A combination of LongSAGE with Solexa sequencing is well suited to explore the depth and the complexity of transcriptome

<p>Abstract</p> <p>Background</p> <p>"Open" transcriptome analysis methods allow to study gene expression without <it>a priori </it>knowledge of the transcript sequences. As of now, SAGE (Serial Analysis of Gene Expression), LongSAGE and MPSS (Massively Parallel Signature Sequencing) are the mostly used methods for "open" transcriptome analysis. Both LongSAGE and MPSS rely on the isolation of 21 pb tag sequences from each transcript. In contrast to LongSAGE, the high throughput sequencing method used in MPSS enables the rapid sequencing of very large libraries containing several millions of tags, allowing deep transcriptome analysis. However, a bias in the complexity of the transcriptome representation obtained by MPSS was recently uncovered.</p> <p>Results</p> <p>In order to make a deep analysis of mouse hypothalamus transcriptome avoiding the limitation introduced by MPSS, we combined LongSAGE with the Solexa sequencing technology and obtained a library of more than 11 millions of tags. We then compared it to a LongSAGE library of mouse hypothalamus sequenced with the Sanger method.</p> <p>Conclusion</p> <p>We found that Solexa sequencing technology combined with LongSAGE is perfectly suited for deep transcriptome analysis. In contrast to MPSS, it gives a complex representation of transcriptome as reliable as a LongSAGE library sequenced by the Sanger method.</p

A role of melanin-concentrating hormone producing neurons in the central regulation of paradoxical sleep

BACKGROUND: Peptidergic neurons containing the melanin-concentrating hormone (MCH) and the hypocretins (or orexins) are intermingled in the zona incerta, perifornical nucleus and lateral hypothalamic area. Both types of neurons have been implicated in the integrated regulation of energy homeostasis and body weight. Hypocretin neurons have also been involved in sleep-wake regulation and narcolepsy. We therefore sought to determine whether hypocretin and MCH neurons express Fos in association with enhanced paradoxical sleep (PS or REM sleep) during the rebound following PS deprivation. Next, we compared the effect of MCH and NaCl intracerebroventricular (ICV) administrations on sleep stage quantities to further determine whether MCH neurons play an active role in PS regulation. RESULTS: Here we show that the MCH but not the hypocretin neurons are strongly active during PS, evidenced through combined hypocretin, MCH, and Fos immunostainings in three groups of rats (PS Control, PS Deprived and PS Recovery rats). Further, we show that ICV administration of MCH induces a dose-dependant increase in PS (up to 200%) and slow wave sleep (up to 70%) quantities. CONCLUSION: These results indicate that MCH is a powerful hypnogenic factor. MCH neurons might play a key role in the state of PS via their widespread projections in the central nervous system

A Very Large Number of GABAergic Neurons Are Activated in the Tuberal Hypothalamus during Paradoxical (REM) Sleep Hypersomnia

We recently discovered, using Fos immunostaining, that the tuberal and mammillary hypothalamus contain a massive population of neurons specifically activated during paradoxical sleep (PS) hypersomnia. We further showed that some of the activated neurons of the tuberal hypothalamus express the melanin concentrating hormone (MCH) neuropeptide and that icv injection of MCH induces a strong increase in PS quantity. However, the chemical nature of the majority of the neurons activated during PS had not been characterized. To determine whether these neurons are GABAergic, we combined in situ hybridization of GAD67 mRNA with immunohistochemical detection of Fos in control, PS deprived and PS hypersomniac rats. We found that 74% of the very large population of Fos-labeled neurons located in the tuberal hypothalamus after PS hypersomnia were GAD-positive. We further demonstrated combining MCH immunohistochemistry and GAD67 in situ hybridization that 85% of the MCH neurons were also GAD-positive. Finally, based on the number of Fos-ir/GAD+, Fos-ir/MCH+, and GAD+/MCH+ double-labeled neurons counted from three sets of double-staining, we uncovered that around 80% of the large number of the Fos-ir/GAD+ neurons located in the tuberal hypothalamus after PS hypersomnia do not contain MCH. Based on these and previous results, we propose that the non-MCH Fos/GABAergic neuronal population could be involved in PS induction and maintenance while the Fos/MCH/GABAergic neurons could be involved in the homeostatic regulation of PS. Further investigations will be needed to corroborate this original hypothesis

The waking brain: an update

Wakefulness and consciousness depend on perturbation of the cortical soliloquy. Ascending activation of the cerebral cortex is characteristic for both waking and paradoxical (REM) sleep. These evolutionary conserved activating systems build a network in the brainstem, midbrain, and diencephalon that contains the neurotransmitters and neuromodulators glutamate, histamine, acetylcholine, the catecholamines, serotonin, and some neuropeptides orchestrating the different behavioral states. Inhibition of these waking systems by GABAergic neurons allows sleep. Over the past decades, a prominent role became evident for the histaminergic and the orexinergic neurons as a hypothalamic waking center

La narcolepsie (approches transcriptomiques et physiopathologiques)

La narcolepsie se caractérise par des attaques de sommeil diurnes, des insomnies nocturnes, des crises de cataplexies (perte de tonus musculaire). Cette maladie serait due à une attaque autoimmune conduisant à la dégénérescence de neurones éveillants, les neurones à Hypocrétine ou Orexine. Quelle est la cible de l attaque autoimmune responsable de la narcolepsie ? Pour répondre à cette question, nous avons cherché à déterminer quels étaient les transcrits spécifiques des neurones à hypocrétine en comparant leur transcriptome à celui des neurones à MCH intacts chez les sujets narcoleptiques. Ainsi nous avons développé une méthode novatrice d analyse transcriptomique à haut débit. Les neurones à MCH sont des régulateurs du sommeil paradoxal, ont ils un rôle dans les manifestations anormales de ce sommeil chez les sujets narcoleptiques ? Nous avons réalisé une caractérisation neuroanatomique de ces cellules, puis nous avons évalué le taux de MCH dans le liquide céphalorachidien de sujets atteints d hypersomnieNarcolepsy is characterized by diurnal sleep attacks, nocturnal insomnia, cataplexy (loss of muscle tone). In this pathology, an autoimmune attack is said to be responsible for the loss of a wake active neuronal population, Hypocretin or Orexin neurons. What is the target of the autoimmune attack which is responsible for narcolepsy? We have searched for specific hypocretin transcripts by comparing hypocretin transcriptome to MCH transcriptome, a second neuronal population unaffected by narcolepsy. For this, we have developed an innovative approach of high flow transcriptomic analysis. MCH neurons are paradoxical sleep regulator : are these cells implicated in abnormal manifestations of this kind of sleep in narcoleptic patients ? We have carried out a neuroanatomical characterization of these cells, then we have evaluated the MCH levels in the cerebrospinal fluid of hypersomniac subjectsLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Radioanatomie en coupes de l'épaule (application en chirurgie orthopédique)

BESANCON-BU Médecine pharmacie (250562102) / SudocSudocFranceF

Puberté Précoce dans la narcolepsie de type 1: A qui la faute? la perte d'orexine et/ou l'état neuroinflammatoire?

International audienc

Neuroanatomical and neurochemical systems involved in paradoxical sleep (PS) generation

International audienc

Role of MCH Neurons in Paradoxical (REM) Sleep Control

International audienc

Neuroanatomical and neurochemical systems involved in paradoxical sleep (PS) generation

International audienc