and primordium hippocampi) and in the olfactory bulbs. In the diencephalic habenular nuclei there was a rich GABAergic innervation, and immunoreactive neurons were observed in the dorsal thalamus. In the hypothalamus the GABA immunoreactivity was found in the preoptic area, the paraventricular organ and in the hypothalamo-hypophysial complex. In the preoptic area of the frog some GABA-immunoreactive CSF-contacting cells were shown. In the optic tectum immunolabeled neurons were present in all the cellular layers. A rich GABAergic innervation characterized both the fibrous layers of the tectum and the neuropil of the tegmentum and interpeduncular nucleus. In the cerebellum, in addition to the Purkinje cells showing a variable immunopositivity, some immunoreactive cell bodies appeared in the central grey. Abundant immunolabeled nerve fibers in the acoustico-lateral area and some immunopositive neurons in the region of the raphe nucleus were observed. In conclusion, the GABAergic central systems, well-developed in the amphibian species studied, were generally characterized by close similarities to the pattern described in mammals.Dedicated to Professor Valdo Mazzi (Dipartimento di Biologia Animale, Università di Torino), in honor of his 70th birthday     

Characterization of the human nicotinic acetylcholine receptor subunit alpha (alpha) 9 (CHRNA9) and alpha (alpha) 10 (CHRNA10) in lymphocytes

Though the nicotinic acetylcholine receptor (nAChR) subunits alpha9 and alpha 10 have been thoroughly characterized within hair cells of the organ of Corti in the inner ear, prior studies have shown that they are also expressed in lymphocytes. In this report, we sought to more definitively characterize the nAChR subunits alpha9 and alpha10 within various populations of human lymphocytes. Using a combination of techniques, including RT-PCR, single-cell RT-PCR, Northern and western blot analysis, and immunofluorescence, expression of both alpha9 and alpha 10 was demonstrated in purified populations of T-cells (CD3+, CD4+, CD8+ and the Jurkat, MT2 and CEM T-cell lines) and B-cells (CD19+, CD80+ and EBV-immortalized B-cells). Single-lymphocyte recording techniques failed to identify an ionic current in response to applied acetylcholine in either T-cells or B-cells. These results clearly demonstrate the presence of these nicotinic receptor subunits within several populations of human lymphocytes, implicating their role in the immune response. However, a lack of demonstrated response to applied acetylcholine using standard single-cell recording techniques suggests a physiology different than that seen in hair cells of the inner ear.     

Morph switching in a dimorphic population of <Emphasis Type="Italic">Triturus alpestris</Emphasis> (Amphibia,Caudata)

The usual life cycle of Alpine newts comprises an aquatic larval stage and a terrestrial juvenile and adult stage. However, some populations differ from this pattern in exhibiting facultative paedomorphosis where some individuals reach sexual maturity while retaining larval traits such as gills and gill slits. While paedomorphic newts can, in some circumstances, initiate metamorphosis, once a newt has commenced metamorphosis, the state is irreversible. Because the frequency of this switching from one morph to the other has never been quantified in the wild, we attempted to estimate switching rate and survival by carrying out a 3-year monitoring survey of a population inhabiting an alpine lake. While morph switching did occur in this population, it involved a relatively low proportion of the paedomorphs (approx. 12%), suggesting that metamorphosis is not favoured in the study population. The hypothesis of paedomorphic advantage was not supported since neither survival nor body condition differed between morphs. The ontogenetic pathway of wild Alpine newts is thus characterised by two forks in the developmental pathway. The first occurs during the larval stage (metamorphosis vs. paedomorphosis), and the second occurs in paedomorphic adults (switching for metamorphosis vs. continuation of the paedomorphic lifestyle). Such a two-level decision process may allow individuals to cope with environmental uncertainty.     

The juxtaglomerular apparatus in the mesonephros of Newt (Triturus cristatus)

Summary 1)As in mammals, the juxtaglomerular apparatus of the Newt (Triturus cristatus) is composed by cells of the media of the afferent glomerular arteriole and by cells of the intermediary tubule. 2) The cells of the media of the glomerular arteriole are of two different types: granular and agranular cells. 3) The intermediary tubule is formed by dark and light cells. 4) Part of interrenal body is located close to glomerular arteriole and intermediary tubule.This work was supported by grant of Consiglio Nazionale delle Ricerche of Italy (C.N.R.) N. 115/815/04677.     

Structure and activity of alpha-conotoxin PeIA at nicotinic acetylcholine receptor subtypes and GABA(B) receptor-coupled N-type calcium channels

α-Conotoxins are peptides from cone snails that target the nicotinic acetylcholine receptor (nAChR). RgIA and Vc1.1 have analgesic activity in animal pain models. Both peptides target the α9α10 nAChR and inhibit N-type calcium channels via GABA(B) receptor activation, but the mechanism of action of analgesic activity is unknown. PeIA has previously been shown to inhibit the α9α10 and α3β2 nAChRs. In this study, we have determined the structure of PeIA and shown that it is also a potent inhibitor of N-type calcium channels via GABA(B) receptor activation. The characteristic α-conotoxin fold is present in PeIA, but it has a different distribution of surface-exposed hydrophobic and charged residues compared with Vc1.1. Thus, the surface residue distribution, rather than the overall fold, appears to be responsible for the 50-fold increase in selectivity at the α3β2 nAChR by PeIA relative to Vc1.1. In contrast to their difference in potency at the nAChR, the equipotent activity of PeIA and Vc1.1 at the GABA(B) receptor suggests that the GABA(B) receptor is more tolerant to changes in surface residues than is the nAChR. The conserved Asp-Pro-Arg motif of Vc1.1 and RgIA, which is crucial for potency at the α9α10 nAChR, is not required for activity at GABA(B) receptor/N-type calcium channels because PeIA has a His-Pro-Ala motif in the equivalent position. This study shows that different structure-activity relationships are associated with the targeting of the GABA(B) receptor versus nAChRs. Furthermore, there is probably a much more diverse range of conotoxins that target the GABA(B) receptor than currently realized.     

Insect nicotinic acetylcholine receptor gene families: from genetic model organism to vector,pest and beneficial species

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in the insect nervous system and are targets of a major group of insecticides, the neonicotinoids. Analyses of genome sequences have shown that nAChR gene families remain compact in diverse insect species, when compared to their mammalian counterparts. Thus, Drosophila melanogaster and Anopheles gambiae each possess 10 nAChR genes while Apis mellifera has 11. Although these are among the smallest nAChR gene families known, receptor diversity can be considerably increased by alternative splicing and mRNA A-to-I editing, thereby generating species-specific subunit isoforms. In addition, each insect possesses at least one highly divergent nAChR subunit. Species-specific subunit diversification may offer promising targets for future rational design of insecticides that act on particular pests while sparing beneficial insects. Electrophysiological studies on cultured Drosophila cholinergic neurons show partial agonist actions of the neonicotinoid imidacloprid and super-agonist actions of another neonicotinoid, clothianidin, on native nAChRs. Recombinant hybrid heteromeric nAChRs comprising Drosophila Dα2 and a vertebrate β2 subunit have been instructive in mimicking such actions of imidacloprid and clothianidin. Unitary conductance measurements on native nAChRs indicate that more frequent openings of the largest conductance state may offer an explanation for the superagonist actions of clothianidin.     

Base docking model of the homomeric alpha7 nicotinic receptor-beta-amyloid(1-42) complex

A homology model of the human alpha7 nicotinic receptor was constructed based on the acetylcholine-binding protein crystal structure. Subsequently, the three-dimensional structure of the complex between the alpha7 nicotinic receptor and the 42-amino acid beta-amyloid peptide was obtained for the first time with the aid of the ESCHER program, a well-known method for protein-protein docking. The final complex showed that the most important interactions occur between the residues V12-K28 from the peptide and the loop C of the receptor. The model agrees with many experimental data, and may be used as a base model for further detailed studies in order to gain insight into the binding and dynamics of the complex at molecular level and their correlation with the memory impairments characteristic of the Alzheimer's disease.