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International audience; Synchronization of circadian rhythms to the 24-h light/dark (L/D) cycle ă is associated with daily rearrangements of the neuronal-glial network of ă the suprachiasmatic nucleus of the hypothalamus (SCN), the central ă master clock orchestrating biological functions in mammals. These ă anatomical plastic events involve neurons synthesizing vasoactive ă intestinal peptide (VIP), known as major integrators of photic signals ă in the retinorecipient region of the SCN. Using an analog-sensitive ă kinase allele murine model (TrkBF616A), we presently show that the ă pharmacological blockade of the tropomyosin-related kinase receptor type ă B (TrkB), the high-affinity receptor of brain-derived neurotrophic ă factor (BDNF), abolished day/night changes in the dendrite enwrapping of ă VIP neurons by astrocytic processes (glial coverage), used as an index ă of SCN plasticity on electron-microscopic sections. Therefore, the ă BDNF/TrkB signaling pathway exerts a permissive role on the ă ultrastructural rearrangements that occur in SCN under L/D alternance, ă an action that could be a critical determinant of the well-established ă role played by BDNF in the photic regulation of the SCN. In contrast, ă the extent of glial coverage of non-VIP neighboring dendrites was not ă different at daytime and nighttime in TrkBF616A mice submitted to TrkB ă inactivation or not receiving any pharmacological treatment. These data ă not only show that BDNF regulates SCN structural plasticity across the ă 24-h cycle but also reinforce the view that the daily changes in SCN ă architecture subserve the light synchronization process.

International audience; The mammalian circadian clock, whose central component is located in the suprachiasmatic nucleus of the hypothalamus (SCN), orchestrates rhythmic events in metabolism, physiology and behavior. Adaptation of the organism to its environment requires precise adjustment of the clock to the 24 h astronomical time, primarily by the light/dark cycle. Photic synchronization acts on both the molecular loops which trigger circadian oscillations and the phasing of the multiple SCN cellular oscillators whose coordination permits elaboration of the rhythmic message that will be distributed throughout the organism. It is concomitant with structural plastic events characterized by day/night rearrangements of the SCN neuronal-glial network. The two main sources of SCN efferents, namely the VIP (vasoactive intestinal peptide)-synthesizing neurons which are major integrators of photic signals and the AVP (arginine-vasopressin)-synthesizing neurons which are known to importantly contribute to conveying rhythmic messages to brain targets, are involved in these mechanisms. Over the light/dark cycle, they indeed undergo ultrastructural changes in the extent of their membrane coverage by glial, axon terminal and/or somato-dendritic elements. These structural rearrangements appear to be dependent on light entrainment, as the rhythmic expression in SCN of glial fibrillary acidic protein (GFAP), a marker for brain astrocytes whose changing expression has proved to be a reliable index of neuronal-glial plasticity, is disrupted under constant darkness. Glucocorticoid hormones, which are known as important endocrine outputs of the clock, are required to maintain amplitude of the SCN GFAP rhythm to normal values, indicating that they modulate astrocytic plasticity within the SCN and, therefore, nycthemeral changes of the configuration of its neuronal-glial network. The view that such plastic events may subserve synchronization of the clock to the light-dark cycle is reinforced by other data showing that the daily fluctuations of circulating glucocorticoids actually are involved in modulation of light effects, contributing to the resistance of the circadian timing system to variations of the photoperiod. It is thus proposed that the capacity of the clock to integrate cyclic variations of the environment rely on the inherent capacity of the SCN to undergo neuronal-glial plasticity.

International audience; no abstract

International audience; Background: Stem cell-based therapies are an attractive option to promote regeneration and repair defective tissues and organs. Thanks to their multipotency, high proliferation rate and the lack of major ethical limitations, "olfactory ecto-mesenchymal stem cells" (OE-MSCs) have been described as a promising candidate to treat a variety of damaged tissues. Easily accessible in the nasal cavity of most mammals, these cells are highly suitable for autologous cell-based therapies and do not face issues associated with other stem cells. However, their clinical use in humans and animals is limited due to a lack of preclinical studies on autologous transplantation and because no well-established methods currently exist to cultivate these cells. Here we evaluated the feasibility of collecting, purifying and amplifying OE-MSCs from different mammalian genera with the goal of promoting their interest in veterinary regenerative medicine. Biopsies of olfactory mucosa from eight mammalian genera (mouse, rat, rabbit, sheep, dog, horse, gray mouse lemur and macaque) were collected, using techniques derived from those previously used in humans and rats. The possibility of amplifying these cells and their stemness features and differentiation capability were then evaluated. Results: Biopsies were successfully performed on olfactory mucosa without requiring the sacrifice of the donor animal, except mice. Cell populations were rapidly generated from olfactory mucosa explants. These cells displayed similar key features of their human counterparts: a fibroblastic morphology, a robust expression of nestin, an ability to form spheres and similar expression of surface markers (CD44, CD73). Moreover, most of them also exhibited high proliferation rates and clonogenicity with genus-specific properties. Finally, OE-MSCs also showed the ability to differentiate into mesodermal lineages. Conclusions: This article describes for the first time how millions of OE-MSCs can be quickly and easily obtained from different mammalian genera through protocols that are well-suited for autologous transplantations. Moreover, their multipotency makes them relevant to evaluate therapeutic application in a wide variety of tissue injury models. This study paves the way for the development of new fundamental and clinical studies based on OE-MSCs transplantation and suggests their interest in veterinary medicine.

International audience

Paraspeckles are nuclear bodies form around the long non-coding RNA, Neat1, and RNA-binding proteins. While their role is not fully understood, they are believed to control gene expression at a post-transcriptional level by means of the nuclear retention of mRNA containing in their 3’-UTR inverted repeats of Alu sequences (IRAlu). In this study, we found that, in pituitary cells, all components of paraspeckles including four major proteins and Neat1 displayed a circadian expression pattern. Furthermore the insertion of IRAlu at the 3’-UTR of the EGFP cDNA led to a rhythmic circadian nuclear retention of the egfp mRNA that was lost when paraspeckles were disrupted whereas insertion of a single antisense Alu had only a weak effect. Using real-time video-microscopy, these IRAlu were further shown to drive a circadian expression of EGFP protein. This study shows that paraspeckles, thanks to their circadian expression, control circadian gene expression at a post-transcriptional level. DOI: http://dx.doi.org/10.7554/eLife.14837.001, eLife digest Many biological features of animals, including body temperature and hormone levels, follow daily rhythms that repeat every 24 hours. These so-called circadian rhythms are driven by an internal body clock and are essential for the organism to adapt to the daily cycle of light and dark. Circadian rhythms also take place inside individual cells – for example, the amount of a given protein in a cell often rises and falls over each 24-hour period. To generate these daily fluctuations, the processes used to make proteins based on the instructions encoded within a gene must be carefully controlled. Genes are first copied or ‘transcribed' into intermediate molecules called messenger RNAs (mRNAs). These mRNA molecules must then travel out of the cell’s nucleus before they can be de-coded to produce proteins. This means that daily fluctuations in mRNA and protein levels could occur because the rate at which the DNA is transcribed fluctuates or because controlling the steps that occur after transcription. However it is not clear how much these post-transcriptional steps contribute to circadian rhythms inside cells. Recently, structures called paraspeckles were seen inside the nucleus. These structures are made from a long RNA molecule that does not code for a protein, and a number of proteins that can bind mRNA molecules. Paraspeckles are thought to prevent certain mRNAs from leaving the nucleus and therefore stop them from being decoded to make proteins. Torres et al. have now investigated whether paraspeckles may play a role in circadian rhythms. Torres et al. looked at the long non-coding RNA and several proteins that are known to be components of paraspeckles in cells taken from the pituitary glands of rats using a variety of techniques. These cells were chosen because they were known to have a working circadian clock. The analysis showed that the levels of these components, as well as the number of paraspeckles within the nucleus, changed over the course of a daily cycle. Torres et al. then confirmed that mRNAs containing a sequence that is known to recruit mRNAs to paraspeckes (the IRAlu sequence) could be also retained in the nucleus or released with a circadian rhythm. This pattern was lost when the paraspeckles were disrupted. These findings suggest that daily fluctuations in protein levels can be post-transcriptionally controlled by paraspeckles rhythmically retaining mRNAs in the nucleus. Future studies could explore whether it may be possible to control circadian rhythms by targeting the paraspeckles, which could help to improve conditions where the internal body clock goes wrong. DOI: http://dx.doi.org/10.7554/eLife.14837.002

International audience; The 5XFAD mice are an early-onset transgenic model of Alzheimer's disease (AD) in which amyloid plaques are first observed between two and four months of age in the cortical layer five and in the subiculum of the hippocampal formation. Although cognitive alterations have been described in these mice, there are no studies that focused on the onset of hippocampus-dependent memory deficits, which are a hallmark of the prodromal stage of AD. To identify when the first learning and memory impairments appear, 5XFAD mice of two, four, and six months of age were compared with their respective wild-type littermates using the olfactory tubing maze, which is a very sensitive hippocampal-dependent task. Deficits in learning and memory started at four months with a substantial increase at six months of age while no olfactory impairments were observed. The volumetric study using magnetic resonance imaging of the whole brain and specific areas (olfactory bulb, striatum, and hippocampus) did not reveal neuro-anatomical difference. Slight memory deficits appeared at 4 months of age in correlation with an increased astrogliosis and amyloid plaque formation. This early impairment in learning and memory related to the hippocampal dysfunction is particularly suited to assess preclinical therapeutic strategies aiming to delay or suppress the onset of AD.

International audience; Stem cells are attractive tools to develop new therapeutic strategies for a variety of disorders. While ethical and technical issues, associated with embryonic, fetal and neural stem cells, limit the translation to clinical applications, the nasal stem cells identified in the human olfactory mucosa stand as a promising candidate for stem cell-based therapies. Located in the back of the nose, this multipotent stem cell type is readily accessible in humans, a feature that makes these cells highly suitable for the development of autologous cell-based therapies. However, preclinical studies based on autologous transplantation of rodent olfactory stem cells are impeded because of the narrow opening of the nasal cavity. In this study, we report the development of a unique method permitting to quickly and safely biopsy olfactory mucosa in rats. Using this newly developed technique, rat stem cells expressing the stem cell marker Nestin were successfully isolated without requiring the sacrifice of the donor animal. As an evidence of the self-renewal capacity of the isolated cells, several millions of rat cells were amplified from a single biopsy within four weeks. Using an olfactory discrimination test, we additionally showed that this novel biopsy method does not affect the sense of smell and the learning and memory abilities of the operated animals. This study describes for the first time a methodology allowing the derivation of rat nasal cells in a way that is suitable for studying the effects of autologous transplantation of any cell type present in the olfactory mucosa in a wide variety of rat models.

International audience; Extracellular regulated kinase (ERK) signalling is believed to play roles in various aspects of circadian clock mechanisms. In this study, we show in rat that the nuclear versus cytoplasmic intracellular distribution of the phosphorylated forms of ERK1/2 (P-ERK1/2) in the central clock, namely the suprachiasmatic nucleus (SCN), is proportionally constant across the light/dark cycle while the spatial distribution and neurochemical phenotype of cells expressing these activated forms are time-regulated according to a daily rhythm and light-regulated. P-ERK1/2 was exclusively found in neuronal elements. At daytime, it was detected throughout the dorsoventral extent of the SCN, partly within neurons synthesizing either arginine-vasopressin or vasoactive intestinal peptide (VIP). At night time, it was segregated in the ventrolateral aspect of the nucleus, within a cluster of cells 45% of which were gastrin-releasing peptide (GRP) neurons with or without co-localization with VIP. After a light pulse at night, expression of P-ERK1/2 increased in GRP neurons but also appeared in a population of neurons that stained for VIP only. These data show that the GRP neurons are closely associated with ERK1/2 activation at night and point to the importance of ERK1/2 signalling not only in intra-SCN transmission of photic information but also in maintenance of neuronal rhythms in the SCN.

International audience; The main known function of the pineal gland in mammals is the temporal synchronization of physiological rhythms to seasonal changes of day length (photoperiod). In rat, the transcription factor activating protein-1 (AP-1) displays a circadian rhythm in its DNA binding in the pineal gland, which results from the rhythmic expression of Fra-2. We postulated that, if AP-1 is an important component of pineal gland functioning, then variations in photoperiodic conditions should lead to an adaptation of the AP-1 binding rhythm. Here we show that AP-1 binding patterns adapt to variations in lighting conditions, in the same way as the rhythm of arylalkylamine-N-acetyltransferase (AA-NAT) activity. This adaptation appeared to result from photoperiodic adaptation of the rhythmic fra-2 gene expression and was reflected by an adapted delay between the onset of night and the acrophase of the nocturnal peak. We further showed that photoperiodic adaptation of both the AP-1 binding and AA-NAT activity rhythms resulted from adapted changes in adrenergic inducibility of both variables at night onset. We finally provided evidence that AP-1 shared with the CREM gene encoding the transcriptional repressor protein inducible cAMP early repressor (ICER) the ability to be hypersensitive or subsensitive to adrenergic stimuli, depending on prior photoperiod.

International audience; no abstract

International audience; We have previously reported that selective axotomy of serotoninergic neurons produced by an intraventricular injection of 5, 7‐dihydroxytryptamine is followed by an increase in 5‐HT1B binding sites in the suprachiasmatic nucleus of the hypothalamus. This post‐lesion up‐regulation is shown here to be spontaneously reversed after long‐term survival in spite of an incomplete reinnervation of the nucleus. Recovery may be accelerated by fetal raphe transplants that produce more rapid reinnervation.

International audience; Luteinizing hormone‐releasing hormone (LHRH release, which serves as the primary drive to the hypothalamic‐pituitary gonadal axis, is controlled by many neuromediators. Serotonin has been implicated in this regulation. However, it is unclear whether the central effect of serotonin on LHRH secretion is exerted directly on LHRH neurosecretory neurons or indirectly via multisynaptic pathways. The present studies were undertaken in order to examine whether LHRH secretion from immortalized LHRH cell lines is directly regulated by serotonin and, if so, to identify the receptor subtype involved. 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT), a 5‐HT1A/7 receptor agonist, stimulated LHRH release from GT1‐1 cells. This effect was blocked by ritanserin, a 5‐HT2/7 receptor antagonist, but not by SDZ‐216‐525, a 5‐HT1A antagonist. Basal LHRH release was not affected by the 5‐HT2 agonist DOI. Reverse transcription and polymerase chain reaction technique (RT‐PCR) was used in order to identify 5‐HT1A and 5‐HT7 receptor mRNA in immortalized LHRH cell lines. GT1‐1 cells express mRNA for the 5‐HT7, but not the 5‐HT1A receptor subtypes. These results demonstrate a direct stimulatory effect of serotonin on LHRH release via 5‐HT7 receptor.

International audience; Glutamic acid and glycine were quantified in cells and medium of cultured rostral rhombencephalic neurons derived from fetal rats. In the presence of 1 mM Mg2+, NMDA (50 microM) significantly stimulated (by 69%) release of newly synthesized 5‐[3H]hydroxytryptamine ([3H]5‐HT). D‐2‐Amino‐5‐phosphonopentanoate (AP‐5; 50 microM) blocked the stimulatory effect of NMDA. AP‐5 by itself inhibited [3H]5‐HT release (by 25%), suggesting a tonic control of 5‐HT by glutamate. In the absence of Mg2+, basal [3H]5‐HT release was 60% higher as compared with release with Mg2+. AP‐5 blocked the increased [3H]5‐HT release observed without Mg2+, suggesting that this effect was due to the stimulation of NMDA receptors by endogenous glutamate. Glycine (100 microM) inhibited [3H]5‐HT release in the absence of Mg2+. Strychnine (50 microM) blocked the inhibitory effect of glycine, indicating an action through strychnine‐sensitive inhibitory glycine receptors. The [3H]5‐HT release stimulated by NMDA was unaffected by glycine. In contrast, when tested in the presence of strychnine, glycine increased NMDA‐evoked [3H]5‐HT release (by 22%), and this effect was prevented by a selective antagonist of the NMDA‐associated glycine receptor, 7‐chlorokynurenate (100 microM). 7‐Chlorokynurenate by itself induced a drastic decrease in [3H]5‐HT release, indicating that under basal conditions these sites were stimulated by endogenous glycine. These results indicate that NMDA stimulated [3H]5‐HT release in both the presence or absence of Mg2+. Use of selective antagonists allowed differentiation of a strychnine‐sensitive glycine response (inhibition of [3H]5‐HT release) from a 7‐chlorokynurenate‐sensitive response (potentiation of NMDA‐evoked [3H]5‐HT release).

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International audience; Autoregulatory mechanisms affecting serotonin [5‐hydroxytryptamine (5‐HT)] release and synthesis during the early period of development were investigated in dissociated cell cultures raised from embryonic rostral rat rhombencephalon. The presence of 5‐HT1A and 5‐HT1B receptors in serotoninergic neurons was assessed using binding assays. The involvement of 5‐HT1A and 5‐HT1B receptors in the control of the synthesis and release of [3H]5‐HT was studied using biochemical approaches with several serotoninergic receptor ligands. A mean decrease of 30% in [3H]5‐HT synthesis and release was observed in the presence of 5‐HT (10(‐8) M), the 5‐HT1A agonist 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT), the 5HT1B/1A agonist 5‐methoxy‐3‐(1,2,5,6‐tetrahydro‐4‐pyridinyl)‐1H‐indole (RU 24969), the 5‐HT1B agonist 3‐(1,2,5,6‐tetrahydropyrid‐4‐yl)pyrrolo[3,2‐b]pyrid‐5‐one (CP‐93,129), and the 5‐HT(1D/1B) agonist sumatriptan. Inhibition of 5‐HT synthesis and release induced by 8‐OH‐DPAT was blocked by chiral N‐tert‐butyl‐3‐[1‐[1‐(2‐methoxy)phenyl]piperazinyl]‐1‐phenylpropionam ide dihydrochloride quaternary‐hydrate (WAY 100135) (10(7) M) or methyl 4‐[4‐[4‐(1,1,3‐trioxo‐2H‐1,2‐benzoisothiazol‐2‐yl)butyl]‐1‐p iperazinyl]‐1Hindole‐2‐carboxylate (SDZ 216‐525) (10(‐7)M), and that of CP‐93,129 was blocked by methiothepin (10(‐7) M). Paradoxically, extracellular levels of [3H]5‐HT increased in the presence of 8‐OH‐DPAT and RU 24969 at 10(‐6) M. 5‐HT uptake experiments showed that these two agonists interacted with the 5‐HT transporter. 5‐HT1 binding sites (620 fmol/mg of protein) and 5‐HT1A (482 fmol/mg of protein) and 5‐HT1B (127 fmol/mg of protein) receptors were detected in 12‐day in vitro cell cultures. Experiments carried out with tetrodotoxin suggested that 5‐HT1A receptors are located on nerve cell bodies, whereas 5‐HT1B receptors are located on the nerve terminals. We concluded that autoregulatory mechanisms involving 5‐HT1A and 5‐HT1B autoreceptors are functionally mature in cells from rostral raphe nuclei during the early period of development.