Silencing and Un-silencing of Tetracycline-Controlled Genes in Neurons

To identify the underlying reason for the controversial performance of tetracycline (Tet)-controlled regulated gene expression in mammalian neurons, we investigated each of the three components that comprise the Tet inducible systems, namely tetracyclines as inducers, tetracycline-transactivator (tTA) and reverse tTA (rtTA), and tTA-responsive promoters (Ptets). We have discovered that stably integrated Ptet becomes functionally silenced in the majority of neurons when it is inactive during development. Ptet silencing can be avoided when it is either not integrated in the genome or stably-integrated with basal activity. Moreover, long-term, high transactivator levels in neurons can often overcome integration-induced Ptet gene silencing, possibly by inducing promoter accessibility

AMPA receptor channels with long-lasting desensitization in bipolar interneurons contribute to synaptic depression in a novel feedback circuit in layer 2/3 of rat neocortex

A novel, local inhibitory circuit in layer 2/3 of rat somatosensory cortex is described that connects pyramidal cells reciprocally with GABAergic vasoactive intestinal polypeptide-immunoreactive bipolar interneurons. In paired whole-cell recordings, the glutamatergic unitary responses (EPSPs or EPSCs) in bipolar cells evoked by repetitive (10 Hz) stimulation of a pyramidal cell show strong frequency-dependent depression. Unitary IPSPs evoked in pyramidal cells by repetitive stimulation of bipolar cells, on average, maintained their amplitude. This suggests that the excitatory synapses on bipolar cells act as a low-pass filter in the reciprocal pyramid-to-bipolar circuit. The EPSCs in bipolar cells are mediated predominantly by AMPA receptor (AMPAR) channels. AMPARs desensitize rapidly and recover slowly from desensitization evoked by a brief pulse of glutamate. In slices, reduction of AMPAR desensitization by cyclothiazide (50-100 ?M) or conditioning steady-state desensitization induced by application of extracellular AMPA (50 nM) or glutamate (50 ?M) strongly reduced synaptic depression. It is concluded that in the local circuits between pyramidal and bipolar cells the desensitization of AMPARs in bipolar cells contributes to low-pass feedback inhibition of layer 2/3 pyramidal neurons by bipolar cells

Synthèse et caractérisation spectroscopique d hétéro-composés fonctionnalisés (thiols, sélénols, tellurols et phosphine-boranes)

Des thiols, sélénols et tellurols fonctionnalisés et cinétiquement non-stabilisés ont été préparés dans des conditions particulières de réaction sous vide. De façon plus précise, le 3-mercapto-2-propènenitrile, des allényl- et alcynylsélénols, des alcényl- ou cyclopropyl-tellurols ont ainsi pu être isolés et caractérisés par spectroscopie. Les synthèses ont ensuite été adaptées à diverses études spectroscopiques. Les spectres Infrarouge de l éthènetellurol et l éthènylphosphine-borane ont été enregistrés. Les spectres de photoélectron des sélénols et tellurols insaturés ont permis de rationaliser les interactions existant entre la paire libre d électrons de l hétéroatome et l insaturation en fonction de leur éloignement relatif. La complexation des systèmes réactifs a été abordée par la complexation des phosphines primaires par le borane. Une étude générale a été menée sur ces phosphine-boranes en phase gazeuse par diffraction électronique. L influence de la complexation sur les paramètres géométriques (longueurs de liaisons, angles) par rapport aux systèmes libres correspondants a ainsi été précisée.Primary heterocompounds (Thiols, Selenols, Tellurols and Phosphine-boranes) have been prepared in particular conditions on a preparative scale and with a high purity. Allenyl- and alkynylselenols, cyclopropyl- and alkenyltellurols as well as primary , -unsaturated phosphines-boranes have thus been isolated and characterized by spectroscopy. The microwave, infrared or photoelectron spectra of several of these kinetically unstable compounds have been recorded. Thus, by photoelectron spectroscopy and quantum calculations, the type and extent of the interaction between the SeH or TeH group and the unsaturated moiety has been clarified for compounds with gradual increasing distance between the heteroatom and the unsaturated group. On the other hand, a general study was undertaken on the primary phosphine-boranes in gas phase by electronic diffraction. The modification of the interatomic lengths and angles by complexation with borane has been precised.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Impaired NMDA receptor function in mouse olfactory bulb neurons by tetracycline-sensitive NR1(N598R) expression

High Ca2+ permeability and its control by voltage-dependent Mg2+ block are defining features of NMDA receptors. These features are lost if the principal NR1 subunit carries an asparagine (N) to arginine (R) substitution in a critical channel site at NR1 position 598. NR1(R) expression from a single allele in gene-targeted NR1+/R mice is lethal soon after birth, precluding analysis of altered synaptic functions later in life. We therefore employed the forebrain specific small alpha, GreekCaMKII promoter to drive tTA-mediated tetracyclin sensitive transcription of transgenes for NR1(R) and for lacZ as reporter. Transgene expression was observed in cortex, striatum, hippocampus, amygdala and olfactory bulb and was mosaic in all these forebrain regions. It was highest in olfactory bulb granule cells, in most of which Ca2+ permeability and voltage-dependent Mg2+ block of NMDA receptors were reduced to different extents. This indicates significant impairment of NMDA receptor function by NR1(R) in presence of the wild-type NR1 complement. Indeed, even though NR1(R) mRNA constituted only 18% of the entire NR1 mRNA population in forebrain, the transgenic mice died during adolescence unless transgene expression was suppressed by doxycycline. Thus, glutamate receptor function can be altered in the mouse by regulated NR1(R) transgene expression

Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice.

The tet regulatory system in which doxycycline (dox) acts as an inducer of specifically engineered RNA polymerase II promoters was transferred into transgenic mice. Tight control and a broad range of regulation spanning up to five orders of magnitude were monitored dependent on the dox concentration in the water supply of the animals. Administration of dox rapidly induces the synthesis of the indicator enzyme luciferase whose activity rises over several orders of magnitude within the first 4 h in some organs. Induction is complete after 24 h in most organs analyzed. A comparable regulatory potential was revealed with the tet regulatory system where dox prevents transcription activation. Directing the synthesis of the tetracycline-controlled transactivator (tTA) to the liver led to highly specific regulation in hepatocytes where, in presence of dox, less than one molecule of luciferase was detected per cell. By contrast, a more than 10(5)-fold activation of the luciferase gene was observed in the absence of the antibiotic. This regulation was homogeneous throughout but stringently restricted to hepatocytes. These results demonstrate that both tetracycline-controlled transcriptional activation systems provide genetic switches that permit the quantitative control of gene activities in transgenic mice in a tissue-specific manner and, thus, suggest possibilities for the generation of a novel type of conditional mutants

Respiration and parturition affected by conditional overexpression of the Ca2+-activated K+ channel subunit, SK3.

In excitable cells, small-conductance Ca2+-activated potassium channels (SK channels) are responsible for the slow after-hyperpolarization that often follows an action potential. Three SK channel subunits have been molecularly characterized. The SK3 gene was targeted by homologous recombination for the insertion of a gene switch that permitted experimental regulation of SK3 expression while retaining normal SK3 promoter function. An absence of SK3 did not present overt phenotypic consequences. However, SK3 overexpression induced abnormal respiratory responses to hypoxia and compromised parturition. Both conditions were corrected by silencing the gene. The results implicate SK3 channels as potential therapeutic targets for disorders such as sleep apnea or sudden infant death syndrome and for regulating uterine contractions during labor