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Accueil > Agenda > Les séminaires Jean Roche > Tracking cells with fluorescent proteins, from 3-D organization to in vivo (...)

Tracking cells with fluorescent proteins, from 3-D (...)

Lundi 21 novembre 2005,11h, salle Lissitzky.

Bibliographie

*1 : *Proc Natl Acad Sci U S A. 2005 Nov 4 ; [Epub ahead of print] Click here to read <http://www.ncbi.nlm.nih.gov/entrez/...> Click here to read <http://www.ncbi.nlm.nih.gov/entrez/...> *Revealing the large-scale network organization of growth hormone-secreting cells.*

*Bonnefont X* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Lacampagne A* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Sanchez-Hormigo A* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Fino E* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Creff A* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Mathieu MN* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Smallwood S* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Carmignac D* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Fontanaud P* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Travo P* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Alonso G* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Courtois-Coutry N* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Pincus SM* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Robinson IC* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Mollard P* <http://www.ncbi.nlm.nih.gov/entrez/...> .

Department of Endocrinology, Institute of Functional Genomics, Centre National de la Recherche Scientifique, Unite Mixte de Recherche 5203, Institut National de la Sante et de la Recherche Medicale U661, Universities of Montpellier 1 and 2, 141 Rue de la Cardonille, 34094 Montpellier Cedex 05, France ; Institut National de la Sante et de la Recherche Medicale U637, Centre Hospitalier Universitaire Arnaud de Villeneuve, 34295 Montpellier Cedex 05, France ; Division of Molecular Neuroendocrinology, National Institute of Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom ; Montpellier RIO Imaging, Centre de Recherches de Biochimie Macromoleculaire, Formation de Recherche en Evolution 2593, Centre National de la Recherche Scientifique, 1919 Route de Mende, 34293 Montpellier Cedex 05, France.

Pituitary growth hormone (GH)-secreting cells regulate growth and metabolism in animals and humans. To secrete highly ordered GH pulses (up to 1,000-fold rise in hormone levels in vivo), the pituitary GH cell population needs to mount coordinated responses to GH secretagogues, yet GH cells display an apparently heterogeneous scattered distribution in 2D histological studies. To address this paradox, we analyzed in 3D both positioning and signaling of GH cells using reconstructive, two-photon excitation microscopy to image the entire pituitary in GH-EGFP transgenic mice. Our results unveiled a homologous continuum of GH cells connected by adherens junctions that wired the whole gland and exhibited the three primary features of biological networks : robustness of architecture across lifespan, modularity correlated with pituitary GH contents and body growth, and connectivity with spatially stereotyped motifs of cell synchronization coordinating cell activity. These findings change our view of GH cells, from a collection of dispersed cells to a geometrically connected homotypic network of cells whose local morphology and connectivity can vary, to alter the timing of cellular responses to promote more coordinated pulsatile secretion. This large-scale 3D view of cell functioning provides a powerful approach to identify and understand other networks of endocrine cells that are thought to be scattered in situ. Many dispersed endocrine systems exhibit pulsatile outputs. We suggest that cell positioning and associated cell-cell connection mechanisms will be critical parameters that determine how well such systems can deliver a coordinated secretory pulse of hormone to their target tissues.


*2 : *Hum Mol Genet. 2004 Apr 1 ;13(7):771-80. Epub 2004 Feb 12.

Click here to read <http://www.ncbi.nlm.nih.gov/entrez/...> *Amino-acid changes acquired during evolution by olfactory receptor 912-93 modify the specificity of odorant recognition.*

*Gaillard I* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Rouquier S* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Chavanieu A* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Mollard P* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Giorgi D* <http://www.ncbi.nlm.nih.gov/entrez/...> .

IGH, CNRS UPR 1142 rue de la Cardonille, Cedex 5, France.

The sense of smell in mammals can perceive and discriminate a wide variety of volatile odorants. Odorants bind to specific olfactory receptors (ORs) to initiate an action potential that transduces olfactory information to the olfactory cortex. We previously identified the structural motifs of odorant molecules (aliphatic 2- or 3-ketones) required to activate mouse OR912-93 by detection of the odorant response using calcium measurement in transfected cells. In order to study changes in the specificity of this receptor that might have occurred during evolution, we cloned the orthologous genes from six primate species and pig and assayed the encoded receptors for responses to odorants. Primate OR912-93 orthologs share 88-97% sequence identity. All the receptors responded to 2- and 3-heptanone except the squirrel-monkey OR, which responded only to 3-heptanone, and the human and orangutan ORs, which were not functional. Directed mutagenesis allowed us to convert the squirrel-monkey response to that of the other functional 912-93 ORs by substituting three amino acids in the second extracellular loop. Orangutan and human 912-93 ORs regained function after restoration of the arginine residue in the DRY motif required for G-protein activation. However, the human receptor was constitutively activated in the absence of ligand stimulation. Using natural mutants of the OR912-93 receptor, we provide evidence that squirrel-monkeys evolved towards a restriction of the specificity of this receptor and therefore that slight alterations in the sequence of a receptor can induce subtle changes in recognition specificity.


*3 : *FEBS Lett. 2003 Jul 31 ;548(1-3):49-52.

Click here to read <http://www.ncbi.nlm.nih.gov/entrez/...> *Electrical activity in endocrine pituitary cells in situ : a support for a multiple-function coding.*

*Bonnefont X* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Mollard P* <http://www.ncbi.nlm.nih.gov/entrez/...> .

INSERM U469, Centre CNRS-INSERM de Pharmacologie Endocrinologie, 34094 Montpellier Cedex 5, France.

The anterior pituitary is an endocrine gland that controls basic body functions. Pituitary cell functioning depends on membrane excitability, which induces cytosolic calcium rises. Here, we reported the first identification of small-amplitude voltage fluctuations that controlled spike firing in endocrine cells recorded in situ. Three patterns of voltage fluctuations were distinguishable by their durations (1-100 s). These patterns could be ordered on top of each other, namely in response to secretagogues. Thus, pituitary endocrine cells express in situ a cell code in which small-amplitude voltage fluctuations lead to a multimodal arrangement of spike firing, which may finely tune calcium-dependent functions.


*4 : *Endocrinology. 2003 Jun ;144(6):2728-40.

Click here to read <http://www.ncbi.nlm.nih.gov/entrez/...> *Growth hormone-releasing hormone (GHRH) neurons in GHRH-enhanced green fluorescent protein transgenic mice : a ventral hypothalamic network.*

*Balthasar N* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Mery PF* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Magoulas CB* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Mathers KE* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Martin A* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Mollard P* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Robinson IC* <http://www.ncbi.nlm.nih.gov/entrez/...> .

Division of Molecular Neuroendocrinology, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.

The hypothalamic GHRH neurons secrete pulses of GHRH to generate episodic GH secretion, but little is known about the mechanisms involved. We have made transgenic mice expressing enhanced green fluorescent protein (eGFP) specifically targeted to the secretory vesicles in GHRH neurons. GHRH cells transported eGFP from cell bodies in the arcuate nucleus to extensively arborized varicose fiber terminals in the median eminence. Patch clamp recordings from visually identified GHRH cells in mature animals showed spontaneous action potentials, often firing in short bursts up to 10 Hz. GHRH neurons received frequent synaptic inputs, as demonstrated by the recording of abundant inward postsynaptic currents, but spikes were followed by large after-hyperpolarizations, which limited their firing rate. Because many GHRH neurons lie close to the ventral hypothalamic surface, this was examined by wide-field binocular epifluorescence stereomicroscopy. This approach revealed an extensive horizontal network of GHRH cells at low power and individual fiber projections at higher power in the intact brain. It also showed the dense terminal projections of the GHRH cell population in the intact median eminence. This model will enable us to characterize the properties of individual GHRH neurons and their structural and functional connections with other neurons and to study directly the role of the GHRH neuronal network in generating episodic secretion of GH.


*5 : *Trends Endocrinol Metab. 2002 Sep ;13(7):304-9.

Click here to read <http://www.ncbi.nlm.nih.gov/entrez/...> *Hidden face of the anterior pituitary.*

*Fauquier T* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Lacampagne A* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Travo P* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Bauer K* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Mollard P* <http://www.ncbi.nlm.nih.gov/entrez/...> .

INSERM Unit469-IFR3, Montpellier, France.

The traditional view holds that the anterior pituitary is an endocrine gland with a complex and heterogeneous distribution of cells throughout the parenchyma. Thus, a long-distance mode of intraorgan communication is not usually taken into account in our understanding of pituitary functioning. However, recent in situ pituitary studies have begun to unveil a hitherto unknown route of large-scale information transfer within the pituitary. Agranular folliculostellate cells - the sixth type of pituitary cell initially discovered almost half a century ago - are the functional units of a dynamically active cell network wiring the whole gland. Because folliculostellate cells communicate with their endocrine neighbors, this opens the door to considering the pituitary as a cellular puzzle more ordered than was first thought. Hence, cell networking within the pituitary gland could have a privileged role in coordinating the activities of distant cells in both physiological and pathological conditions.


*6 : *Eur J Neurosci. 2002 Feb ;15(3):409-18.

Click here to read <http://www.ncbi.nlm.nih.gov/entrez/...> *A single olfactory receptor specifically binds a set of odorant molecules.*

*Gaillard I* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Rouquier S* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Pin JP* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Mollard P* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Richard S* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Barnabe C* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Demaille J* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Giorgi D* <http://www.ncbi.nlm.nih.gov/entrez/...> .

IGH, CNRS UPR 1142, rue de la Cardonille, 34396 Montpellier cedex 5, France. gaillard igh.cnrs.fr

The sense of smell is mediated by the initiation of action potential in olfactory sensory neurons during odor stimulation. However, little is known about odorant-olfactory receptor (OR) recognition mechanisms. In the present work, we identified the structural motifs of odorant molecules required to activate mouse OR912-93 by detection of the odorant response using calcium measurement in cells transfected with OR and G(alpha)q and G(alpha)15 proteins. The use of sets of odorants led to the identification of ketones with an aliphatic carbon chain length >or= four carbon atoms and a carbonyl group preferentially located in position C2 or C3. The threshold of detection of these odorants is as low as 10(-6)-10(-8)m. No other odorant ligand, out of 70 representatives of the odorant world, was active. The human ortholog of OR912-93 is not functional, suggesting that apart from a stop-mutation located at the 5’-end that was corrected in the construct, it incurred other deleterious mutations during evolution.


*7 : *Proc Natl Acad Sci U S A. 2001 Jul 17 ;98(15):8891-6. Epub 2001 Jul 3.

Click here to read <http://www.ncbi.nlm.nih.gov/entrez/...> Click here to read <http://www.ncbi.nlm.nih.gov/entrez/...> *Folliculostellate cell network : a route for long-distance communication in the anterior pituitary.*

*Fauquier T* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Guerineau NC* <http://www.ncbi.nlm.nih.gov/entrez/...> , *McKinney RA* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Bauer K* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Mollard P* <http://www.ncbi.nlm.nih.gov/entrez/...> .

Institut National de la Sante et de la Recherche Medicale Unite 469, Centre National de la Recherche Scientifique-INSERM de Pharmacologie-Endocrinologie, 141 Rue de la Cardonille, 34094 Montpellier Cedex 5, France.

All higher life forms critically depend on hormones being rhythmically released by the anterior pituitary. The proper functioning of this master gland is dynamically controlled by a complex set of regulatory mechanisms that ultimately determine the fine tuning of the excitable endocrine cells, all of them heterogeneously distributed throughout the gland. Here, we provide evidence for an intrapituitary communication system by which information is transferred via the network of nonendocrine folliculostellate (FS) cells. Local electrical stimulation of FS cells in acute pituitary slices triggered cytosolic calcium waves, which propagated to other FS cells by signaling through gap junctions. Calcium wave initiation was because of the membrane excitability of FS cells, hitherto classified as silent cells. FS cell coupling could relay information between opposite regions of the gland. Because FS cells respond to central and peripheral stimuli and dialogue with endocrine cells, the form of large-scale intrapituitary communication described here may provide an efficient mechanism that orchestrates anterior pituitary functioning in response to physiological needs.


*8 : *Ann N Y Acad Sci. 2000 ;921:410-4.

Click here to read <http://www.ncbi.nlm.nih.gov/entrez/...> *PACAP and VIP induce changes in cytosolic calcium in putative folliculostellate cells of the mouse pituitary.*

*Kunzelmann P* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Creff A* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Bauer K* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Mollard P* <http://www.ncbi.nlm.nih.gov/entrez/...> .

INSERM U469, CCIPE, 141, Rue de la Cardonille, 34094 Montpellier, France. kunzel u469.montp.inserm.fr


*9 : *Endocrinology. 2000 Dec ;141(12):4681-9.

Click here to read <http://www.ncbi.nlm.nih.gov/entrez/...> *A secreted fluorescent reporter targeted to pituitary growth hormone cells in transgenic mice.*

*Magoulas C* <http://www.ncbi.nlm.nih.gov/entrez/...> , *McGuinness L* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Balthasar N* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Carmignac DF* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Sesay AK* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Mathers KE* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Christian H* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Candeil L* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Bonnefont X* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Mollard P* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Robinson IC* <http://www.ncbi.nlm.nih.gov/entrez/...> .

Division of Neurophysiology, National Institute for Medical Research Mill Hill, London, United Kingdom.

In stable transfection experiments in the GH-producing GC cell line, a construct containing the entire signal peptide and the first 22 residues of human GH linked in frame with enhanced green fluorescent protein (eGFP), produced brightly fluorescent cells with a granular distribution of eGFP. This eGFP reporter was then inserted into a 40-kb cosmid transgene containing the locus control region for the hGH gene and used to generate transgenic mice. Anterior pituitaries from these GH-eGFP transgenic mice showed numerous clusters of strongly fluorescent cells, which were also immunopositive for GH, and which could be isolated and enriched by fluorescence-activated cell sorting. Confocal scanning microscopy of pituitary GH cells from GH-eGFP transgenic mice showed a markedly granular appearance of fluorescence. Immunogold electron microscopy and RIA confirmed that the eGFP product was packaged in the dense cored secretory vesicles of somatotrophs and was secreted in parallel with GH in response to stimulation by GRF. Using eGFP fluorescence, it was possible to identify clusters of GH cells in acute pituitary slices and to observe spontaneous transient rises in their intracellular Ca2+ concentrations after loading with Ca2+ sensitive dyes. This transgenic approach opens the way to direct visualization of spontaneous and secretagogue-induced secretory mechanisms in identified GH cells.


*10 : *Endocrinology. 2000 Mar ;141(3):868-75.

Click here to read <http://www.ncbi.nlm.nih.gov/entrez/...> *Rhythmic bursts of calcium transients in acute anterior pituitary slices.*

*Bonnefont X* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Fiekers J* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Creff A* <http://www.ncbi.nlm.nih.gov/entrez/...> , *Mollard P* <http://www.ncbi.nlm.nih.gov/entrez/...> .

INSERM U-469, Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Montpellier, France.

Endocrine cells isolated from the anterior pituitary fire intracellular Ca2+ ([Ca2+]i) transients due to voltage-gated Ca2+ entry. However, the patterns of [Ca2+]i transients within the glandular parenchyma of the anterior pituitary are unknown. Here we describe, using real-time confocal laser microscopy, several spontaneous patterns of calcium signaling in acute pituitary slices prepared from male as well as cycling and lactating female rats. Forty percent of the cells demonstrated a spontaneous bursting mode, consisting of an active period of [Ca2+]i transients firing at a constant frequency, followed by a rest period during which cells were either silent or randomly active. The remaining recordings from endocrine cells either demonstrated random [Ca2+]i transients or were silent. These rhythmic bursts of [Ca2+]i transients, which required extracellular calcium, were detected in lactotrophs, somatotrophs, and corticotrophs within the acute slices. Of significance was the finding that the bursting mode could be adjusted by hypothalamic factors. In slices prepared from lactating rats, TRH recruited more bursting cells and finely adjusted the average duty cycle of [Ca2+]i bursts such that cells fired patterned bursts for approximately 70% of the recording period. Eighty-six percent of these cells were lactotrophs. Thus, the rhythmic [Ca2+]i bursts and their tuning by secretagogues may provide timing information that could encode for one or more cellular functions (e.g. exocytosis and/or gene expression) critical for the release of hormones by endocrine cells in the intact gland.

Bonjour à tous

Voici la bibliographie de l’ invité du séminaire du 21 novembre, Patrick Mollard. ( Contacts Jean-Paul Herman tél : 04 91 69 87 15 )

Olivier Crouzet Bibliothèque universitaire de Médecine-Odontologie ( La Timone) 27, Bd Jean Moulin 13385 Marseille cedex 05 Tel 04 91 32 46 53 - Fax 04 91 79 41 15

Bibliothèque universitaire de Médecine-nord Bd Pierre Dramard 13916 Marseille cedex 20 Tel 04 91 69 89 50 & 04 91 69 89 52 - Fax 04 91 69 89 51

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

Docteur Patrice MOLLARD Dpt. d’endocrinologie de l’Institut de Génomique Fonctionnelle, Cnrs, Inserm et Univ. de Montpellier.

Tracking cells with fluorescent proteins, from 3-D organization to in vivo functioning in the pituitary gland.

1 : Proc Natl Acad Sci U S A. 2005 Nov 4 ; [Epub ahead of print]

Revealing the large-scale network organization of growth hormone-secreting cells.

Bonnefont X, Lacampagne A, Sanchez-Hormigo A, Fino E, Creff A, Mathieu MN, Smallwood S, Carmignac D, Fontanaud P, Travo P, Alonso G, Courtois-Coutry N, Pincus SM, Robinson IC, Mollard P.

Department of Endocrinology, Institute of Functional Genomics, Centre National de la Recherche Scientifique, Unite Mixte de Recherche 5203, Institut National de la Sante et de la Recherche Medicale U661, Universities of Montpellier 1 and 2, 141 Rue de la Cardonille, 34094 Montpellier Cedex 05, France ; Institut National de la Sante et de la Recherche Medicale U637, Centre Hospitalier Universitaire Arnaud de Villeneuve, 34295 Montpellier Cedex 05, France ; Division of Molecular Neuroendocrinology, National Institute of Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom ; Montpellier RIO Imaging, Centre de Recherches de Biochimie Macromoleculaire, Formation de Recherche en Evolution 2593, Centre National de la Recherche Scientifique, 1919 Route de Mende, 34293 Montpellier Cedex 05, France.

Pituitary growth hormone (GH)-secreting cells regulate growth and metabolism in animals and humans. To secrete highly ordered GH pulses (up to 1,000-fold rise in hormone levels in vivo), the pituitary GH cell population needs to mount coordinated responses to GH secretagogues, yet GH cells display an apparently heterogeneous scattered distribution in 2D histological studies. To address this paradox, we analyzed in 3D both positioning and signaling of GH cells using reconstructive, two-photon excitation microscopy to image the entire pituitary in GH-EGFP transgenic mice. Our results unveiled a homologous continuum of GH cells connected by adherens junctions that wired the whole gland and exhibited the three primary features of biological networks : robustness of architecture across lifespan, modularity correlated with pituitary GH contents and body growth, and connectivity with spatially stereotyped motifs of cell synchronization coordinating cell activity. These findings change our view of GH cells, from a collection of dispersed cells to a geometrically connected homotypic network of cells whose local morphology and connectivity can vary, to alter the timing of cellular responses to promote more coordinated pulsatile secretion. This large-scale 3D view of cell functioning provides a powerful approach to identify and understand other networks of endocrine cells that are thought to be scattered in situ. Many dispersed endocrine systems exhibit pulsatile outputs. We suggest that cell positioning and associated cell-cell connection mechanisms will be critical parameters that determine how well such systems can deliver a coordinated secretory pulse of hormone to their target tissues.


2 : Hum Mol Genet. 2004 Apr 1 ;13(7):771-80. Epub 2004 Feb 12.

Amino-acid changes acquired during evolution by olfactory receptor 912-93 modify the specificity of odorant recognition.

Gaillard I, Rouquier S, Chavanieu A, Mollard P, Giorgi D.

IGH, CNRS UPR 1142 rue de la Cardonille, Cedex 5, France.

The sense of smell in mammals can perceive and discriminate a wide variety of volatile odorants. Odorants bind to specific olfactory receptors (ORs) to initiate an action potential that transduces olfactory information to the olfactory cortex. We previously identified the structural motifs of odorant molecules (aliphatic 2- or 3-ketones) required to activate mouse OR912-93 by detection of the odorant response using calcium measurement in transfected cells. In order to study changes in the specificity of this receptor that might have occurred during evolution, we cloned the orthologous genes from six primate species and pig and assayed the encoded receptors for responses to odorants. Primate OR912-93 orthologs share 88-97% sequence identity. All the receptors responded to 2- and 3-heptanone except the squirrel-monkey OR, which responded only to 3-heptanone, and the human and orangutan ORs, which were not functional. Directed mutagenesis allowed us to convert the squirrel-monkey response to that of the other functional 912-93 ORs by substituting three amino acids in the second extracellular loop. Orangutan and human 912-93 ORs regained function after restoration of the arginine residue in the DRY motif required for G-protein activation. However, the human receptor was constitutively activated in the absence of ligand stimulation. Using natural mutants of the OR912-93 receptor, we provide evidence that squirrel-monkeys evolved towards a restriction of the specificity of this receptor and therefore that slight alterations in the sequence of a receptor can induce subtle changes in recognition specificity.


3 : FEBS Lett. 2003 Jul 31 ;548(1-3):49-52.

Electrical activity in endocrine pituitary cells in situ : a support for a multiple-function coding.

Bonnefont X, Mollard P.

INSERM U469, Centre CNRS-INSERM de Pharmacologie Endocrinologie, 34094 Montpellier Cedex 5, France.

The anterior pituitary is an endocrine gland that controls basic body functions. Pituitary cell functioning depends on membrane excitability, which induces cytosolic calcium rises. Here, we reported the first identification of small-amplitude voltage fluctuations that controlled spike firing in endocrine cells recorded in situ. Three patterns of voltage fluctuations were distinguishable by their durations (1-100 s). These patterns could be ordered on top of each other, namely in response to secretagogues. Thus, pituitary endocrine cells express in situ a cell code in which small-amplitude voltage fluctuations lead to a multimodal arrangement of spike firing, which may finely tune calcium-dependent functions.


4 : Endocrinology. 2003 Jun ;144(6):2728-40.

Growth hormone-releasing hormone (GHRH) neurons in GHRH-enhanced green fluorescent protein transgenic mice : a ventral hypothalamic network.

Balthasar N, Mery PF, Magoulas CB, Mathers KE, Martin A, Mollard P, Robinson IC.

Division of Molecular Neuroendocrinology, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.

The hypothalamic GHRH neurons secrete pulses of GHRH to generate episodic GH secretion, but little is known about the mechanisms involved. We have made transgenic mice expressing enhanced green fluorescent protein (eGFP) specifically targeted to the secretory vesicles in GHRH neurons. GHRH cells transported eGFP from cell bodies in the arcuate nucleus to extensively arborized varicose fiber terminals in the median eminence. Patch clamp recordings from visually identified GHRH cells in mature animals showed spontaneous action potentials, often firing in short bursts up to 10 Hz. GHRH neurons received frequent synaptic inputs, as demonstrated by the recording of abundant inward postsynaptic currents, but spikes were followed by large after-hyperpolarizations, which limited their firing rate. Because many GHRH neurons lie close to the ventral hypothalamic surface, this was examined by wide-field binocular epifluorescence stereomicroscopy. This approach revealed an extensive horizontal network of GHRH cells at low power and individual fiber projections at higher power in the intact brain. It also showed the dense terminal projections of the GHRH cell population in the intact median eminence. This model will enable us to characterize the properties of individual GHRH neurons and their structural and functional connections with other neurons and to study directly the role of the GHRH neuronal network in generating episodic secretion of GH.


5 : Trends Endocrinol Metab. 2002 Sep ;13(7):304-9.

Hidden face of the anterior pituitary.

Fauquier T, Lacampagne A, Travo P, Bauer K, Mollard P.

INSERM Unit469-IFR3, Montpellier, France.

The traditional view holds that the anterior pituitary is an endocrine gland with a complex and heterogeneous distribution of cells throughout the parenchyma. Thus, a long-distance mode of intraorgan communication is not usually taken into account in our understanding of pituitary functioning. However, recent in situ pituitary studies have begun to unveil a hitherto unknown route of large-scale information transfer within the pituitary. Agranular folliculostellate cells - the sixth type of pituitary cell initially discovered almost half a century ago - are the functional units of a dynamically active cell network wiring the whole gland. Because folliculostellate cells communicate with their endocrine neighbors, this opens the door to considering the pituitary as a cellular puzzle more ordered than was first thought. Hence, cell networking within the pituitary gland could have a privileged role in coordinating the activities of distant cells in both physiological and pathological conditions.


6 : Eur J Neurosci. 2002 Feb ;15(3):409-18.

A single olfactory receptor specifically binds a set of odorant molecules.

Gaillard I, Rouquier S, Pin JP, Mollard P, Richard S, Barnabe C, Demaille J, Giorgi D.

IGH, CNRS UPR 1142, rue de la Cardonille, 34396 Montpellier cedex 5, France. gaillard igh.cnrs.fr

The sense of smell is mediated by the initiation of action potential in olfactory sensory neurons during odor stimulation. However, little is known about odorant-olfactory receptor (OR) recognition mechanisms. In the present work, we identified the structural motifs of odorant molecules required to activate mouse OR912-93 by detection of the odorant response using calcium measurement in cells transfected with OR and G(alpha)q and G(alpha)15 proteins. The use of sets of odorants led to the identification of ketones with an aliphatic carbon chain length >or= four carbon atoms and a carbonyl group preferentially located in position C2 or C3. The threshold of detection of these odorants is as low as 10(-6)-10(-8)m. No other odorant ligand, out of 70 representatives of the odorant world, was active. The human ortholog of OR912-93 is not functional, suggesting that apart from a stop-mutation located at the 5’-end that was corrected in the construct, it incurred other deleterious mutations during evolution.


7 : Proc Natl Acad Sci U S A. 2001 Jul 17 ;98(15):8891-6. Epub 2001 Jul 3.

Folliculostellate cell network : a route for long-distance communication in the anterior pituitary.

Fauquier T, Guerineau NC, McKinney RA, Bauer K, Mollard P.

Institut National de la Sante et de la Recherche Medicale Unite 469, Centre National de la Recherche Scientifique-INSERM de Pharmacologie-Endocrinologie, 141 Rue de la Cardonille, 34094 Montpellier Cedex 5, France.

All higher life forms critically depend on hormones being rhythmically released by the anterior pituitary. The proper functioning of this master gland is dynamically controlled by a complex set of regulatory mechanisms that ultimately determine the fine tuning of the excitable endocrine cells, all of them heterogeneously distributed throughout the gland. Here, we provide evidence for an intrapituitary communication system by which information is transferred via the network of nonendocrine folliculostellate (FS) cells. Local electrical stimulation of FS cells in acute pituitary slices triggered cytosolic calcium waves, which propagated to other FS cells by signaling through gap junctions. Calcium wave initiation was because of the membrane excitability of FS cells, hitherto classified as silent cells. FS cell coupling could relay information between opposite regions of the gland. Because FS cells respond to central and peripheral stimuli and dialogue with endocrine cells, the form of large-scale intrapituitary communication described here may provide an efficient mechanism that orchestrates anterior pituitary functioning in response to physiological needs.


8 : Ann N Y Acad Sci. 2000 ;921:410-4.

PACAP and VIP induce changes in cytosolic calcium in putative folliculostellate cells of the mouse pituitary.

Kunzelmann P, Creff A, Bauer K, Mollard P.

INSERM U469, CCIPE, 141, Rue de la Cardonille, 34094 Montpellier, France. kunzel u469.montp.inserm.fr


9 : Endocrinology. 2000 Dec ;141(12):4681-9.

A secreted fluorescent reporter targeted to pituitary growth hormone cells in transgenic mice.

Magoulas C, McGuinness L, Balthasar N, Carmignac DF, Sesay AK, Mathers KE, Christian H, Candeil L, Bonnefont X, Mollard P, Robinson IC.

Division of Neurophysiology, National Institute for Medical Research Mill Hill, London, United Kingdom.

In stable transfection experiments in the GH-producing GC cell line, a construct containing the entire signal peptide and the first 22 residues of human GH linked in frame with enhanced green fluorescent protein (eGFP), produced brightly fluorescent cells with a granular distribution of eGFP. This eGFP reporter was then inserted into a 40-kb cosmid transgene containing the locus control region for the hGH gene and used to generate transgenic mice. Anterior pituitaries from these GH-eGFP transgenic mice showed numerous clusters of strongly fluorescent cells, which were also immunopositive for GH, and which could be isolated and enriched by fluorescence-activated cell sorting. Confocal scanning microscopy of pituitary GH cells from GH-eGFP transgenic mice showed a markedly granular appearance of fluorescence. Immunogold electron microscopy and RIA confirmed that the eGFP product was packaged in the dense cored secretory vesicles of somatotrophs and was secreted in parallel with GH in response to stimulation by GRF. Using eGFP fluorescence, it was possible to identify clusters of GH cells in acute pituitary slices and to observe spontaneous transient rises in their intracellular Ca2+ concentrations after loading with Ca2+ sensitive dyes. This transgenic approach opens the way to direct visualization of spontaneous and secretagogue-induced secretory mechanisms in identified GH cells.


10 : Endocrinology. 2000 Mar ;141(3):868-75.

Rhythmic bursts of calcium transients in acute anterior pituitary slices.

Bonnefont X, Fiekers J, Creff A, Mollard P.

INSERM U-469, Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Montpellier, France.

Endocrine cells isolated from the anterior pituitary fire intracellular Ca2+ ([Ca2+]i) transients due to voltage-gated Ca2+ entry. However, the patterns of [Ca2+]i transients within the glandular parenchyma of the anterior pituitary are unknown. Here we describe, using real-time confocal laser microscopy, several spontaneous patterns of calcium signaling in acute pituitary slices prepared from male as well as cycling and lactating female rats. Forty percent of the cells demonstrated a spontaneous bursting mode, consisting of an active period of [Ca2+]i transients firing at a constant frequency, followed by a rest period during which cells were either silent or randomly active. The remaining recordings from endocrine cells either demonstrated random [Ca2+]i transients or were silent. These rhythmic bursts of [Ca2+]i transients, which required extracellular calcium, were detected in lactotrophs, somatotrophs, and corticotrophs within the acute slices. Of significance was the finding that the bursting mode could be adjusted by hypothalamic factors. In slices prepared from lactating rats, TRH recruited more bursting cells and finely adjusted the average duty cycle of [Ca2+]i bursts such that cells fired patterned bursts for approximately 70% of the recording period. Eighty-six percent of these cells were lactotrophs. Thus, the rhythmic [Ca2+]i bursts and their tuning by secretagogues may provide timing information that could encode for one or more cellular functions (e.g. exocytosis and/or gene expression) critical for the release of hormones by endocrine cells in the intact gland.

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