16. Liste détaillée des publications

16.1 Analyse de séquence

Le Novère N, Changeux JP (1995). Molecular evolution of the nicotinic acetylcholine receptor subunit family: an example of multigene family in excitable cells. Journal of Molecular Evolution 40 : 155-172.

Résumé:

An extensive phylogenetic analysis of the nicotinic acetylcholine receptor subunit genes family has been performed by cladistic and phenetic methods. The conserved parts of amino acid sequences have been analyzed by CLUSTALV and PHYLIP software. The structure of the genes was also taken in consideration. The results show that a first gene duplication may have occurred before the appearance of Bilateria. Three subfamilies then appeared: I-the neuronal $\alpha$-bungarotoxin binding site subunits ($\alpha$7, $\alpha$8), III-the neuronal nicotinic subunits ($\alpha$2-$\alpha$6, $\beta$2-$\beta$4) which also contains the muscle acetylcholine binding subunit ($\alpha$1), and IV-the muscle non-$\alpha$ subunits ($\beta$1, $\gamma$, $\delta$, $\epsilon$). The Insecta subunits (subfamily II) could be orthologous to family III and IV. Several tissular switches of expression from neuron to muscle and the converse can be inferred from the extant expression of subunits and the reconstructed trees. The diversification of the neuronal nicotinic subfamily begins in the stem lineage of chordates, the last duplications occurring shortly before the onset of the mammalian lineage. Such evolution parallels the increase in complexity of the cholinergic systems.

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Le Novère N, Changeux JP (1999). The LigandGated Ion Channel Database. Nucleic Acid Research 27 : 340-342.

Résumé:

The Ligand Gated Ion Channels (LGICs) are ionotropic receptors to neurotransmitters. Their physiological effect is carried out by the opening of an ionic channel upon binding of a particular neurotransmitter. These LGICs constitute superfamilies of receptors formed by homologous subunits. A database has been developed to handle the growing wealth of cloned subunits. This database contains nucleic acid sequences, protein sequences, as well as multiple sequence alignments and phylogenetic studies. This database is accessible via the worldwide web (http://www.pasteur.fr/units/neubiomol/LGIC.html), where it is continuously updated. A downloadable version is also available (currently v0.1 (98.06)).

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Le Novère N, Corringer, PJ, Changeux JP. Improved secondary structure prediction of a nicotinic receptor subunit. Incorporation of solvent accessibility and experimental data into a 2D representation. soumis pour publication.

16.2 Physiologie du nAChR

Zoli M, Le Novère N, Hill JA, Changeux JP (1995). Developmental regulation of nicotinic ACh receptor subunit mRNAs in the rat central and peripheral nervous systems. Journal of Neuroscience 15 : 1912-1939.

Résumé:

In the present study we have investigated the anatomical distribution pattern of nAChR $\alpha$3, $\alpha$4, $\beta$2 and $\beta$4 subunit mRNAs during prenatal and perinatal development of the rat CNSand PNS. Three main developmental patterns have been recognized. (1) In the majority of cases studied (caudal brain, spinal cord, dorsal root ganglia, trigeminal and geniculate ganglia) all four subunit mRNAs are initially (E11-13) detected but, during subsequent prenatal development, the level of some of these subunit mRNAs ($\alpha$3 and $\beta$4 in the brain and spinal cord, $\alpha$4 and $\beta$4 in the dorsal root ganglia, $\alpha$4 in the visceral sensory ganglia, and $\alpha$3, $\alpha$4 and $\beta$4 in the somatic sensory ganglia) become undetectable. (2) In the case of the cerebral cortex a pair of subunit mRNAs ($\alpha$3 and $\beta$2) is initially (E12-13) expressed followed by a repression of the $\alpha$3 subunit (E15) and the subsequent (E17-19) induction of the $\alpha$4 subunit. (3) Only some subunit mRNAs are initially (E12-15) expressed in the retina ($\alpha$3-$\alpha$4-$\beta$2-$\beta$4), parasympathetic or sympathetic motor ganglia ($\alpha$3-$\beta$2-$\beta$4), and vestibulo-cochlear ganglia ($\alpha$4-$\beta$2) and their level remains stable throughout prenatal and early postnatal development.

Overall, in most central and peripheral structures the appearance of nAChR subunit mRNAs is precocious and temporally related to the timing of neuronal differentiation. In addition, in several structures the expression of certain subunits ($\alpha$3, $\alpha$4 or $\beta$4) is transient, althought not $\beta$2. Finally, the comparison of the different regional distribution patterns suggests that a limited number of structure-specific receptor isoforms are functional during development of CNS and PNS.

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Picciotto MR, Zoli M, Léna C, Bessis A, Lallemand Y, Le Novère N, Vincent P, Merlo-Pich E, Brûlet P, Changeux JP (1995). Abnormal avoidance learning in mice lacking functional high-affinity nicotine receptor in the brain. Nature 374 : 65-67.

Résumé:

Nicotine affects many aspects of behaviour including learning and memory throught its interactions with neuronal nicotinic acetylcholine receptors (nAChR). Functional nAChR are pentameric proteins containing at least one type of $\alpha$-subunit and one type of $\beta$-subunit. The involvment of a particular neuronal nicotinic subunit in pharmacology and behaviour was examined using gene targeting to mutate $\beta$2, the most widely expressed nAChR subunit in the central nervous system. We report here that high affinity binding sites for nicotine are absent form the brains of mice homozygous for the $\beta$2-subunit mutation. Further, electrophysiological recording from brain slices reveals that thalamic neurons from these mice do not respond to nicotine application. Finally, behavioural tests demonstrate that nicotine no longer augments the performance of $\beta$2^-/- mice on passive avoidance, a test of associative memory. Paradoxically, mutant mice are able to perform better than their non-mutant siblings on this task.

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Bessis A, Salmon AM, Zoli M, Le Novère N, Picciotto M, Changeux JP (1995). Promoter elements conferring neuron-specific expression of the beta2 subunit of the neuronal nicotinic acetylcholine receptor studied in vitro and in transgenic mice. Neuroscience 69 : 807-819.

Résumé:

Several genes encoding subunits of the neuronal nicotinic acetylcholine receptors have been cloned and regulatory elements involved in the transcription of the $\alpha$2 and $\alpha$7-subunit genes have been described. Yet, the detailed mechanisms governing the neuron-specific transcription and the spatio-temporal expression pattern of these genes remain largely uninvestigated. The $\beta$2-subunit is the most widely expressed neuronal nicotinic receptor subunit in the nervous system. We have studied the structural and regulatory properties of the 5' sequence of this gene. A fragment of 1163 bp of upstream sequence is sufficient to drive the cell-specific transcription of a reporter gene in both transient transfection assays and in transgenic mice. Deletion analysis and site-directed mutagenesis of this promoter reveal two negative elements and one positive element. The positively-acting sequence includes one functional E-box. One of the repressor elements is located in the transcribed region and is the NRSE/RE1 sequence already described in promoters of neuronal genes. In this paper, we describe the neuron-specific promoter of the gene encoding the neuronal nicotinic acetylcholine receptor $\beta$2-subunit.

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Le Novère N, Zoli M, Changeux JP (1996). Neuronal nicotinic receptor $\alpha$6 subunit mRNA is selectively concentrated in catecholaminergic nuclei of the rat brain. European Journal of Neuroscience 8 : 2428-2439.

Résumé:

Although the neuronal nicotinic receptor a6 subunit was cloned several years ago, its functional significance remains to be investigated. Here we describe an in situ hybridisation study of the mRNA coding for this subunit in the adult rat central nervous system using oligonucleotide probes. Specific $\alpha$6 mRNA labelling was restricted to a few nuclei throughout the brain; it was particularly high in several catecholaminergic nuclei [the locus cœruleus (A6), the ventral tegmental area (A10) and the substantia nigra (A9)] at levels significantly higher than those found for any other known nicotinic receptor subunit mRNA. Labelling for $\alpha$6 mRNA was also detected at lower levels in the reticular thalamic nucleus, the supramammillary nucleus and the mesencephalic V nucleus. Some cells of the medial habenula (medioventral part) and of the interpeduncular nucleus (central and lateral parts) were also labelled. The distribution of $\alpha$6 mRNA was compared with the distribution of the other known nicotinic acetylcholine receptor subunit mRNAs. In several nuclei, the expression of $\alpha$6 was complementary to those of other a subunits. Moreover, some of the cell groups (such as the substantia nigra, the ventral tegmental area and the locus coeruleus) previously thought to contain mainly $\alpha$3 mRNA in fact were found to contain high levels of $\alpha$6 mRNA. Finally, we found extensive colocalisation of $\alpha$6 and $\beta$3, indicating the possible existence of nicotinic receptor hetero-oligomers containing both subunits. The present results show that a6 is the major nicotinic acetylcholine receptor a subunit expressed in dopaminergic cell groups of the mesencephalon and noradrenergic cells of the locus cœruleus. This suggests the involvment of the $\alpha$6 subunit in some of the major functions of central nicotinic circuits, including modulation of locomotor behaviour and reward.

Marubio LM, Arroyo-Jimenez MM, Cordero-Erausquin M, Léna C, Le Novère N, De Kerchove d'Exaerde A, Huchet M, Damaj MI, Changeux JP. Reduced nicotine-elicited antinociception in mice lacking the neuronal nicotinic alpha-4 subunit. Soumis pour publication.

16.3 Articles de collaboration technique

Li XM, Zoli M, Finnman UB, Le Novère N, Changeux JP, Fuxe, K (1995). A single (-)-nicotine injection causes change with a time delay in the affinity of striatal D2 receptors for antagonist, but not for agonist, nor in the D2 receptor mRNA levels in the rat substantia nigra. Brain Research 679 : 157-167.

Résumé:

The in vitro and in vivo effects of (-)-nicotine on dopamine D₂ receptors in the rat neostriatum have been studied using biochemical binding, in situ hybridization and immunocytochemistry. A single i.p. injection (1 mg/kg) of (-)-nicotine resulted in a reduction of the K_D value of the D₂ antagonist [³H]raclopride binding sites in rat neostriatal membrane preparations at 12 h without any significant change in the B_max value. This action of (-)-nicotine was counteracted by pretreatment 15 min earlier with the nicotine antagonist mecamylamine (1 mg/kg, i.p.). However, the K_D and the B_max values of the D₂ agonist [³H]NPA binding sites in the rat neostriatal membrane preparations were not significantly affected 0.5-45 h after a single i.p. injection with 1 mg/kg of (-)-nicotine. No significant change in neostriatal D₂ receptor mRNA levels was observed at any time interval after the (-)-nicotine injection. No significant change was observed in tyrosine hydroxylase (TH) immunoreactivity in either the substantia nigra or the neostriatum, nor in nigral TH mRNA levels during the time interval studied (4-24 h posttreatment). Furthermore, addition of low (10 nM) or high (1 µM) concentrations of (-)-nicotine in vitro to rat neostriatal membranes did not alter the characteristics of [³H]raclopride or [³H]NPA binding. These results indicate that a single (-)-nicotine injection can produce a selective and delayed increase in the affinity of D₂ receptors for the antagonist, but not for the agonist without modifying the levels of D₂ receptor mRNA, probably via the activation of central nicotinic receptors.

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Beeri R, Le Novère N, Mervis R, Huberman T, Grauer E, Changeux JP, Soreq H (1997). Enhanced hemicholinium binding and attenuated dendrite branching in cognitively impaired acetylcholinesterase-transgenic mice. Journal of Neurochemistry 68 : 441-2451.

Résumé:

In a search for behavioral, neuroanatomical, and metabolic characteristics of Alzheimer's disease that may result from cholinergic malfunction, we used transgenic mice overexpressing acetylcholinesterase (AChE) mRNA and active enzyme in brain neurons. Mapping by in situ hybridization revealed that transgenic and host AChE mRNAs were distributed similarly. In a Morris water maze working memory paradigm, adult transgenic mice did not display the characteristic improvement found in control mice either between and within test days and spend less time than control mice in the platform zone. IN 5-week-old transgenic mice, the basilar dendritic trees of layer 5 pyramidal neurons from the fronto-parietal cortex were essentially as developped as n age-matched contrals. However, branching totally ceased after this age, whereas in control adults it continued up to at least 7 months. Therefore, dendritic arbors became smaller in adult transgenic mice than those of controls. Furthermore, the average number of spines was significantly lower on dendritic branches of 7-months-old but not in 5-week-old transgenics as compared with controls. Binding of tritiated hemicholinium-3, a blocker of the high-affinity choline uptake characteristic of active cholinergic terminals, was over twofold enhanced in the brain of transgenic mice. In contrast, no differences were oberved in the mRNA and ligand binding levels of several different subtypes of nicotinic and muscarinic acetylcholine receptors. These findings suggest that three different hallmarks associated with Alzheilmer's disease -- namely, progressive cognitive failure, cessation of dendritic branching and spine formation, and enhanced high-affinity choline uptake -- are outcomes of cholinergic malfunction.

16.4 Revues

Le Novère N, Bessis A, Léna C, Picciotto MR, Zoli M (1993). Le récepteur nicotinique neuronal de l'acétylcholine: du gène au tabagisme. Médecine/Science 9 : 41-49.

Résumé:

Le récepteur nicotinique musculaire de l'acétylcholine est une protéine allostérique pentamérique, prototype de la superfamille des canaux ioniques activés par des neuromédiateurs. Une famille de récepteurs apparentés est localisée dans le système nerveux. Ces récepteurs, composés de deux sous-unités $\alpha$ engagées dans la liaison de l'acétylcholine, et de trois sous-unités non-$\alpha$, sont les agents moléculaires de la transmission nicotinique neuronale. Une dizaine de gènes codant pour des sous-unités sont actuellement identifiés et transcrits différemment selon la localisation anatomique. Les propriétés électrophysiologiques et pharmacologiques du récepteur dépendent de sa composition en sous-unités et sont modifiées par l'innervation ainsi que par diverses substances comme le Ca²⁺ et l'AMPc. L'expression du récepteur nicotinique neuronal est altérée lors de diverses pathologies.

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Changeux JP, Bessis A, Bourgeois JP, Corringer PJ, Devillers-Thiéry A, Eiselé JL, Kerszberg M, Léna C, Le Novère N, Picciotto M, Zoli M (1996). Nicotinic receptors and brain plasticity. Cold Spring Harbor Symposia of Quantitative Biology LXI : 343-362.

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Changeux JP, Bertrand D, Corringer PJ, Dehaene S, Edelstein S, Léna C, Le Novère N, Marubio L, Picciotto M, Zoli M (1998). Brain nicotinic receptors: structure and regulation, role in learning and reinforcement. Brain Research Reviews 26 : 198-216

Résumé:

The introduction, in the late sixties, of the concepts and methods of molecular biology to the study of the nervous system had a profound impact on the field, primarily through the identification of its basic molecular components. These structures include, for example, the elementary units of the synapse : neurotransmitters, neuropeptides and their receptors, but also ionic channels, intracellular second messengers and the relevant enzymes, cell surface adhesion molecules, or growth and trophic factors. Attempts to establish appropriate causal relationships between these molecular components, the actual organisation of neural networks, and a defined behavior, nevertheless, still must overcome many difficulties. A first problem is the recognition of the minimum levels of organisation, from the molecular, cellular, or multicellular (circuit) to the higher cognitive levels, that determine the given physiological and/or behavioral performance under investigation. A common difficulty (and potential source of errors of interpretation) is to relate a cognitive function to a network organization which does not possess the required structural complexity and vice-versa. Another problem is to distinguish, among the components of the system, those which are actually necessary and those which, taken together, suffice for a given behavior to take place. Identification of such a minimal set of building-blocks may receive decisive insights from the elaboration of neurally plausible formal models that bring together, within a single and coherent `artificial organism', the neuronal network, the circulating activity, and the behavior they determine. In this communication, we shall attempt, still in a preliminary fashion, to bring together : (1) our recent knowledge on the molecular biology of brain nicotinic receptors (nAChRs) and their allosteric properties and (2) integrated behaviors, such as cognitive learning, investigated for instance with delayed-response or passive avoidance tasks that are likely to involve nAChRs in particular at the level of reinforcement (or reward) mechanisms.

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Le Novère N (1998). Aspects théoriques et pratiques en hybridation in situ et autoradiographie réceptorielle. Annales de l'Institut Pasteur, actualités 9 : 167-179.

Résumé:

La neuro-anatomie chimique est une discipline formée à l'interface de la biochimie, au sens large, et de l'anatomie. Elle consiste en la détection, la localisation et la quantification de marqueurs moléculaires dans le système nerveux. Chaque technique de neuro-anatomie chimique apporte des données mais également l'utilisation conjointe des différentes techniques.

Les méthodes de neuro-anatomie chimique in situ permettent une localisation fine de la molécule recherchée atteignant parfois le niveau sub-cellulaire.

Cet article a pour objet les méthodes de localisation sur coupe de tissus. Nous nous focaliserons plus précisément sur l'autoradiographie réceptorielleet l'hybridation in situpar oligonucléotides, deux approches qui présentent des similitudes importantes au niveau fondamental et méthodologique.