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International audience; The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome by identifying their molecular partners, DNA, proteins and/or RNAs. The latter can interact with lncRNA through networks involving proteins, but they can also be engaged in direct RNA/RNA interactions. We, therefore, developed an easy-to-use RNA pull-down procedure that allowed identification of RNAs engaged in direct RNA/RNA interaction with a lncRNA using psoralen, a molecule that cross-links only RNA/RNA interactions. Bioinformatics modeling of the lncRNA secondary structure allowed the selection of several specific antisense DNA oligonucleotide probes with a strong affinity for regions displaying a low probability of internal base pairing. Since the specific probes that were designed targeted accessible regions throughout the length of the lncRNA, the RNA-interaction zones could be delineated in the sequence of the lncRNA. When coupled with a high throughput RNA sequencing, this protocol can be used for the whole direct RNA interactome studies of a lncRNA of interest.

Paraspeckles are nuclear ribonucleic complex formed of a long non-coding RNA, nuclear-enriched abundant transcript one (Neat1) and associated RNA-binding proteins (RBP) whose cellular known functions are to sequester in the nucleus both proteins and RNAs. However, how RNAs are bound to paraspeckles is largely unknown. It is highly likely that binding of RNAs may occur via interactions with RBPs and accordingly, two structures present in the 3’UTR of some RNAs have been shown to allow their association to paraspeckles via protein binding. However, Neat1 could also be involved in the targeting of RNAs through direct RNA-RNA interactions. Using a RNA pull-down procedure adapted to select only RNAs engaged in direct RNA-RNA interactions and followed by RNA-seq we showed that in a rat pituitary cell line, GH4C1 cells, 1791 RNAs were associated with paraspeckles by direct interaction with Neat1. Neat1 was actually found able to bind more than 30% of the total transcripts targeted by the paraspeckles, we have identified in this cell line in a previous study. Furthermore, given the biological processes in which direct RNAs targets of Neat1 were involved as determined by gene ontology analysis, it was proposed that Neat1 played a major role in paraspeckle functions such as circadian rhythms, mRNA processing, RNA splicing and regulation of cell cycle. Finally, we provided evidence that direct RNA targets of Neat1 were preferentially bound to the 5’ end of Neat1 demonstrating that they are located in the shell region of paraspeckles.

Paraspeckles are nuclear membraneless structures composed of a long non-coding RNA, Nuclear-Enriched-Abundant-Transcript-1 and RNA binding proteins, which associate numerous mRNAs. It is therefore believed that their cellular function is to sequester in the nucleus their associated proteins and/or target mRNAs. However, little is known about the molecular determinant in mRNA targets that allow their association to paraspeckles except that inverted repeats of Alu sequences (IRAlu) present in 3’UTR of mRNAs may allow this association. While in a previous study we established the list of paraspeckle target RNAs in a rat pituitary cell line, we didn’t find however, inverted repeated SINEs, the rat equivalent of primate IRAlus in 3’UTR of these RNAs. By developing a candidate gene strategy, we selected a paraspeckle target gene, namely calreticulin mRNA, and we searched for other potential RNA recruitment elements in its 3’UTR, since 3’UTRs usually contain the sequence recognition for nuclear localization. We found a 15-nucleotide sequence, present as a tandem repeat in 3’UTR of this mRNA, which is involved in the nuclear retention by paraspeckles. While being not overrepresented in the 3’UTR of the paraspeckle target RNAs, this recruitment element was present in near 30% of all 3’UTR mRNAs. In addition, since an oligonucleotide containing this sequence binds the paraspeckle protein component HNRNPK, it is proposed that HNRNPK may constitute a bridging protein between paraspeckles and target mRNAs.

International audience; The circadian clock drives daily rhythms of multiple physiological processes, allowing organisms to anticipate and adjust to periodic changes in environmental conditions. These physiological rhythms are associated with robust oscillations in the expression of at least 30% of expressed genes. While the ability for the endogenous timekeeping system to generate a 24-hr cycle is a cell-autonomous mechanism based on negative autoregulatory feedback loops of transcription and translation involving core-clock genes and their protein products, it is now increasingly evident that additional mechanisms also govern the circadian oscillations of clock-controlled genes. Such mechanisms can take place post-transcriptionally during the course of the RNA life cycle. It has been shown that many steps during RNA processing are regulated in a circadian manner, thus contributing to circadian gene expression. These steps include mRNA capping, alternative splicing, changes in splicing efficiency, and changes in RNA stability controlled by the tail length of polyadenylation or the use of alternative polyadenylation sites. RNA transport can also follow a circadian pattern, with a circadian nuclear retention driven by rhythmic expression within the nucleus of particular bodies (the paraspeckles) and circadian export to the cytoplasm driven by rhythmic proteins acting like cargo. Finally, RNA degradation may also follow a circadian pattern through the rhythmic involvement of miRNAs. In this review, we summarize the current knowledge of the post-transcriptional circadian mechanisms known to play a prominent role in shaping circadian gene expression in mammals. This article is categorized under: RNA Processing > Splicing Regulation/Alternative Splicing RNA Processing > RNA Editing and Modification RNA Export and Localization > Nuclear Export/Import.

International audience; Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA's family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co-and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That's the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest. This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest.

International audience; In primary cultures of rat pituitary cells and in a pituitary sommatolactotroph cell line (GH4C1), endogenous core-clock- as well as hormone-genes such as prolactin displayed a rhythmic expression pattern, fitted by a sinusoidal equation in which the period value was close to the circadian one. This is consistent with the presence of a functional circadian oscillator in pituitary cells whose importance was ascertained in GH4C1 cell lines stably expressing a dominant negative mutant of BMAL1. In these cells, both endogenous core-clock- and prolactin-genes no more displayed a circadian pattern. Some genes we recently identified as mouse pituitary BMAL1-regulated genes in a DNA-microarray study, lost their circadian pattern in these cells, suggesting that BMAL1 controlled these genes locally in the pituitary. The intra-pituitary circadian oscillator could then play a role in the physiology of the gland that would not be seen anymore as a structure only driven by hypothalamic rhythmic control.

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

Circadian clocks regulate rhythmic gene expression levels by means of mRNA oscillations that are mainly driven by post-transcriptional regulation. We identified a new post-transcriptional mechanism, which involves nuclear bodies called paraspeckles. Major components of paraspeckles including the long noncoding RNA Neat1, which is the structural component, and its major protein partners, as well as the number of paraspeckles, follow a circadian pattern in pituitary cells. Paraspeckles are known to retain within the nucleus RNAs containing inverted repeats of Alu sequences. We showed that a reporter gene in which these RNA duplex elements were inserted in the 3'-UTR region displayed a circadian expression. Moreover, circadian endogenous mRNA associated with paraspeckles lost their circadian pattern when paraspeckles were disrupted. This work not only highlights a new paraspeckle-based post-transcriptional mechanism involved in circadian gene expression but also provides the list of all mRNA associated with paraspeckles in the nucleus of pituitary cells.

International audience; Although it is known to contain five cell types that synthesize and release hormones with a circadian pattern, the pituitary gland is poorly characterized as a circadian oscillator. By a differential microarray analysis, 252 genes were found to be differentially expressed in pituitaries from Bmal1(-/-) knockout versus wild-type mice. By integrative analyses of the data set with the Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources annotation analysis system, pituitary genes with altered expression in Bmal1(-/-) mice were dispatched among functional categories. Clusters of genes related to signaling and rhythmic processes as well as transcription regulators, in general, were found enriched in the data set, as were pathways such as circadian rhythm, transforming growth factor β (TGFβ) signaling, valine, leucine, and isoleucine degradation, and peroxisome proliferator-activated receptor (PPAR) signaling pathways. Gene Ontology term overrepresentation analyses revealed significant enrichment for genes involved in 10 key biological processes. To determine whether genes with altered expression in Bmal1(-/-) mice were actually circadian genes, we further characterized in the mouse pituitary gland the daily pattern of some of these genes, including core-clock genes. Core-clock genes and genes selected from three identified overrepresented biological processes, namely, hormone metabolic process, regulation of transcription from RNA polymerase II promoter, and cell adhesion, displayed a rhythmic pattern. Given the enrichment in genes dedicated to cell adhesion and their daily changes in the pituitary, it is hypothesized that cell-cell interactions could be involved in the transmission of information between endocrine cells, allowing rhythmic hormone outputs to be controlled in a temporally precise manner.

Rhythmic biological functions in mammals are orchestrated by a circadian timekeeper in the suprachiasmatic nucleus of the hypothalamus (SCN) which precisely adjusts clock outputs to solar time through the process of photic synchronization. Entrainment to the 24-h light-dark cycle is known to act on the molecular loops which trigger circadian oscillations but is also thought to involve day-night adjustments in the intercellular phasing of the multiple component SCN oscillators. This view is supported by data showing that the SCN undergoes important rearrangements of its neuroglial architecture throughout the 24-h cycle. The present paper highlights our data showing in rat that the two main sources of SCN efferents, composed of neurons synthesizing either vasopressin (AVP) or vasoactive intestinal peptide (VIP), are differentially involved in day-night SCN neuroglial plasticity. We found that the synaptic inputs received by the VIP neurons, which are major integrators of photic signals in the retinorecipient SCN subregion, increased during the day while those received by the AVP neurons remained unchanged at day and night. Glutamatergic axons, known to convey photic information from the retina, together with nonglutamatergic axons, contribute to the synaptic remodellings on VIP neurons. Experimental data providing strong indication that these plastic events may subserve synchronization of the clock to the light-dark cycle and that the daily fluctuations of plasma glucocorticoid hormones may act as temporal endocrine signals that may modulate SCN neuroglial plasticity through the rhythmic release of serotonin are also reviewed.

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.

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; Circadian rhythms in mammals are synchronized to the light (L)/dark (D) cycle through messages relaying in the master clock, the suprachiasmatic nucleus of the hypothalamus (SCN). Here, we provide evidence that the SCN undergoes rhythmic ultrastructural rearrangements over the 24-h cycle characterized by day/night changes of the glial, axon terminal, and/or somato-dendritic coverage of neurons expressing arginine vasopressin (AVP) or vasoactive intestinal peptide (VIP), the two main sources of SCN efferents. At nighttime, we noted an increase in the glial coverage of the dendrites of the VIP neurons (+29%) that was concomitant with a decrease in the mean coverage of the somata (-36%) and dendrites (-43%) of these neurons by axon terminals. Conversely, glial coverage of the dendrites of AVP neurons decreased (-19%) with parallel increase in the extent of somatal (+96%) and dendritic (+52%) membrane appositions involving these neurons. These plastic events were concomitant with daily fluctuations in quantitative expression of glial fibrillary acidic protein (GFAP), which were then used as an index of structural plasticity. The GFAP rhythm appeared to be strictly dependent on light entrainment, indicating that structural reorganization of the SCN may subserve synchronization of the clock to the L/D cycle. Other results presented reinforced this view while showing that circulating glucocorticoid hormones, which are known to modulate photic entrainment, were required to maintain amplitude of the GFAP rhythm to normal values.

International audience; The circadian timing system orchestrates daily variations in physiology and behavior through coordination of multioscillatory cell networks that are highly plastic in responding to environmental changes. Over the last decade, it has become clear that this plasticity involves structural changes and that the changes may be observed not only in central brain regions where the master clock cells reside but also in clock-controlled structures. This review considers experimental data in invertebrate and vertebrate model systems, mainly flies and mammals, illustrating various forms of structural circadian plasticity from cellular to circuit-based levels. It highlights the importance of these plastic events in the functional adaptation of the clock to the changing environment.

The circadian clock in the suprachiasmatic nucleus (SCN) controls day-to-day physiology and behavior by sending timing messages to multiple peripheral oscillators. In the pineal gland, a major SCN target, circadian events are believed to be driven exclusively by the rhythmic release of norepinephrine from superior cervical ganglia (SCG) neurons relaying clock messages through a polysynaptic pathway. Here we show in rat an SCN-driven daily rhythm of pineal MAPK activation that is not dependent on the SCG and whose maintenance requires vitamin A as a blood-borne factor. This finding challenges the dogma that SCG-released norepinephrine is an exclusive mediator of SCN-pineal communication and allows the assumption that humoral mechanisms are involved in pineal integration of temporal messages.

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.

The daily rhythm in circulating melatonin is driven by a circadian rhythm in the expression of the arylalkylamine N-acetyltransferase gene in the rat pineal gland. Turning off expression of this gene at the end of night is believed to involve inhibitory transcription factors, among which Fos-related antigen 2 (Fra-2) appears as a good candidate. Circadian rhythms in the expression of three proteins of activating protein-1 (AP-1) complexes, namely, Fra-2, c-Jun, and Jun-D, are shown here to account for circadian variations in AP-1 binding activity. Quantitative variations in the Fra-2 component over the circadian cycle were associated with qualitative variations in protein isoforms. Destruction of the suprachiasmatic nucleus resulted in decreased nocturnal AP-1 activity, showing that AP-1 circadian rhythm is driven by this nucleus. Exposure to light during subjective night and administration of a serotonin 5-HT1A/5-HT7 receptor agonist during subjective day, respectively, induced a 50% decrease and a 50% increase in both AP-1 and Fra-2 expression. These effects were impaired by suprachiasmatic nucleus lesions. These data show that pineal AP-1 binding activity, which results from Fra-2 expression, can be modulated by light and serotonin through the suprachiasmatic nucleus according to a “phase dependence” that is characteristic of the rhythm of clock sensitivity to both zeitgebers.

Short communication on POU1F1, with data on DNA/RNA, on the protein encoded and where the gene is implicated.

In mammals, photic entrainment of circadian rhythms likely involves light- and clock-dependent expression of immediate early genes, including fos-like and jun-like genes, in the rat suprachiasmatic nucleus. Using an electrophoretic mobility shift assay, we evaluated whether the photic regulation of DNA-binding activity and composition of activating protein-1 (AP-1) complexes in the suprachiasmatic nucleus is also dependent on circadian phase. Phase-dependent light inducibility in the expression of fra-2 and c-fos genes and in immunoreactive Fra-2 and c-Fos protein expression was also evaluated, by in situ hybridization and immunocytochemistry. Light's effects on AP-1 DNA-binding differed both qualitatively and quantitatively according to the circadian phase at which light was applied. This phase dependence accounted for by both compartmentalization of proteins involved in constitutive AP-1 complexes within the nucleus or cytoplasm and control of the extent to which the expression of specific complexes was induced. It was then shown that the mechanisms by which the circadian clock gates the photic induction of AP-1 components differed according to the nature of the protein.

Luteinizing hormone-releasing hormone (LHRH release, which serves as the primary drive to the hypothalamic-pituitary gonaldal axis, is controlled by many neuro-mediators. 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.

Previous works have suggested an interactive stimulatory effect of progesterone (P) and serotonin (5-HT) on luteinizing hormone release. The purpose of the present study was to determine whether 5-HT via 5-HT1A receptors interacts with P in the process of luteinizing hormone-releasing hormone (LHRH) release. Using fetal hypothalamic neurons in primary cell cultures the first goal of this study was to determine the effects of 5-HT1A receptor agonists on LHRH secretion. 8-Hydroxy-2 (di-n-propylamino) tetralin (8-OH-DPAT) or ipsapirone (10–5M) significantly stimulated LHRH release. Pharmacological studies have allowed to rule out the possible involvement of α2-or β-adrenore-ceptors, or 5-HT uptake sites, in the stimulatory effect of 8-OH-DPAT on LHRH release, thus demonstrating the specific involvement of 5-HT1A receptors in the stimulation of LHRH release. The second goal was to test the ability of P to stimulate LHRH release from fetal hypothalamic neurons. P (10–6M) applied for 30 or 120 min significantly stimulated LHRH secretion. The maintenance of the stimulation of LHRH release by P after a cycloheximide treatment or by an impermeable analog of P, P-3-BSA, has suggested a nonge-nomic effect of P on LHRH release. The effects of a pretreatment of cells by P on 8-OH-DPAT-induced LHRH release were tested. While 10–7M P alone did not stimulate LHRH release, this concentration of steroid potentiated the LHRH response to 10–5M 8-OH-DPAT. These findings led to the conclusion that P acting at the level of the plasma membrane potentiates the stimulatory effect of 5-HT1A receptor agonists on LHRH release.

Synchronization of circadian rhythms to the 24-h light/dark (L/D) cycle is associated with daily rearrangements of the neuronal-glial network of the suprachiasmatic nucleus of the hypothalamus (SCN), the central master clock orchestrating biological functions in mammals. These anatomical plastic events involve neurons synthesizing vasoactive intestinal peptide (VIP), known as major integrators of photic signals in the retinorecipient region of the SCN. Using an analog-sensitive kinase allele murine model (TrkB(F616A) ), we presently show that the pharmacological blockade of the tropomyosin-related kinase receptor type B (TrkB), the high-affinity receptor of brain-derived neurotrophic factor (BDNF), abolished day/night changes in the dendrite enwrapping of VIP neurons by astrocytic processes (glial coverage), used as an index of SCN plasticity on electron-microscopic sections. Therefore, the BDNF/TrkB signaling pathway exerts a permissive role on the ultrastructural rearrangements that occur in SCN under L/D alternance, an action that could be a critical determinant of the well-established role played by BDNF in the photic regulation of the SCN. In contrast, the extent of glial coverage of non-VIP neighboring dendrites was not different at daytime and nighttime in TrkB(F616A) mice submitted to TrkB inactivation or not receiving any pharmacological treatment. These data not only show that BDNF regulates SCN structural plasticity across the 24-h cycle but also reinforce the view that the daily changes in SCN architecture subserve the light synchronization process.

International audience; Most clock-controlled genes (CCGs) lack the specific E-box response element necessary for direct circadian regulation. This is the case for the prolactin (Prl) gene, the expression of which oscillates in individual lactotrope pituitary cells. To characterize the processes underlying this oscillation, we used a lactotrope cell line (GH4C1 cells). In these cells, Prl gene expression fluctuated significantly during 24 h (P=0.0418). Circadian Prl transcription depended on an interaction between the pituitary-specific transcription factor, PIT-1, and the helicase-like transcription factor (HLTF), a SWI/SNF chromatin remodeler, shown here to bind the Prl promoter on an E-box that differs from the specific E-box preferentially bound by clock proteins. Circadian Prl transcription was further accompanied by marked daily chromatin transitions. While neither HLTF nor PIT-1 was rhythmically expressed, NONO and SFPQ, identified as HLTF-associated proteins by mass spectrometry, displayed a circadian pattern and bound rhythmically to the Prl promoter. Furthermore, NONO and SFPQ were functionally involved in circadian Prl transcription since overexpression of both proteins greatly reduced Prl promoter activity (P

Control of serotonin release and synthesis by amino acid neurotransmitters was investigated in rat rostral and caudal rhombencephalic raphe cells in primary cultures respectively. Endogenous amounts of taurine, glycine, GABA and glutamate were measured in both types of cultures. These amino acids were spontaneously released to the incubating medium. Exogenous taurine (10(-4) M) inhibited release and synthesis of newly formed [3H]serotonin [3H]5-HT from [3H]-tryptophan only in rostral raphe cells. Glycine (10(-3) M) decreased [3H]5-HT release in both types of cells, synthesis being diminished only in rostral raphe cells. Glycine inhibitory effect was totally blocked by strychnine (5 x 10(-5) M). GABA (10(-4) M) reduced [3H]5-HT metabolism in rostral as well as caudal raphe cells. This effect was totally antagonized in caudal and partially in rostral raphe cells by bicuculline (5 x 10(-5) M) a GABAA receptor antagonist. Baclofen (5 x 10(-5) M), a GABAB receptor agonist, induced a decrease of 5-HT release in rostral raphe cells. These observations suggest that monoamine release was entirely mediated by GABAA receptors in caudal raphe cells although GABAA and GABAB receptors were involved in control of 5-HT metabolism in rostral raphe cells. L-glutamate (10(-4) M) stimulated 5-HT metabolism in both types of cells, effect totally blocked by PK26124 (10(-6) M). N-methyl-D-aspartate (10(-4) M) enhanced 5-HT metabolism and the induced-effect was antagonized by the selective N-methyl-D-aspartate receptor antagonist D,L-2 amino-5-phosphonovaleric acid. Quisqualate (10(-5) M) stimulated [3H]5-HT release only in caudal raphe cells. This effect was mimicked by (RS)-a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, a quisqualate "ionotropic" receptor agonist, this increase being blocked by 6,7-dinitroquinoxaline 2,3-dione. These observations suggest that the glutamate stimulating-induced effect on serotonin metabolism is entirely mediated by N-methyl-D-aspartate receptor-type in rostral raphe cells and that quisqualate "ionotropic" receptors are also involved in caudal raphe cells. Taken together these results show that [3H]5-HT metabolism is controlled by taurine, glycine, GABA and glutamate in rhombencephalic raphe cells in primary cultures. However, some difference in amino acid receptor-types involved in the control of serotonin metabolism are observed according to the rostral or caudal origin of raphe cells.

International audience; The daily temporal organization of rhythmic functions in mammals, which requires synchronization of the circadian clock to the 24-h light-dark cycle, is believed to involve adjustments of the mutual phasing of the cellular oscillators that comprise the time-keeper within the suprachiasmatic nucleus of the hypothalamus (SCN). Following from a previous study showing that the SCN undergoes day/night rearrangements of its neuronal-glial network that may be crucial for intercellular phasing, we investigated the contribution of glutamatergic synapses, known to play major roles in SCN functioning, to such rhythmic plastic events. Neither expression levels of the vesicular glutamate transporters nor numbers of glutamatergic terminals showed nycthemeral variations in the SCN. However, using quantitative imaging after combined immunolabelling, the density of synapses on neurons expressing vasoactive intestinal peptide, known as targets of the retinal input, increased during the day and both glutamatergic and non-glutamatergic synapses contributed to the increase (+36%). This was not the case for synapses made on vasopressin-containing neurons, the other major source of SCN efferents in the non-retinorecipient region. Together with electron microscope observations showing no differences in the morphometric features of glutamatergic terminals during the day and night, these data show that the light synchronization process in the SCN involves a selective remodelling of synapses at sites of photic integration. They provide a further illustration of how the adult brain may rapidly and reversibly adapt its synaptic architecture to functional needs.

Preproenkephalin metabolism, in the rat, was studied in primary striatal neurons maintained in a chemically defined medium. Acute treatment (30 min) with forskolin (10 −5 M) or phorbol 12 myristate 13 acetate (10 −7 M) resulted, respectively, in a two- and seven-fold increase in methionine-enkephalin secretion. Chronic treatment with forskolin or phorbol 12 myristate 13 acetate (24 h) induced a 100% increase in methionine-enkephalin content (forskolin) and on the other hand a 50% decrease in methionine-enkephalin (phorbol 12 myristate 13 acetate). Both treatments increased preproenkephalin mRNA levels in a time-dependent manner, this augmentation being observable after 180 min by Northern blot analysis and in situ hybridization. These data indicate that under chronic stimulation, with either forskolin or phorbol 12 myristate 13 acetate, proenkephalin turnover is accelerated. However, after stimulation with phorbol 12 myristate 13 acetate, the more potent methionine-enkephalin secretagogue, increased peptide synthesis is not sufficient to replenish methionine-enkephalin intracellular stores. Preproenkephalin gene transcription was analysed by introducing the preproenkephalin gene promoter fused to the bacterial acetyl chloramphenicol transferase reporter gene into primary neurons. Chronic stimulation (48 h) by forskolin (10 −5 M) or phorbol 12 myristate 13 acetate (10 −7 M) of striatal neurons transfected with this fusion gene increased chloramphenicol acetyltransferase activity six-fold and the two effects were additive. These data suggest that the cyclic AMP and the protein kinase C pathways directly activate preproenkephalin gene transcription.

The hypothalamus of both adult and fetal rats contains a population of cells which can exhibit some features of serotoninergic (5-HT) neurons under certain circumstances. However, their neuronal serotoninergic nature is still controversial. In fact the presence of tryptophan hydroxylase activity has not yet been clearly established. This study attempted to verify whether [3H]5-HT can be synthesized from [3H]tryptophan ([3H]TRP) in hypothalamic cell cultures from 16-day-old fetuses. Data showed that [3H]5-HT was synthesized from [3H]TRP and the amounts of [3H]5-HT increased linearly as a function of time for 60 min. Pargyline markedly increased the quantities of [3H]5-HT and decreased those of [3H]5-hydroxyindole acetic acid, [3H]5-HT synthesis was inhibited by p-chlorophenylalanine, while α-methyl-p-tyrosine had no effect. The present biochemical study shows the presence of an intrinsic 5-HT neuronal system in the hypothalamus of the fetal rat.

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 question of involvement of glucocorticoid hormones as temporal signals for the synchronization of the timekeeping system was addressed in rats with different corticosterone status. The authors showed that adrenalectomy had no effects on the synchronization of wheel-running activity rhythms to a steady-state LD 12:12 cycle, regardless of whether it was compensated for by a corticosterone replacement therapy that either reinstated constant plasma concentrations of the hormone or mimicked its natural rhythm. However, after a 12-h phase shift (daylight reversal), the lack of circulating corticosterone induced a significant shortening of the resynchronization rate (less than 3 days vs. 7 days). Normalization required restoration of a rhythmic corticosterone secretion that was synchronized to the new photoperiod. Under constant darkness, the corticosterone rhythm did not show any synchronizing effect, providing evidence that it participates in entrainment of the locomotor activity rhythm through modulation of light effects. It is proposed that, under stable lighting conditions, circulating glucocorticoids contribute to stabilizing activity rhythms by reinforcing resistance of the circadian timing system to variations of the photoperiod. Experimental evidence that serotonergic neurons are involved in relaying their modulatory effects to the clock is also presented.

La rythmicite circadienne de l’activite de l’adenohypophyse est une composante essentielle de la physiologie de la glande parce que toutes les hormones qu’elle secrete presentent un profil rythmique, pulsatile et/ou circadien. Dans le domaine des rythmes circadiens, il apparait aujourd’hui que les regulations s’exercant au niveau post-transcriptionnel jouent un role preponderant. Dans l’adenohypophyse, nous avons identifie un mecanisme post-transcriptionnel original a la base de la rythmicite d’expression de certains ARNm. Ce mecanisme repose sur la retention nucleaire rythmique d’ARNm par des structures appelees paraspeckles dont l’element structurant est un long ARN non-codant, Neat1 (Nuclear-Enriched-Abundant-Transcript1) egalement connu sous le nom de Men1 (multiple-endocrine-neoplasia1). Nous avons montre que, dans les cellules hypophysaires, tous les composants moleculaires de ces paraspeckles, non seulement Neat1 mais aussi les principaux composants proteiques, presentent un profil d’expression circadienne. Du reste, l’expression de Neat1 est egalement rythmique dans d’autres oscillateurs y compris dans l’horloge circadienne centrale, les noyaux suprachiasmatiques de l’hypothalamus. Nous avons montre que certains ARNm qui sont associes aux differents composants des paraspeckles et dont la rythmicite circadienne est controlee au niveau post-transcriptionnel perdent leur expression circadienne en presence de siRNA dirigees contre Neat1 qui entrainent une destruction des paraspeckles. Le role grandissant que l’on attribue aujourd’hui aux longs ARN non-codants dans la pathogenie de nombreuses maladies et la caracterisation d’un role physiologique joue par un long ARN non-codant dans l’adenohypophyse ouvrent de nouvelles perspectives de strategie possible d’identification de candidats moleculaires participant a l’etiologie de pathologies hypophysaires en particulier de deficits hypophysaires chez l’homme.

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.

Monoaminergic projections to the spinal cord are involved in the modulation of motor, autonomic, and sensory functions. More specifically, the increase of electrical activity of serotonergic neurons in raphe obscurus has been correlated with locomotion in treadmill-trained cats [Jacobs, B.L. and Fornal, C., Trends Neurosci., 9 (1993) 346-352]. In order to test the direct correlation between locomotion and the release of monoamines, microdialysis probes were permanently implanted for 45 days into the ventral funiculus of the spinal cord (white matter) of adult rats. Eight days after implantation, these rats were subjected to an endurant exercise on a treadmill, and dialysis sessions were organized in such a way that microdialysate samples of 15 min duration were collected during pre-, per- and post-exercise periods. Measurements of serotonin, 5-hydroxyindoleacetic acid, dopamine and 3-methoxy-4-hydroxyphenylethylglycol concentration in the extracellular space showed significant increases during locomotion when compared with both pre- and post-exercise values. Histological analysis shows that serotonergic axons were present close to the dialysis probe. These results demonstrate that the implantation of a microdialysis probe in the ventral funiculus, close to a potential target of monoaminergic projections, is a suitable technique for the collection of neuromediators released during spontaneous running.

Serotonin1B (5-HT1B) receptor binding in the suprachiasmatic nucleus (SCN) following impairment of serotoninergic transmission was studied by quantitative autoradiography. Serotonin (5-HT) denervation with 5,7-dihydroxytryptamine (5,7-DHT) caused a significant increase in the density of 5-HT1B receptors in both the ventral (62%) and dorsal (53%) parts of the SCN as early as 3 days after axotomy. The magnitude of this increase did not differ 3, 15 or 21 days post-lesion. An up-regulation of 5-HT1B receptors with similar magnitude was obtained in the two parts of the SCN after inhibition of 5-HT synthesis by chronic parachlorophenylalanine treatment. In this case, up-regulation was shown to be reversible after restoration of 5-HT synthesis with l-5-hydroxytryptophan. These results indicate that 5-HT1B receptor density in the SCN was inversely correlated with 5-HT levels. These plastic properties exhibited by 5-HT1B receptors in the SSN are discussed in relation to the mode of 5-HT transmission and possible localization of the receptors onto the main chemically defined cell populations of the nucleus.

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).

The inter-renal (adrenal) gland of amphibians is composed of chromaffin and steroidogenic cells which can interact through a paracrine mode of communication. We have previously shown that serotonin is present in secretory granules of frog adrenochromaffin cells; concurrently, we have demonstrated that serotonin is a potent stimulator of corticosterone and aldosterone secretion by adrenocortical cells. The aim of the present study was to determine the origin of the amine contained in frog chromaffin cells. Using 3 H-labelled tryptophan as a precursor, we observed the formation of substantial amounts of serotonin and its metabolite 5-hydroxyindoleacetic acid by frog inter-renal slices. Newly synthesized serotonin was secreted into the incubation medium and the release process was enhanced by depolarizing concentrations of KCl. Fluoxetine, an inhibitor of serotonin uptake, caused an increase of 3 H-labelled serotonin in the incubation medium, suggesting that the indoleamine was taken up again by adrenal chromaffin cells. The capacity of the frog inter-renal gland to synthesize serotonin was also demonstrated by incubating inter-renal slices with non-labelled tryptophan or 5-hydroxytryptophan. In these conditions, we observed that the rate of synthesis was higher when 5-hydroxytryptophan was used as a precursor, rather than tryptophan. Taken together, these results indicate that chromaffin cells, which have the capacity for synthesizing and releasing serotonin, behave like authentic serotonergic paraneurons. As far as is known, these data provide the first evidence for the occurrence of tryptophan-5-hydroxylase activity within the adrenal gland.

In order to better understand the mechanisms underlying the reduction in growth hormone (GH) secretion in diabetic rats, we studied hypothalamic somatostatin secretion both in vivo (into hypophysial portal blood) and in vitro (from hypothalamic fragments) 5, 9 and 30 days after induction of diabetes. Experimental diabetes was induced by an intraperitoneal injection of streptozotocin (STZ) at a dose of 65 mg/kg. Basal plasma GH was significantly reduced in diabetic rats at all stages. Somatostatin levels in hypophysial portal blood was unaffected in 5-day STZ-diabetic rats and significantly increased 9 days after STZ administration. Chronic insulin replacement therapy in diabetic animals partly normalized somatostatin levels as well as plasma GH and glucose levels. A good correlation was observed between in vivo and in vitro experiments. Indeed, somatostatin release from hypothalamic fragments did not change 5 days after STZ-induced diabetes and significantly increased 9 and 30 days after STZ administration. The in vitro increase in hypothalamic somatostatin secretion was observed in 10 as well as in 33 mM glucose concentration in the incubation medium. In the same experiment, the in vitro hypothalamic corticotropin-releasing factor secretion was lowered 5 and 9 days after diabetes induction. We conclude that hypothalamic somatostatin release increases in diabetic rats. These changes may contribute to the reduction in GH secretion in these animals. However, since these changes occur after the onset of plasma GH decrease, a factor(s) other(s) than somatostatin may play a causal role in the reduction in GH secretion.

International audience; no abstract

This study aimed at analyzing the regulation of in vitro serotonin expression by neurons taken from different regions of the embryonic rat rhombenceph-alon. We studied the influence of co-culture with alar-plate tissue using immunocytochemical and biochemical methods. Computer-assisted densitometry was used to estimate the co-culture effects on the serotonin content of the cell bodies. The more dynamic aspects of serotonin expression, such as synthesis and release, were studied by measuring (3H)serotonin newly synthe-sized from (3H) tryptophan. The density of the immunostaining was significantly decreased in B1, B2 cells by co-culture with both caudal and rostral alar-plate tissue. For B4–B9 cells, only co-culture with rostral alar-plate tissue produced a significant decrease. The de novo synthesis of serotonin was significantly decreased in B1, B2 neurons co-cultured with caudal alar-plate tissue only. Once again, the B4–B9 cells proved to be less influenced by the experimental conditions, as co-culture with both types of alar-plate tissue produced no significant effect. We concluded that the in vitro expression of serotonin can be modulated by environmental factors, but the relative influence of these factors is very different in rostral versus caudal serotonin expressing cell populations.

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 (10−8M), 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-HT1D/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-phenylpropionamide dihydrochloride quaternary-hydrate (WAY 100135) (10−7M) or methyl 4-[4-[4-(1,1,3-trioxo-2H-1,2-benzoisothiazol-2-yl)butyl]-1-piperazinyl]-1H-indole-2-carboxylate (SDZ 216–525) (10−7M), and that of CP-93, 129 was blocked by methiothepin (10−7M). Paradoxically, extracellular levels of [3H]5-HT increased in the presence of 8-OH-DPAT and RU 24969 at 10−6M. 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.

International audience; The TRH secretory responsiveness of the pancreatic islet cell clusters from newborn rat in organ culture was studied. Basal TRH secretion was stable over a 9‐day period. The response to various secretagogues was tested on day 4. TRH secretion was stimulated by high potassium‐induced depolarization and also through both cAMP and protein kinase‐C dependent pathways. Like insulin, TRH release was stimulated by glucose and arginine and inhibited by somatostatin. These data suggest the existence of a common mechanism for TRH and insulin secretion by the pancreatic beta‐cells.

The local effect of L-glutamic acid (5 x 10(-5) M) on the release of [3H]serotonin continuously synthesized from [3H]tryptophan was examined in the caudate nucleus of 'encephale isole' unanaesthetized cats implanted with push-pull cannula. L-Glutamic acid (5 x 10(-5) M) decreased [3H]serotonin release from nerve terminals of the dorsalis raphe-striatal serotonergic neurons. The effect was antagonized by 2-amino-6-trifluoromethoxybenzothiazole (PK 26124) (10(-6) M), an antagonist of glutamatergic transmission. This effect was mimicked by N-methoxy-D-aspartic acid NMDA (5 x 10(-5) M) and prevented by DL-2-phosphono-valeric acid (APV) (5 x 10(-6) M), indicating that L-glutamic acid decreased serotonin release via a N-methoxy-D-aspartate type receptor. The superfusion of serotonergic nerve terminals in the caudate nucleus with tetrodotoxin prevented the inhibitory L-glutamic acid-induced effect on serotonin release. Furthermore, L-glutamic acid-induced inhibition of [3H]serotonin release was antagonized by bicuculline (5 x 10(-5) M). These data suggest that the glutamatergic receptors involved were not located directly on serotonin nerve terminals. The inhibitory control exerted by L-glutamic acid on serotonergic transmission could involve gamma-aminobutyric acid interneurons. Since no reduction of spontaneous [3H]serotonin release was observed in the presence of bicuculline, GABAergic neurons appeared to exert a phasic influence on serotonin release. Indirect inhibitory presynaptic control on serotonin release mediated by corticostriatal glutamatergic fibers is discussed in light of previous findings.

Using a push-pull cannula technique and an isotopic method for estimating [ 3 H]serotonin continuously synthesized from [ 3 H]tryptophan, the effects of changes in the release of serotonin in the dorsalis raphe nucleus on in vivo release of [ 3 H]serotonin in the cat caudate nucleus were investigated. The increase in the release of serotonin in the dorsalis raphe nucleus caused by local application of parachlorophenylethylamine (10 −6 M) reduced striatal [ 3 H]serotonin release. This inhibition in serotonin release in the striatum was blocked by the prior and continuous local superfusion of the dorsal raphe with methiothepin (10 −6 M), a serotonin autoreceptor antagonist. GABA (5 × 10 −5 M) applied to the dorsalis raphe reduced both local and striatal release of [ 3 H]serotonin. However, picrotoxin (10 −5 M), a OABA A receptor antagonist applied locally in the dorsalis raphe nucleus increased [ 3 H]serotonin release while decreasing striatal [ 3 H]serotonin release. This decrease in serotonin release in the striatum was again blocked by continuous superfusion of the raphe with methiothepin. Furthermore, superfusion of serotonergic cell bodies of the dorsalis raphe nucleus with methiothepin alone never altered local release or striatal release of [ 3 H]serotonin. These data strongly suggest that the release of serotonin from the cell body in the dorsalis raphe nucleus phasically controls release of the amine at the axonal nerve ending through serotonergic autoreceptors located on serotonergic nerve cell bodies in the dorsalis raphe nucleus. The origin of the serotonin released in the dorsalis raphe nucleus and the possibility that this type of regulation could be related to changes in nerve impulse conduction of the serotonergic raphe-striatal system are discussed.

Unilateral kainic acid lesion of the rat centromedian-parafascicular complex (CM-PF) of the thalamus induced a decrease in the 5-hydroxyindole acetic acid/5-hydroxytryptamine ratio both in ipsi and contralateral striatum and substantia nigra, and an increase in both ipsi and contralateral frontal cerebral cortex. No change in apparent serotonin turnover was detected in anterior raphe nuclei. Serotonin synthesis, estimated by measuring 5-hydroxytryptophan accumulation after injection of a decarboxylase inhibitor, was not affected by the CM-PF lesion. The possible pathways involved in the control of serotonin transmission by CM-PF are discussed.

The mechanisms involved in parafascicularis nucleus control on serotoninergic neurons projecting into the caudate nucleus were investigated in “ence´phale-isole” cats. The effects of unilateral stimulation of the parafascicularis nucleus on the release of newly synthesized [3H]serotonin were simultaneously determined in the ipsilateral caudate nucleus and the dorsalis raphe nucleus using push-pull cannulae. The actions of various pharmacological treatments performed either in the caudate nucleus or in the dorsalis raphe nucleus were also examined. The electrical or chemical stimulation of the parafascicularis nucleus induced a decrease in striatal [3H]serotonin release and an increase in [3H]serotonin release in the dorsalis raphe nucleus. The blockade of cholinergic (mecamylamine) and glutamatergic (PK 26124) transmissions at the striatal level did not modify the thalamic stimulation induced effect on serotonin release in the caudate nucleus or in the dorsalis raphe nucleus. However, a decrease induced by parafascicularis nucleus stimulation in serotonin release in the caudate nucleus could not be observed when the autoreceptors present on serotoninergic nerve cell bodies localized in the dorsalis raphe nucleus were blocked by a methiothepin perfusion within the nucleus. These results indicate that the parafascicularis nucleus controls striatal serotonin transmission by inducing changes in the nerve activity of serotoninergic neurons in the dorsalis raphe nucleus via somatodendritic serotonin release and autoreceptors.