ABSTRACT BOOK - hypothalamus.euhypothalamus.eu/wp-content/uploads/BSN-SNE2015_Program_low.pdf · LocalOrganizingCommittee !! Vincent Prévot, Chair Bénédicte Dehouck, Deputy Chair

ABSTRACT BOOK

17:00-‐19:00 Oral Presentations (Salle des Congrès)

Chair: * Rebecca Campbell, University of Otago, New Zealand

Luis Paiva University of Edinburgh, UK Activation of oxytocin neurons following systemic Melanotan-‐II

administration Zane Andrews, Monash University, Australia Glucose metabolism affects ghrelin-‐induced feeding and

motivation Tina Bake, University of Gothenburg, Sweden Ghrelin changes food preference from high fat diet to chow in schedule-‐fed rats D’Agostino, Giuseppe, University of Aberdeen, UK Nucleus of the solitary tract cholecystokinin-‐expressing neurons control appetite Caroline Decourt, INRA, University of Tours, France Design of a selective kisspeptin analog capable of synchronizing

ovulation Alexander Tups, University of Otago, New Zealand Photoperiodic regulation of Wnt signalling in the arcuate nucleus of the Djungarian hamster, Phodopus sungorus Helen Christian, University of Oxford, UK Photoperiod-‐driven remodelling of lactotroph, gonadotroph and folliculostellate cells in the sheep pituitary Francesca Spiga, University of Bristol, UK Experimental and mathematical studies on the dynamics of adrenal glucocorticoid synthesis suggest an intra-‐adrenal negative feedback mechanism that involves activation of the glucocorticoid receptor Lisa Koorneef, Leiden University Medical Centre, the Netherlands A novel mixed glucocorticoid/mineralocorticoid receptor selective modulator reduces obesity and adipose tissue and liver inflammation Stefania Maccari, CNRS, University of Lille, France Early carbetocin treatment prevents metabolic aging induced by early stress in rats

19:15 Welcome Mixer (Atrium)

Local Organizing Committee

Vincent Prévot, Chair

Bénédicte Dehouck, Deputy Chair

Ariane Sharif, Deputy Chair

Samuel Malone, Webmaster

By alphabetical order:

Jean-Claude Beauvillain

Emilie Caron

Konstantina Chachlaki

Irene Cimino

Manon Duquenne

Sarah Gallet

Paolo Giacobini

Anne Loyens

Stefania Maccari

Daniele Mazur

Giuliana Pellegrino

Charlotte Vanacker

Didier Vieau

PROGRAM

19:30 Opening Public Lecture in French

Olivier Kah, Inserm, University of Rennes, France

L’environnement perturbe nos hormones! Salle Quebec, Le Nouveau Siècle, Lille Downtown (Métro Rihour)

Chairs: *Valerie Simmoneaux, CNRS, University of Strasbourg, France * Vincent Prevot, Inserm, University of Lille, France

TUESDAY, SEPTEMBER 2 2

12:00-‐13:00 Arrival – Registration (Name Tags) Reception desk, Salle des Congrès, Pôle Recherche, Medical School 13:00-‐13:30 Welcome Introduction Salle des Congrès Vincent Prevot, Inserm, University of Lille, France 13:30-‐14:30 Mortyn Jones Memorial Lecture

Chair: * Kevin O’Byrne, King’s College London, UK

Allan Herbison, University of Otago, New Zealand Generating Hormone Pulses in Neuroendocrine Circuits

14:30-‐17:00 Glucocorticoids, Stress and memory process Salle des Congrès

Chairs: * Marie-‐Pierre Moisan, INRA, University of Bordeaux, France

* Onno C. Meijer, Leiden University Medical Center The Netherlands

Onno C. Meijer, Leiden University Medical Centre, the Netherlands Selective activation of glucocorticoid receptor pathways: linking gene transcription to cognition Marie-‐Pierre Moisan, INRA, University of Bordeaux, France Corticosteroid binding globulin role in memory processes

15:30-‐16:00 Coffee Break Joyce Yau, University of Edinburgh, UK 11β-‐HSD1 and age-‐associated memory decline Laurent Givalois, Inserm, Montpellier, France Glucocorticoids' role in the hippocampus function impairment associated with Alzheimer’s disease

17:00-‐19:00 Oral Presentations Salle des Congrès Chair: * Rebecca Campbell,

University of Otago, New Zealand Luis Paiva, University of Edinburgh, UK Activation of oxytocin neurons following systemic Melanotan-‐II administration

Zane Andrews, Monash University, Australia Glucose metabolism affects ghrelin-‐induced feeding and motivation

Tina Bake, University of Gothenburg, Sweden Ghrelin changes food preference from high fat diet to chow in schedule-‐fed rats

Giuseppe D’Agostino, University of Aberdeen, UK Nucleus of the solitary tract cholecystokinin-‐expressing neurons control appetite

Caroline Decourt, INRA, University of Tours, France Design of a selective kisspeptin analog capable of synchronizing ovulation

Alexander Tups, University of Otago, New Zealand Photoperiodic regulation of Wnt signalling in the arcuate nucleus of the Djungarian hamster, Phodopus sungorus

Helen Christian, University of Oxford, UK Photoperiod-‐driven remodelling of lactotroph, gonadotroph and folliculostellate cells in the sheep pituitary

Francesca Spiga, University of Bristol, UK Experimental and mathematical studies on the dynamics of adrenal glucocorticoid synthesis suggest an intra-‐adrenal negative feedback mechanism that involves activation of the glucocorticoid receptor

Lisa Koorneef, Leiden University Medical Centre, the Netherlands A novel mixed glucocorticoid/mineralocorticoid receptor selective modulator reduces obesity and adipose tissue and liver inflammation

Stefania Maccari, CNRS, University of Lille, France Early carbetocin treatment prevents metabolic aging induced by early stress in rats

19:15 Welcome Mixer Atrium

WEDNESDAY, SEPTEMBER 2 3

08:00-‐10:00 Prolactin neuroendocrinology: new developments in an old system Salle des Congrès

Chairs: * Dave Grattan, University of Otago, New Zealand

* Paul le Tissier, University of Edinburgh, UK

Julian Davis, University of Manchester, UK Imaging gene transcription dynamics in living cells – from cells to tissue

Nadine Binart, Inserm, Le Kremlin-‐Bicêtre, France Mechanisms for prolactin regulation of reproduction

Rosemary Brown, University of Otago, New Zealand Prolactin actions in the brain

Agnes Martin, Inserm, Montpellier, France Plasticity in the neuroendocrine regulation of prolactin secretion during lactation

10:00-‐10:15 Coffee Break 10:15-‐11:15 Annual General Meetings -‐ BSN (Salle des Congrès) -‐ SNE (Amphi B) 11:15-‐14:00 Poster Session 1 Halls & Atrium

13:00-‐14:00 Lunch 14:00-‐16:00 Hypothalamic networks controlling puberty onset Salle des Congrès

Chairs: * Allan Herbison, University of Otago, New Zealand

* Vincent Prevot, University of Lille, France

Jane Robinson, University of Glasgow, UK Neural effects of blocking puberty with a long term GnRH agonist

Ariane Sharif, Inserm, University of Lille, France Astrogenesis in the postnatal hypothalamus: a new mechanism involved in female sexual maturation

Kevin O’Byrne, King’s College London, UK Puberty timing: is the hypothalamus controlled by the amygdala

Valerie Simonneaux, CNRS, University of Strasbourg, France Is seasonal reactivation of reproduction a yearly puberty?

16:00-‐16:15 Coffee Break

16:15-‐18:15 Symposium Early Stage Researchers (ESRs) 16:15-‐16:45 Alison Douglas Lecture Salle des Congrès

Chairs: * Matei Bolborea, University of Warwick, UK

* Michelle Bellingham, University of Glasgow, UK Gareth Leng, University of Edinburgh, UK Oxytocin in the brain: some misconceptions

16:40-‐18:00 "Speed Networking" event Salle Phinaert

Chairs: * Gisela Helfer, University of Aberdeen, UK

* Cristina Sáenz de Miera, University of Strasbourg, France

Short debates chaired by an ESR of the organising committee at each table. Rotation between tables every 10/15 minutes

Table 1-‐ EDITORS: Julian Mercer, Journal of Neuroendocrinology, UK Tim Geach, Nature Endocrinology Reviews, USA

Table 2 – PRIVATE SECTOR: Jean A. Boutin, SERVIER, France Elena Velarde, Madrid, Spain

Tables 3 and 4 – ESTABLISHED RESEARCHERS: Marian Jöels, UMC Utrecht, The Netherlands Fran Ebling, University of Nottingham, UK Paula Brunton, University of Edinburgh, UK Neil Evans, University of Glasgow, UK

Tables 5 and 6 – JUNIOR RESEARCHERS: Hugues Dardente, University of Tours, France Karolina Skibicka, University of Gothenburg, Sweden Gabi Wagner, University of Tromsø, Norway Alexander Tups, University of Otago, New Zealand

18:30-‐19:30 Jacques Benoit Memorial Lecture Chair: * Vincent Prevot, Inserm, University of Lille, France

Marian Joels, UMC Utrecht, The Netherlands Is early life stress always bad news?

20:30 Gala Diner La Halle au Sucre, Vieux Lille

THURSDAY, SEPTEMBER 2 4

FRI DAY, SEPTEMBER 2 5

8:45-‐11:00 Early life origins of obesity: programming of hypothalamic development and function

Salle des Congrès Chairs: * Laura Dearden, University of Cambridge, UK

* Susan E Ozanne, University of Cambridge, UK

Nina Balthasar, University of Bristol, UK The paraventricular hypothalamus in glucose-‐sensing, maternal programming and over-‐nutrition

Sophie Steculorum, Max Planck Institute, Cologne, Germany Novel regulator of orexigenic AgRP-‐neurons

Coffee Break (15 min)

Patricia Parnet, INRA, University of Nantes, France Early life nutrition and long term appetite regulation

Barry Levin, Rutgers New Jersey Medical School, USA Critical Amylin-‐Leptin Interactions Influencing Hypothalamic Development

11:00-‐14:00 Poster Session 2 13:00-‐14:00 Lunch 14:00-‐16:30 SNE & BSN Young Investigator Awards Salle des Congrès SNE Lecture

Chair: * Vincent Prevot, Inserm, University of Lille, France

Cristina Saenz de Miera, CNRS, University of Strasbourg, France Maternal photoperiod programs hypothalamic thyroid hormone deiodinase expression and seasonal reproduction in Siberian hamsters

Mick Harbuz lecture Chair: * Giles Yeo, University of Cambridge, UK

Bryn Owen, Imperial College, London, UK The physiology and pharmacology of the fasting hormone FGF21

SERVIER Symposium Chairs: * Valerie Simonneaux, CNRS,

University of Strasbourg, France * Jean A Boutin, SERVIER, France

Céline Cansell, University of Aberdeen, UK Nutritional triglycerides act on mesolimbic structures to regulate the rewarding and motivational aspect of feeding

Sarah Geller, University of Lausanne, Switzerland Hypothalamic astrocytes possess a higher glycolytic phenotype compared to cortical astrocytes: A potential role in the regulation of energy balance and reproduction function

Irene Cimino, Inserm, University of Lille, France Novel Role for Anti-‐Müllerian Hormone in the Regulation of GnRH Neuron Excitability and Hormone Secretion

Acknowledgments

Thank you… … to Bruno and Sophie Lesage (Inserm, Lille) for their everyday involvement in the organization of this meeting and for handling all its financial aspects. Their dedication and diligence made this conference possible. We would also like to thank Laeticia Coudert (SFR DN2M, Lille) for her help in preparing the Public Lecture and editing the program.

Foundation Obélsique Scholarship

Caroline Alfaïa, University of Tours, France

Amélie Borie, Inserm, University of Montpellier, France

Fernando Cazarez Marquez, CNRS, Institute of Cellular and Integrative Neuroscience, Strasbourg, France

Sophie Croizier, The Saban Research Institute, Children Hospital of Los Angeles, University of Southern California, USA

Lyes Derouiche, INRA, CNRS, University of Tours, France

Gabriela Ferreira de Medeiros, INRA, CNRS, University of Tours, France

Delphine Franssen, University of Liège, Belgium

Vincent Hellier, University of Liège, Belgium

Jerome Lannes, CNRS, Inserm, Paris Diderot University, France

OphéliaLe Thuc, CNRS, University of Nice Sophia Antipolis, France

Sébastien Milési, University of Strasbourg, France

Sarah Nicolas, CNRS, University of Nice Sophia Antipolis, France

Brooke Tata, Inserm, Paris Diderot University, France

Hadi Zarif, CNRS, Pierre and Marie Curie University, France

Mortyn Jones Memorial Lecture—Wednesday, September 23

L0101: Herbison Generating Hormone Pulses in Neuroendocrine Circuits

Allan E. Herbison

Centre for Neuroendocrinology, University of Otago School of Medical Sciences, Dunedin, New Zealand

Neuroendocrine hormones are released from the pituitary gland into the circulation in an episodic manner. The mechanisms through which neural circuits generate these pulsatile patterns of hormone release are variably understood amongst the different neuroendocrine axes. For example, our understanding of how episodic gonadotropin-‐releasing hormone (GnRH) release occurs remains poor despite decades of investigation. In this case, the scattered distribution of the GnRH neuron cell bodies within the basal forebrain has made it particularly difficult to investigate these neurons as a functional unit. As such, several theories explaining GnRH pulse generation have arisen involving intrinsic and/or extrinsic mechanisms. Recent studies have highlighted the importance of kisspeptin in pulse generation in addition to the unusual "dendronic" nature of GnRH neuron projections to median eminence. Studies using a range of approaches including GCaMP6-‐based calcium imaging and in vitro/in vivo optogenetics are now providing direct evidence than an arcuate kisspeptin neuron projection to the GnRH neuron distal dendron can generate pulses of GnRH secretion. Thus, an extrinsic pattern generator may be necessary for the generation of pulsatility within the neuroendocrine reproductive axis.

Alison Douglas Lecture—Thursday, September 24

L0201: Leng Oxytocin in the brain: some misconceptions

Gareth Leng

Centre for Integrative Physiology University of Edinburgh EH9 8XD

Oxytocin in the brain is a potent appetite regulating peptide, has multiple roles in reproductive functions, and has interesting effects on social behaviours. In these actions it functions as an autoregulator of oxytocin cell activity, is released at nerve endings in the caudal brainstem and spinal cord, and acts as a “neurohormone” within the hypothalamus following dendritic secretion. These are all now well established (1); much more controversial are the purported effects of intranasal oxytocin administration (2). I will talk about the gaps in our understanding of apparently established roles, as well as about the much greater problems in interpreting the outcomes of behavioural experiments with intranasal oxytocin.

1. Leng G, Pineda Reyes R, Sabatier N, Ludwig M. (2015) The posterior pituitary: from Geoffrey Harris to our present understanding. J Endocrinol. in press

2. Leng G, Ludwig M. (2015) Intranasal oxytocin: myths and delusions. Biol Psychiatry in press

Jacques Benoit Memorial Lecture—Thursday, September 24

L0202: Joels Is early life stress always bad news?

Marian Joëls

Dept. Translational Neuroscience, Brain Center Rudolf Magnus, UMC Utrecht, The Netherlands

In response to potential threats, the body initiates a stress response, involving activation of the hypothalamo-‐pituitary-‐adrenal axis. This has impact on both peripheral organs and the brain. Generally the stress response is very beneficial because it promotes adaptation. This certainly holds true for the cognitive domain. Thus, shortly after stress, vigilance and emotional responses are strongly stimulated, in part due to activation of the amygdala and striatum; this allows us to rapidly respond to the situation at hand. Some hours later, resources are redistributed in favor of higher cognitive regions, such as the dorsal hippocampus and prefrontal cortex, which collectively help to rationalize and contextualize stressful events. These adaptive responses may become maladaptive when occurring repetitively or at a young age, when brain circuits are fully in development. In accordance, stress experienced early in life is a well-‐documented risk factor for vulnerability to both psychiatric and neurological disorders. We use various animal models to delineate the sequential changes in brain structure and function as a consequence of early life adversity, and to unravel critical steps and mechanisms. It will be argued that the long-‐term consequences of early life stress are not invariably bad but depend on the conditions in which individuals have to perform later in life. Moreover, pharmacological or environmental intervention applied after early life adversity can to some extent lastingly reverse changes in brain structure and function due to early life stress. Finally, examples will be given that variations in genetic background may make individuals more or less susceptible to the effects of challenges early in life.

Young Investigator SNE Lecture—Friday, September 25

L0301: Saenz de Meira Maternal photoperiod programs hypothalamic thyroid hormone deiodinase expression and seasonal reproduction in Siberian hamsters

Cristina Sáenz de Miera

Département de Neurobiologie des Rythmes, Institut des Neurosciences Cellulaires et Intégratives, University of Strasbourg France. Department of Integrative Environmental Physiology, School of Biological Sciences, University of Aberdeen, UK

Mammals perceive seasons via the photoperiodic changes in circulating melatonin and adapt their biological function accordingly. Melatonin acts through a hypothalamic network involving thyroid stimulating hormone subunit β (TSHβ) in the pars tuberalis, type 2 (Dio2) and 3 (Dio3) thyroid hormone deiodinases in the tanycytes. Maternal melatonin reaches the fetus via the placenta and is known to affect fetal photoperiodic responses. We used pregnant female Siberian hamsters (Phodopus sungorus) placed under long (LP: 16h light (L):8h dark (D)) or short (SP: 8L:16D) photoperiod until pups’ weaning to investigate the programming effect of maternal melatonin on the hypothalamic mechanisms regulating seasonal physiology. At weaning, half of the offspring from both groups were transferred to intermediate photoperiod (IP; 14L:10D) and their testicular development and hypothalamic genes expression was studied when the animals were 50 days old. Offspring gestated in SP showed increased testicular development when exposed to IP as compared to the IP exposed animals gestated in LP. In IP exposed animals, TSH expression in the pars tuberalis was identical but there was a strong change in the expression of deiodinases only in the SP gestated animals suggesting a profound alteration of the hypothalamic thyroid system by the melatonin signal received during gestation. This alteration appears to affect the expression of glial and proliferation markers and thus the structural plasticity of the mediobasal hypothalamus. Therefore, it is possible that hypothalamic thyroid hormone levels mediate the differential reproductive responses observed in IP via tanycyte and glial structural changes initiated during pregnancy. Our results show that the photoperiod experienced during gestation is able to program the adult offspring hypothalamic gene expression in response to the same melatonin input.

Young Investigator Mick Harbuz Lecture—Friday, September 25

L0302: Bryn Owen The physiology and pharmacology of the fasting hormone FGF21

Bryn Owen

Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA ; Section of Investigative Medicine, Imperial College, London, UK Fibroblast growth factor 21 (FGF21) is a fasting-‐induced hormone that regulates the adaptive starvation response and extends lifespan in mice. Analogs of FGF21 are currently undergoing clinical evaluation for the treatment of obesity and type 2 diabetes. Mechanistically, FGF21 acts on the brain to induce the hypothalamic-‐pituitary-‐adrenal (HPA) axis, which contributes to both its physiological and pharmacological actions. However, chronic activation of the HPA axis is associated with alterations in behavior. Here we show that in mice, FGF21 reduces sweet preference and social interaction, both measures of reward behavior. These effects require the FGF21 co-‐receptor beta-‐Klotho in the central nervous system (CNS). FGF21 also reduces sweet preference in cynomolgus monkeys. These findings raise the possibility that FGF21 administration could affect reward behavior in humans.

Symposium Speaker Abstracts—Wednesday, September 23

S0101: Meijer Selective activation of glucocorticoid receptor pathways: linking gene transcription to cognition

Onno C. Meijer

Dept. Internal Medicine/Endocrinology, Leiden University Medical Center, PO Box 9600, Leiden, the Netherlands

Glucocorticoid hormones affect brain function in a cell-‐ and context specific manner that is ill understood. Their mineralo and glucocorticoid receptors (MRs & GRs) act in large measure as transcription factors, in interaction with other transcription factors and with nuclear receptor coregulator proteins. These diverse signalling pathways may be selectively targeted in order to either elucidate mechanisms of glucocorticoid signalling, or to develop drugs in stress-‐related psychopathology. With a single gene approach, we manipulated the expression of Steroid Receptor Coactivator-‐1 (SRC-‐1), and tested its involvement in GR-‐mediated regulation of the Crh gene in mice. SRC-‐1 knockout mice showed that SRC-‐1 is necessary for both up-‐ and downregulation of Crh via GR. Using local manipulation of SRC-‐1 splice variants using exon skipping we could induce resistance for the (anxiogenic) GR-‐mediated upregulation Crh in the amygdala. A wider approach for selective targeting is the use of selective receptor modulators. These receptor ligands allow interaction with only a part of all downstream coregulators. We compared two SGRMs that induce largely overlapping yet distinct patterns of GR-‐coregulator intractions. While one SRGM acted as a strong GR agonist in a passive avoidance memory task (potentiating memory), the other acted as GR antagonist. Datasets such as these should allow to link GR to the signaling pathways responsible for specific glucocorticoid effects.

S0102: Moisan Corticosteroid binding globulin role in memory processes

Marie-‐Pierre Moisan

INRA, Bordeaux, France

Corticosteroid Binding Globulin (CBG) is a glycoprotein synthesized in liver and secreted in the blood where it binds with a high affinity but low capacity glucocorticoid hormones, cortisol in humans and corticosterone in laboratory rodents. In mammals, 95% of circulating glucocorticoids are bound to either CBG (80%) or albumin (15%) and only the 5% free fraction is able to enter the brain. The importance of CBG in glucocorticoid biology is revisited since genetic studies in pigs, rats and humans have recently highlighted that the gene encoding CBG is the major factor affecting cortisol/corticosterone variability in these species. We generated a mouse line totally devoid of CBG (Cbg k.o.) to better understand the role of CBG in the regulation of the hypothalamic-‐pituitary-‐adrenal axis and the consequences of its deficiency on brain functions. We found that Cbg k.o. mice display a reduced pool of circulating glucocorticoids and a blunted rise of these hormones after stress. We showed that this hyporesponse is caused by an increased clearance of blood glucocorticoids rather than a differential adrenal sensitivity. The glucocorticoid hyporesponse of the Cbg k.o. mice was found associated with altered emotional reactivity and memory function. We have reported that male Cbg ko mice display more despair-‐like behaviors than wt mice, what was observed in several depression models such as the forced swim test, the tail suspension test and the learned helplessness paradigm. These results were confirmed in female Cbg ko mice, although a regulation of CBG by estrogens introduces a layer of complexity. We have also demonstrated that CBG, through its role in regulating glucocorticoid delivery to the brain, is indirectly involved in memory processes. Cbg k.o. mice are insensitive to the impairment in memory retrieval induced by stress, which can be restored by infusing glucocorticoids directly in the hippocampus. In this context, we are now further investigating the impact of GC hyporesponse to stress on different memory-‐related paradigms. By using various behavioral tests involving different brain regions, we showed that CBG deficiency impacts hippocampus-‐dependent but not amygdala-‐dependent memory processes.

Symposium Speaker Abstracts—Wednesday, September 23

S0103: Yau 11β-‐HSD1 and age-‐associated memory decline

Joyce Yau

University of Edinburgh, Edinburgh, United Kingdom

Long-‐term exposure to elevated circulating glucocorticoid (GC) levels correlates with impaired hippocampal-‐dependent memory in a subgroup of aged individuals. A component part of brain GC concentrations is derived from the local regeneration of active GCs from their inactive forms by 11β-‐hydroxysteroid dehydrogenase type 1 (11β-‐HSD1). The hippocampus, an area with abundant glucocorticoid receptors (GRs) and 11β-‐HSD1, is particularly vulnerable to the effects of stress and ageing. Our studies highlight the important contribution of brain 11β-‐HSD1 regenerated GCs in age-‐associated memory decline. Thus, life-‐long deficiency of 11β-‐HSD1 protects against spatial memory impairments in aged mice despite elevated plasma corticosterone (CORT) levels. Moreover, 11β-‐HSD1-‐deficiency also protects against stress-‐induced memory deficits in adult mice. The improved cognition in aged hsd11b1-‐/-‐ mice associates with lower intra-‐hippocampal CORT levels during learning and memory, a switch from predominant activation of ‘anti-‐cognitive’ GRs to predominant ‘pro-‐cognitive’ MRs, and enhanced hippocampal long-‐term potentiation (LTP). Importantly, short-‐term treatment of aged cognitively impaired C57BL/6J mice with a CNS active 11β-‐HSD1 inhibitor reverses the impaired spatial memory. 11β-‐HSD1 is therefore a promising novel target for the treatment of age-‐associated cognitive decline in humans.

S0104: Givalois Glucocorticoids' role in the hippocampus function impairment associated with Alzheimer's disease

Laurent Givalois

Inserm, Montpellier, France

Alzheimer’s disease (AD) is the most common cause of dementia in the elderly and is characterized by a progressive impairment in cognitive functions, resulting from synapse and nerve cell destruction in the brain. AD symptoms include memory loss, alteration of the individual’s personality and failure to communicate or perform routine tasks. It was shown that in AD patients, cognitive deficits and psychological symptoms were associated with an early deregulation of the hypothalamic-‐pituitary-‐adrenal (HPA) axis, as well as elevated levels of glucocorticoids in plasma and cerebrospinal fluid. The HPA axis, which is highly involved in the stress response, triggers the adrenal cortex to release glucocorticoids. These steroid hormones readily cross the BBB and bind to glucocorticoid receptors (GR). These nuclear receptors are necessary for normal cellular activity and crucial for many CNS functions, including learning and memory. Both stress and increased glucocorticoids exposure were shown to induce cognitive impairments, trigger amyloid precursor protein (APP) misprocessing, reduce amyloid-‐β peptide (Aβ) clearance, increase Aβ levels and stimulate tau hyperphosphorylation and its neuronal accumulation. These observations validate a key role of glucocorticoids in the etiology of AD, especially cognitive decline and associated psychological symptoms. In an acute rodent model of AD, used to screen the therapeutic potential of new molecules, we evidenced a deregulation of the HPA axis activity, reinforcing the hypothesis of its involvement in the etiology of AD and providing evidence for the establishment of a vicious circle whereby the pathology increases the secretion of glucocorticoids, which further increase the pathology. Using new selective modulators of GR in this acute model of AD reverses amyloid toxicity, thus reinforcing the evidence of glucocorticoids and GR involvement in AD and demonstrating a promising therapeutic potential for this new family of molecules.

Symposium Speaker Abstracts—Thursday, September 24

S0201: Davis Imaging gene transcription dynamics in living cells – from cells to tissue

Julian RE Davis

Faculty of Medical & Human Sciences, University of Manchester, UK

Production of prolactin and growth hormone by the pituitary gland displays both acute and long-‐term responses, and requires flexibility of transcriptional behaviour. This has been extensively studied in the past using standard biochemical measurements of RNA and protein concentrations in large populations of cells. Luminescent and fluorescent reporter genes have recently enabled quantitative microscopic study of individual living cells, and reveal dramatic variability in the level of gene expression between different individual cells and across tissues. Using pituitary GH3 cells expressing either luciferase or destabilised EGFP under the control of the human prolactin gene locus, we have found that prolactin promoter activity is highly pulsatile in individual living cells. The majority of cells display cyclical promoter activation with an average cycle length of 11 hours. Using binary switch modelling we determined the lengths of transcriptionally active and inactive periods, and found a minimum ‘off’ period of 3h, implying that a refractory period before transcriptional initiation could generate the transcriptional cycles seen. In order to assess transcription dynamics in normal pituitary cells, we generated transgenic rats in which a recombinant BAC comprising the entire hPRL locus (160kbp) controlled Luc or dEGFP expression. Transgenic pituitary cells that had been enzymatically dispersed and grown in primary culture displayed similar dynamic transcription patterns to those seen in the GH3 cell line. Similar behaviour was found in intact fetal pituitary tissue, with uncoordinated pulsatile patterns of gene transcription, but in the intact adult pituitary gland there was more coordinated expression between cells, probably mediated through gap junctional communication. In summary, hormone gene expression in isolated pituitary cells is highly dynamic and cyclical. The patterns change during pituitary development, and are modulated by the hormonal environment. The mechanisms of these effects remain unclear but dynamic transcriptional responses may have important implications for physiological control and possibly in pathological states such as pituitary adenomas. Statistical modelling of these dynamic patterns has generated new hypotheses about the control of gene expression in vivo.

S0202: Binart Mechanisms for prolactin regulation of reproduction

Nadine Binart

Inserm, Le Kremlin-‐Bicêtre, France

Hyperprolactinemia induced hypogonadotropic anovulation (PRL-‐HA) is a major cause of amenorrhea secondary to hypothalamic GnRH deficiency in women. This gonadotropic deficiency has been proposed to result from direct suppression of prolactin (PRL) on GnRH release but its mechanism remains unknown. Because GnRH neurons do not express unequivocally the PRL receptor, and are stimulated by kisspeptin (Kp) neurons which do express PRL receptors, we hypothesized that GnRH deficiency in this condition could be due to a decrease in Kp secretion. We developed a hyperprolactinemic female mouse model mimicking the human pathology and analyzed the ability of Kp administration to restore gonadotropin secretion and cyclicity. We demonstrated that hypothalamic Kp expression was diminished and that Kp administration restored hypothalamic GnRH release, gonadotropin secretion, and ovarian cyclicity suggesting that Kp neurons play a major role in PRL-‐HA. Altogether with the recent demonstration that Kp neurons express high levels of PRL receptor, our data suggest that PRL excess acts directly on Kp neurons to suppress Kp secretion and downstream GnRH secretion. Therefore, Kp neurons appear to be the missing link between hyperprolactinemia and GnRH deficiency. To evaluate the effect of Kp administration on human gonadotropic-‐ovarian axis, two hyperprolactinemic women with cabergoline resistant microadenomas (<6 mm) presenting with chronic secondary amenorrhea were studied. Measurement of LH, FSH and free a-‐subunit pulsatility, serum estradiol and inhibin B levels was performed. Infusion of Kp induced a significant increase of two gonadotropins pulsatile secretion, and FAS with a dramatic rise in their amplitude. A rapid and very significant increase, in mean LH, FSH, but also in ovarian estradiol, inhibin B circulating levels occurred. Therefore, we demonstrate that Kp administration can stimulate short term gonadotropin secretion in women with PRL-‐HA. This exploratory study suggests that Kp could be an alternative therapeutic approach to restore ovulation and fertility in hyperprolactinemic women resistant to dopamine agonists. Long-‐term clinical trials will be necessary to confirm the therapeutic relevance of Kp in clinical practice.


S0203: Brown Prolactin actions in the brain Rosemary Brown

University of Otago, New Zealand

Prolactin is an anterior pituitary hormone that has a broad range of physiological functions, including several mediated through actions in the brain. These actions are mediated through the prolactin receptor, which is widely expressed throughout the hypothalamus. Prolactin signaling is mediated by the transcription factor signal transducer and activator of transcription (STAT) 5, and we have shown that immunohistochemical labelling of phosphorylated STAT5 (pSTAT5) is a reliable maker of activated prolactin receptors in the brain. pSTAT5 expression in the brain can be induced by peripherally administered prolactin, demonstrating that prolactin crosses the blood brain barrier and enters the brain, but the mechanism by which it does this is not completely understood. It has been hypothesised that the prolactin receptor in the choroid plexus, where extremely high levels of expression are observed, may serve as a transporter by binding prolactin in the blood and releasing it in the cerebrospinal fluid. By measuring transport of 125I-‐labelled prolactin into the brain in mice lacking the prolactin receptor, we have shown that the prolactin receptor is not required for transport. We have also shown that lactation, a period when levels of prolactin are chronically elevated, is accompanied by an increase in the rate of prolactin transport into the brain, leading to an increase in the number of neurons responding to prolactin. During pregnancy and lactation, a critical adaptation that occurs in the maternal brain is the establishment of appropriate behaviour to enable the mother to feed and nurture offspring. We have shown that prolactin plays a critical role in the initiation of these behaviours, by removing prolactin receptors specifically from the medial preoptic nucleus, a region known to be important in maternal behaviour.

S0204: Martin Plasticity in the neuroendocrine regulation of prolactin secretion during lactation

Agnes Martin

Inserm, Montpellier, France

The PRL secretion in the blood is finely tuned, differently depending on the physiological demand in order to assure its physiological role. We begin to have a better insight into the mechanism at stake in modulating both the timing and quantity of secreted hormones. It involves regulations all along the hypothalamo-‐pituitary axis. One of the main regulators of PRL secretion are the Tuberoinfundibular neurons of the arcuate nucleus (TIDA). TIDA exert a negative feedback on PRL by secretion of Dopamine by (TIDA) at the median eminence (ME) into the portal blood. We will discuss their role in converting ultrashort electrical signal based information from neurons into long lasting hormonal secretion by endocrine cells. We show a robust long-‐term organization of TIDA neurons electrical activity with a harmonization of their firing rates converting single electrical units lasting few msec (action potential) into burst of co-‐activation lasting several minutes. This effect of “loose” coordination is conveyed to the physiological output of the TIDA i.e the secretion of DA. By live amperometry both in vitro and in vivo, we show that at the level of the ME DA secretion is organised in hierarchically-‐organized buildings blocks which recur over long term (weeks). This system could well apply to most of the physiological PRL pattern except lactation. During this specific state of chronic hyperprolactinemia we showed that the electrical properties of the TIDA where not changed and that TIDA were certainly not electrically silenced. To allow for this long term change, TIDA undergo tremendous phenotypical plasticity, switching from dopamine to enkephalin secreting neurons. We believe because of their anatomical and functional properties, they provides a unique model allowing to study the plasticity of the neurohemal interface between the brain and the organism.


S0205: Robinson Neural effects of blocking puberty with a long term Gonadotrophin Releasing Hormone agonist (GnRHa)

Jane Robinson

Institute of Biodiversity, Animal Health and Comparative Medicine, The University of Glasgow, Glasgow, UK

GnRH is widely recognised as a peptide hormone that controls the reproductive axis. Recent publications have also revealed roles for GnRH in non-‐reproductive tissues including the heart, skin, kidney and smooth muscle as well as areas of the brain that regulate cognitive function. Long term treatments with GnRHa are employed to block puberty in a range of medical abnormalities in children including central precocious puberty, idiopathic short stature, and early onset Gender Identity Disorder. However, the effects of delaying puberty with long term agonist exposure on behaviour and the functioning of areas of the brain that control behavioural like the amygdala and hippocampus, are largely unexplored. In the current studies we have used an ovine model in which sheep were exposed to a GnRHa from just prior to the time of normal puberty until one year of age to determine effects on risk taking behaviour, spatial orientation and emotional regulation. Male and female same sex twins were included in the studies; one as a control and one given monthly GnRHa implants (Zoladex). GnRHa blocked the pubertal process in both sexes. Post mortem brain tissue was collected at 11 months of age and functional studies conducted to explore sex and treatment differences on morphometric measurements of global and regional brain volume as well as differential gene expression and synaptic plasticity in the amygdala and hippocampus. Both these brain regions have been shown to contain receptors for GnRH. Our studies have shown that long term GnRHa treatment that blocks puberty in both sexes alters brain development, the response to stress and important cognitive functions. Supported by funding from the BBSRC, BSN, Norwegian Research Council, Norwegian School of Veterinary Medicine and a gift of Zoladex from Astra Zeneca.

S0206: Sharif Astrogenesis in the postnatal hypothalamus: a new mechanism involved in female sexual maturation

Ariane Sharif

Inserm, University of Lille, France

The initiation of mammalian puberty requires an increased pulsatile secretion of GnRH from specialized neurons of the hypothalamus. This increase is brought about by coordinated changes in both transsynaptic and glial inputs to GnRH neurons. Here, we show that the timely onset of puberty in female rodents involves the genesis of astrocytes during the infantile period in the environment of GnRH neurons. Our results suggest that a significant fraction of GnRH neurons recruit newly generated cells born on postnatal day 8 in their immediate vicinity. These newborn cells differentiate into astrocytes and their morphological association with GnRH neurons remains visible in adult animals. Local inhibition of cell proliferation in the surroundings of GnRH neuron cell bodies during the infantile period by stereotaxic injection of beads releasing the anti-‐mitotic paclitaxel markedly delayed puberty. Experiments carried out in mice deficient in both erbB1 and erbB4 signaling in astrocytes, which exhibit impaired sexual development and mature reproductive function, showed that erbB signalling was required for the long-‐term maintenance of the association between astrocytes born during the infantile period and GnRH neurons. Altogether, our results raise the exciting possibility that the birth of new astrocytes morphologically and functionally connected to GnRH neurons is a key maturational event required to initiate GnRH secretion in female rodents.


S0207: O’Byrne Puberty timing: is the hypothalamus controlled by the amygdala?

Kevin T O’Byrne and Xiao Feng Li

Division of Women’s Health, Faculty of Life Sciences & Medicine, King’s College London, Guy’s Campus, London SE1 1UL, UK

Although it is recognised that hypothalamic kisspeptin plays an integral role in the GnRH pulse generating neural network, and is obligatory for puberty, the upstream regulatory mechanisms that drive the GnRH pulse generator to trigger the onset of puberty have remained stubbornly elusive, as have those underlying its advance or delay by stress. The amygdala, a key limbic brain structure commonly known for its role in higher-‐order emotional processing, is implicated in pubertal timing and stress-‐induced suppression of LH pulse frequency. We have recently shown that lesions of the medial amygdala (MeA) and more specifically its posterodorsal subnucleus (MePD), dramatically advances puberty via mechanisms independent of changes in body weight, but involving intrinsic GABA and glutamate receptor signalling. We have also shown that kisspeptin signalling in this extra-‐hypothalamic structure surprisingly exerts a profound influence on GnRH pulse generator frequency and alters pubertal timing. Additionally, activation of MePD corticotrophin-‐releasing factor type 2 receptor (CRF-‐R2) by the endogenous ligand urocortin 3 delays puberty. These finding might suggest that amygdala kisspeptin interacting with local GABA/glutamate neural circuits form a higher-‐order regulator of the hypothalamic GnRH pulse generator that is critical for triggering puberty.

S0208: Simonneaux Is seasonal reactivation of reproduction a yearly puberty?

Valérie Simonneaux, Cristina Saenz de Miera, David Hazlerigg*

Institut des Neurosciences Cellulaires et Intégratives, CNRS UPR 3212, Strasbourg, France, *Faculty of Biosciences, Fisheries and Economy, University of Tromsø, Norway

Puberty in youth is characterized by a full activation of the reproductive axis. However, throughout adulthood most mammals are further submitted to phases of activation and inactivation of reproduction according to seasons. It has been proposed that central mechanisms involved in seasonal reactivation could be compared to a recurrent annual puberty. The major progresses made recently in our understanding of the neuroendocrine control of seasonality now allow revisiting this hypothesis. The annual change in the nocturnal production of the pineal hormone melatonin is the main driver of seasonal reproduction. Whether mammals are long-‐day (like hamsters) or short-‐day (like sheep) breeders, the large production of melatonin in winter leads to an inhibition of TSH production in the pars tuberalis. TSH has been shown to activate deiodinase2 in the tanycytes surrounding the basal part of the 3rd ventricle, leading to an increased production of thyroid hormone (TH) locally in the basal hypothalamus. In seasonal hamsters, the spring activation of the TSH/TH pathway increases the synthesis of RFRP and kisspeptin, two hypothalamic RF-‐amide peptides critical for the activation of GnRH neuron and downstream pituitary-‐gonadal axis. In new born (day 0) and young (day 15) Siberian hamsters, expression of TSH in the pars tuberalis and deiodinase2 in the tanycytes is elevated and remains stable until adulthood. The developmental timing of genes coding for RFRP and kisspeptin is different: Rfrp mRNA is not detected at birth but rises constantly until adulthood whereas Kiss1 mRNA is detected at birth, increases strongly when gonadal development peaks (day 24), and then decreases in adults as a consequence of the negative sex steroid feedback. In conclusion, the comparison of the neuroendocrine pathway regulating seasonal and pubertal activation of central reproduction in rodents indicates that the former relies mainly on the TSH/TH/RFRP pathway whereas the later relies mainly on the kisspeptin neurons. Interestingly however, the kinetic of this developmental reproductive pathway is modulated by the photoperiod perceived by the gestating female.

Symposium Speaker Abstracts—Friday, September 25

S0301: Balthasar The paraventricular hypothalamus in glucose-‐sensing, maternal programming and over-‐nutrition

Nina Balthasar

University of Bristol, UK

Within the hypothalamus, the paraventricular hypothalamus (PVH) is a key integrator and modulator of metabolic and cardiovascular function, mainly due to its anatomical connectivity to both neuroendocrine and autonomic output. The PVH is thus a likely CNS structure to be modulated by glucose state, diet and maternal diet and a probable mediator of downstream metabolic and cardiovascular consequences – good and bad. Here we will discuss our data identifying significant PVH glucose-‐sensitivity both in terms of transcriptome and electrophysiological changes in addition to offspring PVH transcriptome alteration downstream of early life high-‐fat/high-‐sugar diet exposure. To assess mechanisms underlying these transcriptomic changes we have furthermore assessed diet-‐mediated changes to the PVH epigenome. In summary, we will demonstrate significant glucose-‐, diet-‐ and maternal diet-‐mediated PVH transcriptome and epigenome plasticity.

S0302: Steculorum Novel regulator of orexigenic AgRP-‐neurons

Sophie Steculorum

Max Planck Institute, Cologne, Germany

Activation of orexigenic AgRP-‐expressing neurons in the arcuate nucleus of the hypothalamus potently promotes feeding, thus defining new regulators of AgRP-‐neuron activity could uncover potential novel targets for obesity treatment. We demonstrate that AgRP-‐neurons express the purinergic receptor 6 (P2Y6), which is activated by uridine-‐diphosphate (UDP). In vivo, UDP induces ERK-‐phosphorylation and cFos-‐expression in AgRP-‐neurons and promotes action potential firing of these neurons in brain slice recordings. Consequently, central application of UDP promotes feeding and this response is abrogated upon pharmacological or genetic inhibition of P2Y6 as well as upon pharmacogenetic inhibition of AgRP-‐neuron activity. In obese animals, hypothalamic UDP-‐content is elevated as a consequence of increased circulating uridine concentrations. Collectively, we revealed a regulatory pathway in obesity, where peripheral uridine increases hypothalamic UDP-‐concentrations, which in turn promote feeding via PY6-‐dependent activation of AgRP-‐neurons.

Symposium Speaker Abstracts—Friday, September 25

S0303: Parnet Early life nutrition and long term appetite regulation

Patricia Parnet

UMR 1280 Physiologies des Adaptations Nutritionnelles, Université de Nantes, France

Feeding behaviour is a complex character whose role is the regulation of food intake in order to meet the physiological needs of the body. A strict regulation at the physiological level is maintained by the central nervous system which controls the energy homeostasis and peripheral organs involved in food intake and energy storage and expenditure. But eating is to a large extent influenced by social and emotional cues which are largely depending on the socio-‐economic context and the familial environment. It is now admitted that early environment plays a major role in the establishment of feeding behaviour both at the physiological and psychological level. Among environmental parameters, early nutrition, including maternal nutrition before and during pregnancy and during lactation, and infant nutrition during the first months and years of life are essential for a normal fetal development and for an optimal setting up of the complex physiological interactions that will drive food intake regulation throughout the life course. In the last few decades, evidence has been accumulating from both human cohort studies and experimental animal models that shed a light on several biological mechanisms underlying this early influence. Some physiological and molecular mechanisms by which early experience, as early as during fetal life, may have long term influence have been identified. Whether, in what time-‐window and how, these effects can be reversed in under investigations. Knowledge in the field of nutritional programming is progressing but faces the difficulty of a high complexity and interaction between many parameters belonging to various fields spanning physiology, metabolism but also psychology and sociology.

S0304: Levin Critical Amylin-‐Leptin Interactions Influencing Hypothalamic Development

Barry E. Levin

Neurology Service, VA Medical Center, E. Orange, NJ and Department of Neurology, Rutgers, New Jersey Medical School, Newark, NJ, USA

Rats selectively bred to develop diet-‐induced obesity (DIO) become hyperphagic and obese when fed a high fat diet. DIO rats have an inborn reduction in leptin receptor signaling in the ventromedial hypothalamus (VMH: arcuate (ARC) + ventromedial nucleus (VMN)). This includes reduced Lepr-‐b expression and leptin receptor binding and leptin-‐induced pSTAT3 expression, anorexia and thermogenesis. They also have defective development of leptin-‐dependent AgRP and aMSH ARC to paraventricular nucleus (PVN) axonal outgrowth, pathways which develop during the first two postnatal weeks. Co-‐administration of leptin and the pancreatic b-‐cell-‐derived peptide, amylin, act synergistically to produce weight loss in obese DIO rats and humans. This effect is mediated by amylin-‐induced increase in VMH leptin signaling. Amylin increases leptin signaling by stimulating VMH microglial IL-‐6 production which then interacts with its IL-‐6/gp130 receptor to co-‐activate pSTAT3 downstream of the Lepr-‐b receptor. In fact, IL-‐6 incubation with DIO VMH neurons corrects both their defective Lepr-‐b expression and leptin-‐induced excitation. To assess whether amylin might correct defective DIO VMH leptin signaling and ARC-‐PVN pathway development, we treated DIO neonates with amylin (300mg/kg) twice daily from P0-‐16. This treatment reduced body weight gain, increased ARC and VMN leptin-‐induced pSTAT3 expression and markedly enhanced PVN AgRP and aMSH fiber density. However, amylin-‐induced IL-‐6 production and enhanced leptin signaling appear to be required only for enhanced AgRP ARC-‐PVN pathway development since IL-‐6 knockout mice have a >50% decrease in density of PVN AgRP but not aMSH fibers compared to wildtype mice. Thus, amylin appears to increase ARC-‐PVN pathway development by an IL-‐6-‐leptin-‐dependent (AgRP) and a direct neurotrophic (aMSH) process.

Young Investigator SERVIER Symposium—Friday, September 25

S0305: Cansell Nutritional triglycerides act on mesolimbic structures to regulate the rewarding and motivational aspect of feeding

Céline Cansell

Central control of feeding behaviour and energy expenditure Research Team, CNRS, Paris-‐Diderot University, France

Circulating triglycerides (TGs) normally increase after a meal but are altered in pathophysiological conditions, such as obesity. Although TG metabolism in the brain remains poorly understood, several brain structures express enzymes that process TGenriched particles, including mesolimbic structures. For this reason, and because consumption of high-‐fat diet alters dopamine signaling, we tested the hypothesis that TG might directly target mesolimbic reward circuits to control reward-‐seeking behaviors. We found that the delivery of small amounts of TG to the brain through the carotid artery rapidly reduced both spontaneous and amphetamine-‐induced locomotion, abolished preference for palatable food and reduced the motivation to engage in food-‐seeking behavior. Conversely, targeted disruption of the TG-‐hydrolyzing enzyme lipoprotein lipase specifically in the nucleus accumbensn increased palatable food preference and food-‐seeking behavior. Finally, prolonged TG perfusion resulted in a return to normal palatable food preference despite continued locomotor suppression, suggesting that adaptive mechanisms occur. These findings reveal new mechanisms by which dietary fat may alter mesolimbic circuit function and reward seeking.

S0306: Geller Hypothalamic astrocytes possess a higher glycolytic phenotype compare to cortical astrocytes: A potential role in the regulation of energy balance and reproduction function

Sarah Geller, Luc Pellerin

Department of Physiology, University of Lausanne, Switzerland

The ability of the hypothalamus to detect changes in plasma glucose concentration is critical for the regulation of energy balance and impact the regulation of reproduction function. Among the mechanisms involved in hypothalamic glucosensing, a metabolic interaction between glial cells and neurons via lactate transfer has been proposed. Unlike for cortex, the astrocyte-‐neuron lactate shuttle and its regulation has not been thoroughly characterized in the hypothalamus. Knowing that hypothalamus and cortex have different functions, one may wonder whether the degree of glycolytic metabolism and the metabolic response of hypothalamic glial cells are the same than cortical astrocytes. Indeed, previous studies have revealed differences for other metabolic pathways (e.g. glutamate metabolism, long-‐chain fatty acid metabolism) between hypothalamic and cortical astrocytes. The aim of this project is to characterize and compare glycolytic metabolism and its regulation in hypothalamic astrocytes relative to cortical astrocytes in mouse primary cultures. By biochemical analysis, we highlighted that, over time, hypothalamic astrocytes consume more glucose and release more lactate than cortical astrocytes in basal condition. Furthermore, qPCR, Western blot and immunochemistry analysis showed that expression of key actors involved in cellular metabolism (such as transporters isoform of glucose and monocarboxylates, the enzymes isoform of hexokinase and pyruvate kinase) differs between hypothalamic and cortical astrocytes. Our data show that higher glycolytic phenotype of hypothalamic astrocytes would be pyruvate kinase (PKM2) /monocarboxylate transporter MCT4 signaling dependent. Indeed, our results revealed that PKM2 enzyme translocated more in hypothalamic astrocytes nuclei as compared to cortical cells. Thus, this enzyme acts as a transcriptional factor which regulates the expression of glycolytic metabolism actors. In an interesting manner, our results also showed that the regulation of the metabolism of these two types of astrocytes differs. In contrast to cortical astrocytes, exposure to glutamate did not enhance glucose utilization and lactate production in hypothalamic astrocytes. This study suggests that hypothalamic astrocytes possess a different metabolic phenotype compared to cortical astrocytes, a characteristic which could take part in the specific sensitivity of the hypothalamus to fuel sensing.

Young Investigator SERVIER Symposium—Friday, September 25

S0307: Cimino Novel Role for Anti-‐Müllerian Hormone in the Regulation of GnRH Neuron Excitability and Hormone Secretion

Irene Cimino

Inserm, University of Lille, France

Anti-‐Müllerian hormone (AMH) plays crucial roles in sexual differentiation and gonadal functions. However, the possible extra-‐gonadal effects of AMH on the hypothalamic-‐pituitary-‐gonadal axis remain unexplored. Here we demonstrate that a significant subset of GnRH neurons both in mice and humans express the AMH receptor, and that AMH potently activates the GnRH neuron firing in mice. Combining in vivo and in vitro experiments, we show that AMH increases GnRH-‐dependent LH pulsatility and secretion, strengthening the notion of a central action of AMH on GnRH neurons. Increased LH pulsatility is an important pathophysiological feature in many cases of Polycystic Ovary Syndrome (PCOS), the most common cause of female infertility, in which circulating AMH levels are also often elevated. However, the origin of this dysregulation remains unknown. Our findings suggest that AMH-‐dependent regulation of GnRH release could be critically involved in the pathophysiology of fertility and could hold therapeutic potential for treating PCOS.

Oral Presentation Abstracts—Thursday, September 24

O1: Paiva Activation of oxytocin neurons following systemic Melanotan-‐II administration seeking

Luis Paiva, Mike Ludwig

University of Edinburgh, UK

Central oxytocin release is involved in social behaviours in animals and humans. Furthermore, it has been suggested that changes in central oxytocin levels might be linked to mental disorders such as anxiety and depression. Systemic administration of oxytocin has been proposed as a potential therapeutic option, but oxytocin does not cross the blood brain barrier in physiologically significant amounts. An alternative approach to systemic administration of oxytocin is to stimulate central release of oxytocin. It has been shown that central injections of alpha-‐melanocyte stimulating hormone (α-‐MSH) are a potent stimulus to induce central (dendritic) oxytocin release while inhibiting systemic secretion. Thus, experiments were conducted to test the hypothesis that systemic administration of a synthetic melanocortin agonist (Melanotan-‐II, MT-‐II) is able to induce oxytocin neuron activation using the immediate early gene product c-‐Fos protein as a marker for neuronal activation. We analysed Fos expression combined with oxytocin immunohistochemistry in response to intravenous and intranasal MT-‐II. Our results show that MT-‐II intravenously administrated (1mg/kg) induces Fos expression in oxytocin and vasopressin neurons in magnocellular neurons of the supraoptic (SON) and in the paraventricular (PVN) nucleus; however, there was no increase in Fos expression in the parvocellular subdivisions of the PVN, and circumventricular organs (OVLT, SFO). Fos expression induced by Melanotan-‐II was reduced by central administration of the melanocortin antagonist SHU-‐9119. In contrast, there were no significant changes in Fos expression when MT-‐II was given intranasally (1µg/rat and 30µg/rat). These results suggest that intravenous administration of Melanotan-‐II can be an effective pharmacological tool to induce central activation of oxytocin neurons. Whether Melanotan-‐II is able to induce central oxytocin release however is still to be determined.

O2: Andrews Glucose metabolism affects ghrelin-‐induced feeding and motivation Sarah Lockie, Romana Stark, Sarah Ch’ng, Andrew Lawrence, Zane Andrews

Monash University, University of Melbourne, Australia

Neuroendocrine studies typically examine feedback actions of single exogenous hormones in the brain. However, feedback information is more than a rise or fall of a single hormone. Metabolic feedback is a dynamic physiologic fluctuation of nutrients, such as glucose and fatty acids; and hormones such as insulin, leptin and ghrelin. Thus, we tested whether glucose metabolism affects ghrelin-‐induced feeding behaviour. To modulate endogenous glucose metabolism we injected mice (ip) with glucose (2.25g/kg) or 2-‐deoxyglucose (250mg/kg) to simulate hyper and hypoglycaemia respectively. 30 minutes later we injected ghrelin in different experiments ip, ICV, or into the ventral tegmental area (VTA) and measured food intake after 2 hours. In all experimental paradigms, glucose suppressed ghrelin-‐induced feeding whereas 2-‐deoxyglucose potentiated ghrelin-‐induced feeding. Moreover, we examined cfos activation in NPY GFP neurons after ip glucose or 2-‐deoxyglucose combined with ICV ghrelin administration. ICV ghrelin significantly increased cfos in identified NPY neurons but neither glucose nor 2-‐deoxyglucose affected ghrelin-‐induced NPY/cfos coexpression despite the significant effects on feeding behaviour. Finally we examined if ip glucose modulates ghrelin-‐induced motivation. Mice were trained to lever press to obtain sucrose (10% w/v) in operant conditioning chambers. While ICV ghrelin significantly increased lever-‐pressing for sucrose, this was significantly attenuated by pretreatment with ip glucose. These studies show that the neuroendocrine actions of ghrelin on feeding behaviour depend on glucose metabolism. The ability of glucose metabolism to influence food intake is independent from ghrelin-‐induced cfos expression. These results highlight that metabolic feedback is an integration of multiple nutrient and hormonal cues, an observation that is physiologically relevant to multiple neuroendocrine feedback systems.


O3: Bake Ghrelin changes food preference from high fat diet to chow in schedule-‐fed rats

Tina Bake, Kim T. Hellgren, Suzanne L. Dickson

The Sahlgrenska Academy at the University of Gothenburg, Institute of Neuroscience and Physiology, Department of Physiology/Endocrinology, Sweden

Ghrelin is a gut peptide released from the empty stomach that increases food intake. It has been linked to food-‐related behaviours such as food motivation, food reward and food anticipatory activity. Ghrelin levels in the blood are linked to meal pattern, increasing prior to feeding. To mimic human meal eating behaviour in animals we used a scheduled feeding (SF) paradigm in which rodents have 2h-‐access to high fat diet (HFD) in addition to ad libitum chow. Previous studies with this paradigm have shown that both rats and mice will rapidly adapt their feeding behaviour and binge-‐eat on HFD. Here we sought to investigate the role of ghrelin during binge-‐like meal eating induced by SF. We utilised a combination of two different animal models: pharmacologically manipulated rats via administration of ghrelin or genetically modified mice lacking GHS-‐R1A. For acute ICV or intra-‐VTA injections of either ghrelin or vehicle, rats were surgically implanted with guide cannulas and then habituated to SF for at least 2 weeks prior injections. GHS-‐R1A-‐KO mice and wildtype (WT) littermates were scheduled-‐fed for 4 weeks. Remarkably and unexpectedly, we found that injecting ghrelin ICV or intra-‐VTA resulted in a shift in food preference from high fat diet towards chow during the SF period without altering total daily energy consumption. However an increase of body weight was observed after ICV ghrelin. A fasting challenge also led to an increase in chow intake during the SF session but HFD intake did not reduce. GHS-‐R1A-‐KO mice were able to adapt and maintain large meals of HFD in a similar fashion as WT mice suggesting that ghrelin signalling may not have a critical role in acquisition or maintenance in this kind of feeding behaviour. In conclusion, ghrelin appears to act as a modulating factor for binge-‐like eating behaviour by shifting the food preference towards a healthier choice (from HFD to chow), effects that were divergent from fasting.

Supported by EC (Nudge-‐it, 607310).

O4: D’Agostino Nucleus of the solitary tract cholecystokinin-expressing neurons control appetite Giuseppe D’Agostino, David J. Lyons, Lora K. Heisler

Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen, UK

The nucleus of the solitary tract (NTS) is a principal gateway for meal-‐related signals entering the brain from the periphery and is comprised of cells with diverse neurochemical identities. We identified a distinct subset of NTS neurons that express cholecystokinin (NTS-‐CCK) and whose function was previously not known. We observed that NTS-‐CCK neurons are responsive to nutritional states and that chemogenetic manipulation of this subset of neurons produces profound metabolic effects. Specifically, chemogenetic activation (hM3Dq-‐mCherry) of NTS-‐CCK neurons suppresses appetite and rapidly reduces body weight with a mechanism involving downstream activation of CCK receptors. Cell-‐specific anterograde tracing revealed that NTS-‐CCK neurons innervate discrete hindbrain and hypothalamic regions, including the paraventricular nucleus of the hypothalamus (PVH). Moreover, in vivo optogenetic activation of NTS-‐CCK axon terminals reveal the satiating function NTS-‐CCK neurons to be mediated by a NTS-‐CCK→PVH pathway. Optogenetic activation of this circuit reduces food consumption and also encodes positive valence. Thus, our data provide an evidence base for a novel CCKergic circuit originating from the NTS that transmit satiating and appetitive information to the hypothalamus to regulate appetite.


O5: Decourt Design of a selective kisspeptin analog capable of synchronizing ovulation

Caroline Decourt1, Vincent Robert1, Didier Lomet1, Karine Anger1, Mathieu Galibert2, Jean-‐Baptiste Madinier2, Philippe Marceau2, Agnès Delmas2, Alain Caraty1, Vincent Aucagne2, Massimiliano Beltramo1 1UMR Physiologie de la Reproduction et des Comportements (INRA, UMR85; CNRS, UMR7247; Université François Rabelais Tours; IFCE) F-‐37380 Nouzilly, France; 2Centre de Biophysique Moléculaire (CNRS UPR 4301) F-‐45071 Orléans cedex 2, France.

GnRH secretion is central to reproduction control and the neuropeptide kisspeptin (Kp) is the most potent GnRH secretagogue. Hence, Kp system is an appealing target to develop new methods to manage reproduction and heal related pathologies. Endogenous Kp isoforms have short in vivo half-‐life and continuous administration is required to obtain the wanted effects. However, in livestock management and in human therapy a single injection is preferable to continuous administration. To meet this need we designed analogs of the 10 amino acid isoform of Kp with improved pharmacokinetics and pharmacodynamics. To this aim we combined various modifications improving resistance to degradation and reducing renal clearance. Our effort produced analogs that compared to Kp10 have equal efficacy, similar or better potency, and a prolonged half-‐life in blood serum. Several compounds were active by intramuscular injection at very low doses (5 to 15 nmoles/ewe) and LH level was still higher than basal nine hours after analog injection. When injected during the breeding season, in ewes pretreated with flugestone acetate (FA) for 14 days, the best Kp analog produced a superior synchronization of LH surge compared to available treatment (pregnant mare serum gonadotropin). In presence of a ram treated ewes showed all behavioral signs of estrus. Ovulations triggered by Kp analog were fertile as indicated by the rate of pregnancy (7 out of 10) obtained after servicing. Experiments are ongoing to assess if analogs could also induce ovulation during the non-‐breeding season. In conclusion we generated a Kp analog with improved pharmacokinetics and pharmacodynamics capable of inducing, after a single intramuscular injection, synchronized fertile ovulations in ewes pretreated with FA. This molecule holds a strong potential to improve management of livestock reproduction and possibly to treat human reproductive disorders due to reduced GnRH secretion.

O6: Tups Photoperiodic regulation of Wnt signalling in the arcuate nucleus of the Djungarian hamster, Phodopus sungorus

Alisa Boucsein1, Jonas Benzler1, Cindy Hempp1, Sigrid Stöhr1, Gisela Helfer2, Alexander Tups1,3 1Department of Animal Physiology, Faculty of Biology, Philipps University of Marburg, Marburg, Germany ; 2Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen, Scotland, UK ; 3Department of Physiology, Centre of Neuroendocrinology and Brain Health Research Centre, University of Otago, Dunedin, New Zealand

The Wnt pathway, well characterized in cellular development, was shown to play an important role in the adult central nervous system. We previously identified the Wnt pathway as a novel integration site of the adipokine leptin in mediating its neuroendocrine control of metabolism in obese mice. Here, we investigated whether Wnt signalling plays an important role on seasonal body weight regulation and in the hypothalamus of the Djungarian hamster (Phodopus sungorus), a seasonal mammal that exhibits profound annual changes in leptin sensitivity. We furthermore investigated whether crucial components of the Wnt pathway are regulated in a circadian manner. We first examined mRNA expression of key components of the Wnt pathway in the arcuate nucleus of hamsters acclimated to either long day (LD) or short day (SD) photoperiod by in situ hybridization. We detected elevated expression of the genes WNT-‐4, Axin-‐2, Cyclin-‐D1 and SFRP-‐2 during LD compared to SD, as well as a diurnal regulation of the genes Axin-‐2, Cyclin-‐D1 and DKK-‐3. Next, we investigated the effect of photoperiod as well as leptin on the activation of the Wnt co-‐receptor LRP-‐6 by immunohistochemistry. The number of phosphorylated (activated) LRP-‐6-‐(Ser1490)-‐immunoreactive cells in the arcuate nucleus was elevated during LD relative to SD, as well as in animals from both photoperiods that were treated with leptin (2 mg/kg bod weight) compared to controls. These findings suggest that differential Wnt signalling is associated with seasonal body weight regulation and is partially regulated in a diurnal manner in the adult brain. Furthermore they provide further evidence that this pathway plays a key role in the neuroendocrine regulation of body weight and integration of the leptin signal.


O7: Christian Photoperiod-‐driven remodelling of lactotroph, gonadotroph and folliculostellate cells in the sheep pituitary

Akriti Nanda, Katarzyna Miedzinska, Shona Wood, Alan McNeilly, Andrew Loudon, Helen Christian

Department of Physiology, Anatomy and Genetics, University of Oxford

Sheep exhibit circannual rhythms in circulating concentrations of prolactin which regulate lactation, reproduction and seasonal molts. Chronic hypersecretion of prolactin during long photoperiod is controlled by an intra-‐pituitary mechanism regulated by melatonin, which acts at MT1 receptors in the pars tuberalis to stimulate release of a paracrine prolactin-‐releasing factor. The present study examined the effects of short and long photoperiod on the ultrastructure of lactotroph, gonadotroph and FS cells and their distribution. Sheep (n=4 per group) were housed in artificial light dark cycles, either 8:16h light⁄dark cycle for short photoperiod (SP) or 16:8h light⁄dark cycle for long photoperiod (LP) for 4 weeks. Pituitary glands were collected and prepared for quantitative electron microscopy. It was found that lactotrophs in LP had greater cytoplasmic area (P<0.05), with more dilated rough endoplasmic reticulum (rough ER; P<0.05) and showed a reduced number of PRL granules per micron cytoplasmic area compared to lactotrophs in SP suggesting increased PRL synthesis and release. In LP, secretory granules showed a polarisation towards a neighbouring capillary and lactotrophs were more frequently found adjacent to a capillary compared to SP. There was no significant difference in the number of lactotrophs adjacent to FS cells in LP vs SP. Gonadotrophs in SP showed greater amounts of dilated rough ER consistent with greater amounts of LH and FSH synthesis and release in SP vs LP. FS cells in SP were significantly larger, contained more rough ER and a higher proportion of chromatin was pale (transcriptionally active) in contrast to FS cells in LP displaying greater amounts of dense heterochromatin (transcriptionally inactive). These findings demonstrate plasticity in the morphology of lactotrophs, gonadotrophs and FS cells with photoperiod which reflect the functional changes in PRL and LH secretion.

O8: Spiga Experimental and mathematical studies on the dynamics of adrenal glucocorticoid synthesis suggest an intra-adrenal negative feedback mechanism that involves activation of the glucocorticoid receptor Francesca Spiga1, Zidong Zhao1, Lorna Smith1, Georgina Hazel1, Jamie Walker2, Rita Gupta2, John Terry2 and Stafford L Lightman1 1University of Bristol, Henry Wellcome Laboratories for Integrative Neuroscience & Endocrinology, Bristol, UK ; 2University of Exeter, Wellcome Trust Centre for Biomedical Modelling and Analysis & College of Engineering, Mathematics and Physical Sciences, Exeter, UK

The activity of the hypothalamic-‐pituitary-‐adrenal (HPA) axis is characterised by an ultradian (pulsatile) pattern of glucocorticoid secretion that is critical for optimal transcriptional, neuroendocrine and behavioural responses to glucocorticoids. We have investigated the molecular mechanisms underlying the origin of glucocorticoid ultradian rhythm within the rat adrenal gland during basal conditions and in response to an immunological stressor. Here we show that basal ultradian rhythm of glucocorticoids depends on highly dynamic processes within adrenocortical steroidogenic cells, including rapid phosphorylation of hormone-‐sensitive lipase (HSL), a proteins involved in cholesterol metabolism, as well as rapid transcription of steroidogenic genes, including StAR and MRAP, and activation of nuclear receptors that regulate steroidogenic genes expression, including TORC, SF-‐1, Nur77 and Dax-‐1. We then show that stress-‐induced disruption of these dynamics leads to abnormal glucocorticoid secretion, as observed in disease and critical illness in both human and the rat. Finally, by using mathematical modelling we show that activation of the glucocorticoid receptor appears to be an important factor in the dynamic regulation of these processes via an intra-‐adrenal negative feedback mechanism.


O9: Koorneef A novel mixed glucocorticoid/mineralocorticoid receptor selective modulator reduces obesity and adipose tissue and liver inflammation

L.L. Koorneef1,2, J.K. van den Heuvel1,2, M.R. Boon1,2, I.M. Mol1,2, N. van de Velde1,2, H. Hunt3, P.C.N. Rensen1,2, O.C. Meijer1,2 1Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Leiden, The Netherlands ; 2Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, The Netherlands ; 3Corcept Therapeutics, Menlo Park, California, USA

Excessive glucocorticoid exposure is associated with obesity and related disorders, as is evidenced by the extreme case of Cushing’s disease. Accordingly, antagonism of the glucocorticoid receptor (GR) by means of mifepristone (RU486) markedly lowers obesity in men and mice, yet pure antagonism may lead to side-‐effects (pro-‐inflammation). In this study we tested the efficacy of CORT 118335 (C118335), a selective modulator type drug (exerting both agonism and antagonism) at GR, with antagonistic effects via mineralocorticoid receptor (MR). 10-‐week old C57BL/6J mice were fed a high-‐fat diet for 3 weeks and treated with C118335, RU486 or vehicle. Both RU486 and C118335 reduced body weight gain and fat mass. C118335 reduced liver oil red O staining (-‐84 %, P<0,01), indicating reduced lipid content. Moreover, C118335 reduced F4/80 staining in liver (-‐21%, P<0,05) and white adipose tissue (WAT) (-‐41%, P<0,05) indicating reduced macrophage infiltration. RU486 did not significantly reduce liver and WAT fat mass and macrophage content. Both RU486 and C118335 improved glucose tolerance, tested via ivGTT, and lowered basal plasma glucose levels. Thymus weight was reduced by RU486 (-‐48%) and C118335 (-‐46%), whereas spleen weight was only reduced by C118335 (-‐44%). Moreover, the GR target gene FKBP5 was increased in liver and WAT, but not in brown adipose tissue, further indicating tissue specific GR modulation. Both C118335 and RU486 reduce diet-‐induced obesity development and improve glucose metabolism. C118335 reduces hepatic steatosis and inflammation in liver and WAT, which may be due to GR agonism and/or MR antagonism. Selective modulation of the GR combined with MR may be a promising target for combating obesity and related disorders.

O10: Maccari Early carbetocin treatment prevents metabolic aging induced by early stress in rats

S. Maccari1,3, E. Gatta1,3, H. Bouwalerh1,3, L. Deruyter1,3, G. Vancamp1,3, S. Morley-‐Fletcher1,3, J. Mairesse1,3 , F. Nicoletti2,3

1Glycobiology of Stress-‐related Diseases team, UMR 8576 University of Lille1/CNRS, Villeneuve d’Ascq, France; 2IRCCS Neuromed, Pozzilli, Italy ; 3 International Associated Laboratory “Prenatal Stress and Neurodegenerative Diseases” France/Italy (Glycobiology of Stress-‐related Diseases team, UMR 8576 University of Lille1/CNRS, Villeneuve d’Ascq, France; and Sapienza University of Rome, Rome, Italy)

Oxytocin, often referred to as the “love hormone", has a central anti-‐stress role by reducing the activation of the hypothalamic–pituitary–adrenal (HPA) axis, the major neuroendocrine system that controls response and resilience to stress. The animal model of perinatal stress (PRS) in rat programs the offspring to develop an anxious-‐/depressive-‐like phenotype characterized by a prolonged HPA response to stress during adulthood. Moreover, PRS rats display cognitive decline and metabolic dysfunction, often associated with anticipated ageing. This pathological programming is induced in PRS rats by prenatal overexposure to maternal glucocorticoids combined with reduced maternal behavior during the early postnatal life. We wanted to assess whether the involvement of the mother and pup oxytocin systems in the programming of the PRS phenotype is persistent during aging. We examined the long-‐term effects of a subchronic treatment with oxytocin during the early postpartum period. Stressed and control unstressed mothers were injected with an oxytocin receptor agonist, carbetocin (1mg/kg, i.p.), during the first week of the postpartum period. In PRS aged rats, i.e the 20 month-‐old offspring of dams exposed to gestational stress, we found that postpartum carbetocin treatment prevented anxiety-‐like behavior, spatial memory impairments, and deficits in social behavior. Early carbetocin treatment also prevented the increase in plasmatic glycaemia in aged PRS rats. We then focused on a post-‐translation modification, O-‐linked N-‐acetylglucosamine (O-‐GlcNAc), which directly correlates with the availability of extracellular glucose. Nuclear O-‐GlcNAc dynamically targets key transcriptional and epigenetic regulators. Interestingly, hippocampal nuclear O-‐GlcNAc levels were affected in aged offspring of dams treated with carbetocin. In conclusion, the oxytocinergic system plays a crucial role in early life by shaping emotional, cognitive and metabolic features. Thus, targeting the central oxytocinergic system could represent a new potential strategy for the treatment of emotional and social aspects of psychiatric disorders.

Poster Session 1—Thursday, September 24

P1: Electrophysiological properties of tanycytes from the mouse hypothalamus Clasadonte Jerome and Prevot Vincent

Inserm, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Jean-‐Pierre Aubert Research Centre, U1172, 59045 Lille, France. Tanycytes are polarized cells which occur along the walls and floor of the third ventricle in the hypothalamus, with one side contacting the cerebrospinal fluid and the other sending a single process towards the brain parenchyma. Although morphology of tanycytes has been studied extensively, little is known about their electrophysiological properties. Whole-‐cell patch-‐clamp recordings and injection techniques were used in acute hypothalamic slices from mouse to study tanycytes located near the arcuate nucleus of the hypothalamus (ARH). With physiological extracellular concentrations of potassium (3.2 mM), tanycytes had a very negative resting membrane potential (– 84.00 ± 2.65 mV) and exhibited a near-‐Nernstian response to changes in extracellular concentrations of potassium (from 0 to 20 mM). Their membrane responses to injection of square current pulses (from – 50 to + 50 pA) indicated an incredibly low input resistance (51.30 ± 18.01 MΩ) and a lack of action potential firing. Injection of Lucifer Yellow or biocytin in a single tanycyte via the patch-‐clamp pipette revealed that these cells were dye-‐coupled and sent a single and long process towards the ARH. Bath application or focal delivery (via a puff pipette) of the two neurotransmitters, GABA and glutamate (10 mM), induced a strong and reversible membrane depolarization (5-‐15 mV), suggesting the presence of functional GABA and glutamate receptors in these cells. Taken together, our data indicate that tanycytes located near the ARH form a network of highly interconnected cells, probably via gap junctions, and share similar electrophysiological properties with the glial cells, astrocytes, and may therefore have similar functions such as the sensing of neuronal activity and the uptake of extracellular potassium released from neighboring neurons.

P2: Antibody mediated inhibition of the FGFR1c isoform induces a catabolic lean state in Siberian hamsters Samms, Ricardo J 1,2,Lewis, Jo E 1, Lory, Alex 1, Fowler, Maxine J 1, Cooper, Scott 1, Warner, Amy 1, Emmerson, Paul 2, Adams, Andrew C 2, Luckett, Jeni C 3, Perkins, Alan C 3, Wilson,Dana 4, Barrett, Perry 4, Tsintzas, Kostas 1, Ebling, Francis JP 1 1 School of Life Sciences, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham NG7 2UH, UK. 2 Lilly Research Laboratories, Indianapolis, USA. 3 School of Medicine, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham NG7 2UH, UK. 4 Rowett Institute for Nutrition and Health, University of Aberdeen, Greenburn Road, Aberdeen AB21 9SB, UK.

Hypothalamic tanycytes are considered to function as sensors of peripheral metabolism. To facilitate this role they express a wide range of receptors, including fibroblast growth factor receptor 1 (FGFR1), which is reported to play a central role in the regulation of glucose and energy homeostasis. Using a monoclonal antibody (IMC-‐H7) that selectively antagonizes the FGFR1c isoform, we investigated possible actions of FGFR1c in a natural animal model of adiposity, the Siberian hamster. Infusion of IMC-‐H7 into the third ventricle suppressed appetite and increased energy expenditure. Likewise, peripheral treatment with IMC-‐H7 dose-‐dependently decreased appetite and body weight, and increased energy expenditure and fat oxidation. A greater reduction in body weight and caloric intake was observed in response to IMC-‐H7 during the long day fat state as compared to the short day lean state. This enhanced response to IMC-‐H7 was observed in calorically-‐restricted hamsters suggesting that it is the central photoperiodic state rather than the peripheral adiposity that determines the response to FGFR1c antagonism. Hypothalamic thyroid hormone availability is controlled by tanycytes and is the key regulator of seasonal cycles of energy balance. Therefore, we determined the effect of IMC-‐H7 on hypothalamic expression of the deiodinase enzymes, DIO2 and DIO3, which are local regulators of thyroid hormone availability. The reductions in food intake and body weight were always associated with decreased expression of DIO2 in the hypothalamic tanycyte cell layer. These data provide further support for the notion the tanycytes are an important component of the mechanism by which the hypothalamus integrates central and peripheral signals to regulate energy intake and expenditure

P3: Tanycyte-‐neurones communication in mice hypothalamus Bolborea Matei 1, Kasparov Sergey 2 and Dale Nicholas 1 1-‐ School of Life Sciences University of Warwick Coventry, UK; 2-‐ School of Physiology and Pharmacology University of Bristol Bristol, UK Appetite and body weight is finely controlled by the hypothalamus. Tanycytes are specialised glial cells lining the third ventricle of mammalians’ brain. These cells have a specific cellular morphology, which allows them to have a key role into the hypothalamic functions. Their cell bodies are part of the ependymal layer and their single process penetrate deeply into the hypothalamic parenchyma reaching the arcuate nucleus, the ventromedial hypothalamus and the dorsomedial hypothalamus. Tanycytes are capable to sense nutrients such as glucose and amino acids from the cerebrospinal fluid (CSF). This leads to the idea that these glial cells are important for the regulation of body weight by sensing the CSF from the periphery and communicating with neurones of the hypothalamus. Tanycytes respond to nutrients using specific receptors and consequently induce an influx of extracellular Ca2+ in the cell. This Ca2+ influx leads to release of ATP, and triggers a P2Y1 receptor-‐mediated Ca2+ wave, which travels between tanycytes. Here, we propose that tanycytes also communicate with hypothalamic neurones. However, how tanycytes rely the message to the central nuclei of feeding is not yet fully understood. We propose to describe this new neural network that involves tanycytes-‐nutrient sensing and neurones regulating the appetite. We created the first tanycytes optogenetic tools to be able to finely stimulate these cells and mime its responses to nutrients. We observed that optostimulation of tanycytes in acute hypothalamic slices induces depolarisation of neurones of the arcuate nucleus.


P4: Umami taste signalling in hypothalamic tanycytes Lazutkaite, Greta; Dale, Nicholas University of Warwick Hypothalamic tanycytes are glial cells that line the wall of the 3rd ventricle and send processes into the brain parenchyma. They may help to inform the arcuate nucleus and ventromedial hypothalamus about nutrient availability in the cerebrospinal fluid (CSF). Tanycytes can sense glucose, most likely via the sweet taste receptor (a T1R2/T1R3 heterodimer) originally described in the taste buds of the tongue. A receptor for another taste modality – umami – is part of the same gene family as the sweet taste receptor and comprises the T1R1/T1R3 heterodimer. We have therefore studied whether tanycytes might be able to detect amino acids via the umami taste receptor. We used Fura-‐2 imaging of intracellular Ca2+ to assess the responses of tanycytes in acutely prepared brain slices to L-‐amino acids such as arginine, lysine, alanine, serine and proline. Tanycytes responded to all of these agonists in order of potency (Arg>Lys>Ala>Ser>Pro). We found that the responses to these amino acids could be enhanced by prior application of IMP, a known allosteric modulator of the T1R1/T1R3 receptor. The responses to amino acids required extracellular Ca2+ and could also be blocked if the internal stores of Ca2+ were depleted by cyclopiazonic acid. The responses to amino acids were also blocked by a combination of a selective P2Y1 receptor antagonist (MRS2500) and a non-‐selective P2 antagonist (PPADS). The ATP release appeared to come via connexin hemichannels as it was blocked by high doses of carbenoxolone (100 µM) and the mimetic peptide GAP26, but only weakly affected by the relatively selective pannexin blockers (probenecid and 10 µM carbenoxolone). Our results show the first non-‐neuronal mechanism for amino acid detection and the first example of signaling by the umami taste receptor in the mammalian brain. Full understanding of the amino acid sensitivity of tanycytes may help to devise new strategies for countering the rising tide of excessive weight gain and obesity.

P5: The TSH/T3 dependent hypothalamic pathway in seasonal reproduction Milesi S, Ciancia M, Beymer M, Laran-‐Chich MP, Simmoneaux V, Klosen P Université de Strasbourg, CNRS In mammals, seasonal reproduction is important to have the best survival of the progeny. Melatonin is secreted only during the night, thus the short nights of summer result in a short duration melatonin peak, while the long winter nights produce a long melatonin peak. These seasonal changes in melatonin secretion link the photoperiod to physiological regulations. In hamsters, the short summer melatonin peak relieves the melatonin inhibition of TSH production in the pituitary pars tuberalis, thus increasing TSH production and release. This TSH then acts on tanycytes to stimulate Deiodinase 2 (Dio2) expression. Dio2 converts tetraiodothyronine (T4) to its active form triiodothyronine( T3). This T3 appears to stimulate kisspeptin and RFRP3 expression, two well known regulators gonadotropic activity. Furthermore, this reactivates the gonadotropic axis. Studying the photoperiodic reactivation of the gonadotropic axis, we noted an increase of FSH/LH before the reactivation of RFRP3 and Kisspeptin expression. Moreover, acute intracerebroventricular injection of TSH induces an increase of circulating testosterone levels after only 4 hours. Both of these observations suggest the existence of a short activation pathway of GnRH secretion not involving Kisspeptin and/or RFRP neurons. This pathway might involve direct action of tanycytes on GnRH terminals, a type of interaction known to be important in the control of the ovarian cycle. Our future goal is to confirm the existence of a short TSH dependent control of the gonadotropic axis involving tanycytes and study its role in the seasonal activation of the gonadotropic axis.

P6: It’s all in the timing: Temporal dynamics of hypothalamic gene expression changes over one year in the Siberian hamster in natural photoperiod 1Barrett, Perry; #Petri, Ines, 2Diedrich, Victoria; 3Herwig, Annika; 2Steinlechner Stephan 1Rowett Institute for Nutrition and Health, Bucksburn Aberdeen AB21 9SB; 2University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, Germany; 3Zoological Institute Martin-‐Luther-‐King Platz 3 20146 Hamburg ,Germany The Siberian hamster is an exemplar for the pre-‐emptive physiological adaptations made by seasonal mammals. In shortening days of autumn the hamster reduces body mass and regresses reproductive organs. Following the winter solstice while day length duration is still less than a critical duration to signal long days, hamsters increase body mass and recrudesce the reproductive organs to become reproductively competent by late February. This reversal of physiology ahead of stimulatory long days is known as the photorefractory response and is evidence of a circannual timing mechanism. The mechanism underpinning the adaptation to seasons is thought to be localised changes in thyroid hormone from hypothalamic tanycytes driving seasonal transcriptional responses in the hypothalamic-‐neuroendocrine system. Using in situ hybridization we have investigated gene expression changes in the hypothalamus over 1 year of hamsters born and raised in natural photoperiod. The data reveal a remarkable temporal co-‐ordination of gene expression changes. The most notable changes are in components of the hypothalamic thyroid hormone system which show a temporally restricted peak of anabolic thyroid hormone type 2 deiodinase (Dio2) expression in June and catabolic thyroid hormone type 3 deiodinase (Dio3) expression in October. Woven between these peaks is an ascendance of the thyroid hormone transporter MCT8 to peak in September followed by a gradual decline during winter and spring of the following year. In addition to these changes, Gpr50 expression shows a temporally restricted peak of expression in July. Other genes, such as Vgf and Srif show a peak and trough during the course of the year. Together the temporally co-‐ordinated gene expression changes may help to explain the basis of the circannual timer, but also raise new questions about the seasonal regulatory mechanism


P7: Cell neogenesis in the hypothalamus: a new mechanism of control of the reproductive function? Pellegrino Giuliana; Vincent Prevot; Marc Baroncini; Ariane Sharif UMR-‐S 1172, JPARC, Laboratoire « Développement et plasticité du cerveau neuroendocrine », Université de Lille Adult neurogenesis in mammals has been well documented in the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG). However, there are a growing number of studies that documented neurogenesis and gliogenesis in the postnatal hypothalamus and most of these studies have been conducted to understand its role in the regulation of energy homeostasis. However this is not the only function regulated by the hypothalamus, neurogenesis and gliogenesis have been described also in the hypothalamic regions controlling GnRH function so our work aims to explore the possibility that the generation of new cells within the postnatal hypothalamus may represent a new mechanism of plasticity involved in the control of reproductive function through modulation of GnRH neuronal function. The specific aims are: to determine whether hypothalamic cell neogenesis is implicated in the physiological modulation of the activity of the GnRH system during key stages of the postnatal life; to determine whether gonadal hormones are the physiological determinants that modulate the activity of stem cells within the neuroendocrine hypothalamus. We expect this work to provide significant insights into the mechanisms of cerebral plasticity at adult age and their possible role in the control of a major physiological function: reproduction.

P8: Photoperiod effects on the hypothalamic neurogenic niche in sheep. Lucile Butruille, Martine Batailler, Daniele Mazur, Vincent Prévot, Martine Migaud UMR PRC INRA -‐ CNRS -‐ Université de Tours -‐ IFCE, Equipe «Microenvironment and Dynamic of Neuroendocrine Network », 37380 NOUZILLY, France In mammals recent studies have demonstrated the presence of an adult neurogenic niche in the hypothalamus, a key region that controls physiological functions, such as reproduction. In sheep, a long lived mammalian model, the existence of a neurogenic niche has also been shown in the hypothalamus and DCX+ cells were found in the vicinity of this hypothalamic neurogenic niche, indicating the presence of numerous adult-‐born neurons in this structure. In this seasonal model, reproduction is characterized by alternation a period of reproduction during short days and a period of sexual rest during long days. We have recently reported seasonal increases in both proliferation rates and DCX’s expression in the hypothalamus during short days. This study aims at evaluating (i) whether the markers of the various niche cell types are also sensitive to the photoperiod by comparing their expression between short and long days (ii) the migratory potential of the sheep hypothalamic neuroblasts. Through an immunohistochemical approach, we showed a variation of the labeling for neural stem cells and basal lamina markers according to the photoperiod. This data suggest that photoperiod drives cytoarchitectural rearrangements within the sheep hypothalamic neurogenic niche. Electron microscopy analysis are currently performed to determine to which extend the cytoarchitecture of the cells lining the third ventricle is affected by photoperiod. Next, a neuroimaging approach using micron-‐sized iron oxide particles (MPIOs) injection was developed to explore the migratory potential of the hypothalamic neuroblats. In a pilot experiment we showed that MPIOs are incorporated by cells lining the third ventricle and detected by RMI, indicating that the use of MPIOs and RMI for the detection of a possible migration route in the hypothalamus is feasible in sheep. Once the hypothalamic migratory path determined we will identify the phenotype of the newly born neurons by immunohistochemistry approach.

P9: Seasonal variation in the expression of doublecortin and potential migratory features of neuroblasts in the adult sheep hypothalamus Batailler, Martine; Derouet, Laura; Butruille, Lucile ; Migaud, Martine INRA, UMR 85 Physiologie de la Reproduction et des Comportements, F-‐37380 Nouzilly, France ; CNRS, UMR7247, F-‐37380 Nouzilly, France ; Université de Tours, F-‐37041 Tours, France ; Institut Français du Cheval et de l’Equitation (IFCE), F-‐37380 Nouzilly, France Adult neurogenesis is a process consisting in the generation of new neurons from adult neural stem cells and as such represents a remarkable illustration of the brain structural plasticity abilities. The hypothalamus, a brain region that plays a key role in the neuroendocrine regulations including reproduction, metabolism or food intake, homes neural stem cells within a specialized zone defined as the hypothalamic neurogenic niche located in the arcuate nucleus and the median eminence. In adult sheep, a seasonal species, recent studies have revealed photoperiod-‐dependent changes in the hypothalamic cell proliferation rate. In addition, doublecortin (DCX), a microtubule associated protein expressed in immature migrating neurons, is highly present in the vicinity of the hypothalamic neurogenic niche. With the aim to further explore the mechanism underlying adult sheep hypothalamic neurogenesis we show changes in the density of DCX-‐positive cells according to the photoperiodic conditions at various time points of the year, further suggesting that new neuron generation is seasonally regulated. We also demonstrate that cyclin-‐dependant kinase-‐5 (Cdk5) and p35, two proteins involved in DCX phosphorylation, are co-‐expressed with DCX in young hypothalamic neurons and are capable of in vivo interaction. Additionally, we reveal the rostro-‐caudal extend of DCX labelling on hypothalamic sagittal planes where DCX-‐positive cells are found within the most rostral nuclei of the hypothalamus, including the preoptic area. Altogether, our results support the hypothesis of the existence of a migratory route taken by the new born neurons in the adult hypothalamus that is responsive to seasonal stimuli in sheep brain.


P10: The interplay between social/reproductive stimuli, Adult Neurogenesis and the GnRH system Sara Trova (1,2,3), Livio Oboti (5), Giuliana Pellegrino (1,3), Roberta Schellino (,2), Paolo Giacobini (3,4), Paolo Peretto (1,2) 1.Department of Life Sciences and Systems Biology, University of Turin, Via Accademia Albertina 13 ,10123 Torino, Italy 2. Neuroscience Institute Cavalieri Ottolenghi (NICO), Regione Gonzole 10, Orbassano, 10043 Torino, Italy; 3. INSERM, Laboratory of Development and Plasticity of the Postnatal Brain, Jean-‐Pierre Aubert Research Center, Unité 837, Lille, France; 4. School of Medicine, UDSL, Lille, France 5 Center for Neuroscience Research. Children's National Health System. Washington, D.C., USA

Multiple experimental evidences have shown that Adult Neurogenesis (AN) plays critical roles in encoding social/reproductive stimuli and in turn in modulating a fundamental aspect of life -‐ the propagation of the species. A continuous neurogenesis is required for sex-‐specific activities both in male and female mice (Sakamoto et al., 2011) and it is in turn modulated by gonadal and adenohypophyseal hormones, such as prolactin (PRL) and luteinizing hormone (LH; Larsen et al., C.M., 2012). In these processes, the functional role played by AN in integrating external and internal signals, and the intrinsic mechanisms underlying the interplay between AN and hormones are still poorly understood. Here, by investigating the responses of AN in female mice to sexually experienced or to young sexually inexperienced male mice odors, we found that pheromonal-‐dependent modulation of AN starts around puberty, a developmental critical period characterized by increased activity of gonadotropin releasing hormone secretion. To investigate a possible direct interplay between GnRH activity and AN, we firstly performed quantitative PCR analyses on the total OB of mice that revealed the presence of mRNA of GnRH receptor, Estrogen receptor alpha and beta, and Androgen receptor. Secondly, using a transgenic mice model with an impaired GnRH function (GnRH::Cre; DicerloxP/loxP mice) we show that a dysfunction in GnRH System increases the number of newly-‐born neurons integrated in the Main Olfactory Bulb. Our results suggest that the intriguing co-‐modulation between brain plasticity and the endocrine system, necessary for adjusting the physiological homodynamic equilibrium to each particular life event (i.e. reproduction), could be in part mediated by a direct/indirect co-‐regulation between AN and GnRH System.

P11: Amygdala kisspeptin population: non-‐hypothalamic control of the gonadotropic axis and olfactory function. Rafael Pineda Reyes1, Robert P. Millar2 and Mike Ludwig1

1Centre for Integrative Physiology, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK 2Mammal Research Institute, Department of Zoology and Entomology, University of Pretoria, Pretoria 0001, South Africa

In mammals reproduction is under the control of the gonadotropic axis. Hypothalamic GnRH neurons are the main element of this axis and kisspeptins and their receptors were identified as potent regulators of the axis. Hypothalamic kisspeptin neurons are mainly found in the arcuate and the anteroventral periventricular nuclei. A third population of kisspeptin neurons is located in the amygdala, but their connectivity with other brain regions and their involvement in the control of GnRH neurons was unknown. Using viral specific-‐cell gene expression in combination with immunofluorescence histochemistry we show here that the amygdala kisspeptin neurons project to GnRH neurons in the preoptic area. We also determined the precise localization and number of kisspeptin neurons in the rat medial amygdala and their innervation by the two major systems involved in social behaviors, the vasopressin and dopamine system. The amygdala receives inputs from the olfactory system and here we show that the amygdala kisspeptin neurons project back to the accessory olfactory bulb. Our data suggest that amygdala kisspeptin neurons integrate odour and social behavior information to the GnRH neurons of the gonadotropic axis to coordinate the endocrine axis with appropriate behavior. Acknowledgements : This work was supported by the Newton International Fellowship program awarded to RPR (Ref. NF130516), co-‐funded by the Royal Society and the British Academy, and the British Society for Neuroendocrinology (Project Support Grant)

P12: Multiple markers reveal different features of sex differences in HVC neurogenesis Olesya T. Shevchouk, Gregory F. Ball, Charlotte A. Cornil and Jacques Balthazart University of Liege, Belgium and University of Maryland, College Park, MD In oscines, neurogenesis in the song control nucleus HVC is sensitive to changes in hormonal and social environment. We used three independent markers to investigate effects of social context on HVC neurogenesis in male and female canaries. Males were castrated, implanted with testosterone and housed either alone (M), with a female (M-‐F) or with another male (M-‐M) while females were implanted with 17β-‐estradiol and housed alone (F) or with a male (F-‐M). All subjects received injections of BrdU at the start of the social manipulation, 21 days before brain collection, and EdU 10 days before brain collection. Cells incorporating the two thymidine analogs and expressing doublecortin (DCX) which labels young newborn neurons, were quantified in the HVC of each subject. No effect of social condition was found on the numbers of either BrdU+ or EdU+ and only a subtle effect on the number of DCX+ cells. Further comparisons were performed between males and females regardless of social condition. In terms of number of cells per section, males had more EdU+ and BrdU+ cells in the lateral ventricle dorsal of HVC, suggesting higher rates of proliferation, and also more EdU+ in HVC. In the equivalent analysis for BrdU+ cells no sex difference was found. The total number of DCX+ neurons per section recruited to HVC was equal in males and females, but since HVC is smaller in females, the density of DCX+ neurons was higher in this sex. These data suggest that while initial survival of newborn neurons in HVC is higher in males, in the long-‐term the females have equal numbers and higher densities of newborn neurons. A lack of effect of social context is likely due to the short day lengths the birds were exposed to during this study, indicating that social effects on the song control system are gated by photoperiod. In addition, these data suggest that proliferation, recruitment and survival of new neurons can be independently affected by environmental conditions.


P13: Membrane-‐estrogen receptor alpha does not seemed to be involved in the control of reproductive behavior but might influence sexual differentiation Taziaux Mélanie1, Ceuleers Meg-‐Anne1, Arnal Jean-‐François2, Lenfant Françoise2, Cornil Charlotte A1. 1. GIGA Neurosciences, University of Liege, Liège, Belgium 2. Inserm U1048-‐I2MC-‐ Equipe 9, Institut des Maladies Métaboliques et Cardiovasculaires,Toulouse, France

Besides the slow and long-‐term control they exert on physiologyl and behavior through the transcriptional action of their ligand-‐activated nuclear receptors, estrogens also activate membrane-‐initiated effects resulting in more rapid and transient actions. Yet, the receptors involved in these actions are often unknown. Here we sought to determine the role of the membrane fraction of estrogen receptor alpha (ERα) in the control of male and female sexual behavior using the C451A-‐ERα mouse model whose ERα is unable to translocate to and signal from the membrane while retaining its transcriptional activity due to a mutation in the palmitoylation site of ERα. Because defects in estrogen signaling may impair normal sexual differentiation of brain and behavior, we also investigated the effect of the mutation on the expression profile of markers of brain sexual differentiation. Male sexual behavior did not differ between the three genotypes in castrated males chronically implanted with capsule delivering testosterone. Similarly, no genotype difference was found for lordosis behavior in females implanted with a chronic capsule delivering estradiol and injected with progesterone 3 hours before testing. A second group of mice gonadectomized and injected for 2 weeks with EB were compared for the number of cells immunoreactive (ir) for tyrosine hydroxylase (TH) and kisspeptin (Kp) in the anteroventral periventricular nucleus (AVPv), two cell populations known to be more abundant the in females than males. No genotype difference was found in the number of TH-‐immunoreactive (TH-‐ir) cells. The analysis of the number of Kp-‐ir cells revealed not genotype difference in females while KO males were found to express more Kp-‐ir cells than wild-‐type males suggesting that this membrane receptor might play a role in the sexual differentiation of the brain. Further experiments are currently performed to confirm this observation.

P14: Effects of neural invalidation of estrogen receptor β in mice. Lydie Naulé1, Vincent Robert2, Clarisse Marie-‐Luce1, Caroline Parmentier1, Mariangela Martini2, Hélène Hardin-‐Pouzet1, Valérie Grange-‐Messent1, Matthieu Keller2, Isabelle Franceschini2, Sakina Mhaouty-‐Kodja1 1 Neuroscience Paris Seine, Team “Neuroplasticity of Reproductive Behaviors”, CNRS UMR 8246, INSERM U 1130, UPMC, Paris, France. 2

Physiologie de la reproduction et des comportements, UMR 7247 INRA-‐CNRS-‐Université de Tours-‐Haras Nationaux, Nouzilly, France

Sexual differentiation of the central nervous system is governed by sex steroid hormones (testosterone and its metabolites). During the perinatal period, estradiol, originating from neural aromatization of testicular testosterone, masculinizes and defeminizes the neural structures underlying the regulation of the hypothalamus-‐pituitary-‐gonad (HPG) axis and the expression of reproductive behaviors. In females, the ovaries are inactive during the fetal period, and the brain is protected from the masculinizing effects of perinatal estradiol through the action of the α-‐fetoprotein. Recent studies suggested an active role of estradiol liberated from the postnatal ovaries in the establishment of puberty and feminization of neural circuits involved in the expression of female sexual behavior. In adulthood, estradiol has an activational role in the regulation of the HPG axis and reproductive behaviors. Estradiol acts via two nuclear estrogen receptors (ER) α and ERβ. Many studies showed that neural ERα is essential for the maturation and regulation of the HPG axis, as well as the expression of sexual behavior. Global knockout of ERβ showed either a minor or a major defect of female fertility. The present study aimed to determine the relative contribution of neural ERβ in estradiol-‐induced effects in female mice. For this purpose, we generated and characterized a mouse model selectively lacking ERβ in the nervous system, by using a conditional mutagenesis Cre-‐loxP approach. This strategy allows to distinguish between the peripheral and central effects of this receptor. Female mice lacking the neural ERβ were analyzed for pubertal onset and expression levels of molecular factors involved in pubertal maturation. At adulthood, mutant female mice were tested for fertility, oestrous cyclicity, hormonal levels and expression of sexual behavior and anxiety state level. The results, obtained in this context, will be presented.

P15: Effect of adult exposure to low doses of Phthalates on peripheral and neural responses in male mice Poissenot K.a, Moussu C. a, Robert V. a, Keller M. a, Mhaouty-‐Kodja S1. & Franceschini I. a a INRA, UMR 85 Physiologie de la Reproduction et des Comportements, F-‐37380 Nouzilly, France 1Neuroscience Paris Seine-‐IBPS, Inserm UMR-‐S1130, Université P. et M. Curie, Paris,, France

Di-‐2-‐ethylhexyl phthalate (DEHP) is an endocrine disruptor (EDC) with known anti-‐androgenic activity. The exposure is important and DEHP metabolites are found in urine in humans. A number of such EDCs have been reported to target the developping neuroendocrine circuitry controlling reproductive fonction, including the Kiss1 gene. Moreover, a recent study on Bisphenol A, another EDC with known anti-‐androgenic activity, highlighted a particular sensitivity of the adult male mouse nervous system controlling sexual behavior to this compound following chronic low level exposure (Picot et al., 2014). Such studies raise questions about the effects of adult exposure to low doses of DEHP on reproductive function, neuroendocrine responses, and on the neural circuitry underlying male sexual behavior. Adult male mice were exposed for one month to DEHP at the tolerable daily intake (TDI, 50µg/kg(bw)/day) or the 5 µg/kg(bw)/day dose. The effects were evaluated on a mouse line expressing a GFP reporter under the control of the Kiss1 promoter (Gottsch et al., 2011). This model allowed us to quantify the number of GFP-‐immunoreactive cells (GFP-‐ir), and the proportion of these cells witch express estrogen receptor α (ERα) or androgen receptor (AR) in the preoptic area (POA) and the arcuate nucleus (ARC). The mean density of Erα-‐ir and AR-‐ir cells was further evaluated in brain areas relevant to male sexual behavior. There was no significant effect of DEHP treatment on the number of GFP-‐ir cells and the proportion that co-‐expressed ERα or AR in the POA and in the ARC. Accordingly, DEHP treatment did not affect the weight of the androgen-‐dependant genital tract organs, nor circulating testosterone levels. The density of ERα or AR remained unchanged in the main areas relevant to male sexual behavior following exposure to DEHP at the TDI dose. The potential anti-‐androgenic activity of this adult TDI exposure will be further explored including its physiological and behavioral relevance.


P16: Adult exposure to DEHP affects sexual steroid-‐dependant behaviors at very low doses Dombret C.1, Capela D.1, Malbert-‐Colas A.1, Lavoue A.1, Keller M.2, Franceschini I.2, Mhaouty-‐Kodja S.1. 1Neuroscience Paris Seine-‐IBPS, Inserm UMR-‐S1130, Université P. et M. Curie, Paris,, France ; 2 INRA, UMR 85 Physiologie de la Reproduction et des Comportements, F-‐37380 Nouzilly, France

The male sexual behavior is a complex centrally regulated behavior controlled by sex steroids. During development the male brain is subjected to organisational effects of testosterone, which permanently shapes brain structures through masculinization and defeminization processes. Later, during adulthood, sexual steroids activate these structures to initiate sexual behavior. Endocrine disrupting chemicals (EDCs) are chemicals of natural or human-‐made origin, able to interfere with normal physiology by either altering the levels of hormones, modifying the expression of hormones receptors or changing endogenous signalling pathways triggered by these hormones. EDCs are suspected to play a broad role in dysfunctions and diseases leading to a loss or a reduction of fertility like alteration of the semen quality. EDCs can also induce anomalies of the urogenital tract like testicular dysgenesis syndrome. In this context, we have previously shown that adult, but not perinatal, exposure to bisphenol A (BPA) at low dose alters male sexual behavior through an anti-‐androgenic effect (Picot et al., 2014). In an effort to identify other molecules that alter the neural circuitery supporting the expression of the male sexual behavior, we focused on di (2-‐ethylhexyl) phthalate (DEHP). This plasticizer is the most abundant in PVC-‐based materials and has been shown to have anti-‐androgenic and estrogenic activities in different models. Moreover this molecule is listed in the Priority Substances under the water framework directive (Directives 200/60/CE and 2008/105/CE). In order to evaluate a putative effect of DEHP on male sexual behavior, we treated adult males during one month at doses of 0,5 ug/kg/day, 5 ug/kg/day and 50 ug/kg/day and compared these DEHP-‐treated animals to vehicule-‐treated males. The first two doses are close to the environmental exposure while the last dose corresponds to the tolerable daily intake (TDI) dose (calculated by dividing the no-‐observed-‐adverse-‐effect

P17: Developmental exposure to 17-‐alpha-‐ethinylestradiol alters behavioral and neural outcomes in adult male mice Derouiche Lyes., Keller Mathieu., Duittoz Anne., Pillon Delphine UMR 0085 INRA -‐ CNRS -‐ Université de Tours -‐ IFCE, 37380 NOUZILLY, France Endocrine disrupting chemicals may specifically target the neuroendocrine circuits of the central nervous system and induce deleterious effects on health. Ethinylestradiol (EE2) is a pharmacological estrogen largely used in oral contraceptives and constitutes one of the major pharmaceutical products found as a contaminant in effluent waters. The aim of our study was to demonstrate whether a chronic exposure to environmentally relevant or pharmacological doses (respectively 0.1 and 1 microg/kg/day) of EE2 during the whole development (from mid-‐gestation up to peri-‐puberty) can disturb behavioural and neuroanatomical parameters of reproductive function in adult mice. Mating behaviour, reproductive physiology and then neuroanatomical studies were preformed on control developmentally exposed males to EE2. Our results showed that both doses induced an exacerbated sexual behaviour, evidenced by a decrease in the latency to initiate sexual behaviour and an increase in the number of intromissions. Fertility, plasma testosterone concentrations and testicular histology were not affected. Neuroanatomical analysis showed that exposed males had more Calbindin-‐D28k immunoreactive cells in the hypothalamic sexually dimorphic nucleus (SDN) than controls. No difference was detected in the numbers of kisspeptin neurons in the preoptic periventricular nucleus. Strikingly, the same effects on sexual behaviour and SDN than those depicted in F1 males exposed to EE2 during their development were also observed in their F2 to F4 offspring. Altogether, our results show that a developmental exposure to low doses of EE2 alters mating behaviour and neuroanatomy of the SDN in adult male mice. These disruptions are transmitted to the next generations. We now aim at identifying, by using embryonic murine neural stem cells cultures, the cellular and molecular targets through which EE2 can act during development to induce in adult functional alterations, such as behavioural modifications.

P18: Effect of progestagens on neurodevelopment of zebrafish embryos Elisabeth Pellegrini, Cécile Cruciani, Justyne Feat, Joel Cano, Marie-‐Madeleine Gueguen, Colette Vaillant-‐Capitaine, Olivier Kah Université Rennes 1, IRSET, Neuroendocrine effects of endocrine disruptors

A single nuclear progesterone receptor (Pgr) is widely expressed in the brain of zebrafish, particularly in radial glial progenitor cells (RGCs). This suggests that progestagens (such as progesterone (P4), norethindrone) and/or their metabolites could affect the neurogenic activity of these cells during neurodevelopment. In this context, we used zebrafish embryos in order to study the effects of increasing concentrations of P4 on the expression of several genes potentially involved in brain development such as cyp19a1b (encoding Aro-‐B, the specific enzyme converting androgens into estrogens and selectively expressed in RGCs), pgr, estrogen receptors (esr1, esr2a, and esr2b), the proliferation marker pcna, the anti-‐apoptotic gene bcl2, the pro-‐apoptotic gene bax and the apoptotic gene casp3. Our results show that P4 significantly affects the expression of several genes in the brain of zebrafish larvae. We demonstrate, based on quantitative PCR analysis, that cyp19a1b is upregulated by P4. Inversely, casp3 is downregulated by P4. The inhibitory action of P4 on apoptosis process is confirmed by the TUNEL assay. Given the crucial role of RGCs in neurodevelopment, we suggest that P4 could affect apoptosis process that takes place during early neurogenesis in zebrafish embryo. Using cyp19a1b-‐GFP transgenic zebrafish, we also evaluated the potential estrogenicity of norethindrone, a synthetic progestagenic molecule used in contraceptive pills and found in surface water. Our preliminary results demonstrate that GFP signal is increased in the brain of larvae treated with norethindrone suggesting estrogenic properties of this compound. Although more studies are required, this data suggest that P4 and norethindrone may have effects on neurodevelopment in zebrafish notably on cell proliferation, the fate of newborn cells and their survival. Supported by the ANR PROOF


P19: Alteration of the neuroendocrine control of female puberty by early postnatal Bisphenol A exposure and involvement of GPR151, a potential new regulator of GnRH network Franssen, Delphine 1; Dupuis, Nadine 2; Gerard, Arlette 1; Hennuy, Benoit 3 ; Hanson, Julien 2; Parent, Anne-‐Simone 1; Bourguignon, Jean-‐Pierre 1 1GIGA Neurosciences, Neuroendocrinology Unit, University of Liège, Belgium 2GIGA, Laboratory of Molecular Pharmacology, University of Liège, Belgium 3GIGA, Transcriptomic platform, University of Liège, Liège, Belgium

We studied the effects of early postnatal exposure of female rats to Bisphenol A (BPA) on GnRH release (ex vivo) and pubertal timing. Newborn female rats were exposed from postnatal day (PND) 1 to 15 to vehicle (corn oil) or BPA (25 ng/kg/d or 5 mg/kg/d). After exposure to the low dose of BPA, the age at vaginal opening (VO) was delayed (35.3±0.7 days vs 33.5±0.5 days in controls) while advancement (32.1±0.6 days) was observed after 5 mg/kg/d. The late VO after exposure to 25 ng/kg/d of BPA was preceded by a significantly increased GnRH interpulse interval (52.5±0.8 min vs 44.6±0.7min in controls) at PND 20. By contrast, early VO after exposure to 5 mg/kg/d was preceded by a significantly reduced GnRH interpulse interval (40.3±0.1 min vs 42.8±0.4 min). Gene expression in the retrochiasmatic hypothalamus was assessed by whole exome RNA-‐sequencing on PND20. The most significantly affected gene was GPR151, with dose-‐opposing changes since mRNA levels increased after the low BPA dose and decreased after the high dose. GPR151 is an orphan GPCR with some homology to galanin and kisspeptin receptors. We observed that GPR151 was expressed in the median eminence of pubertal and adult female and male rats where some GnRH nerve terminals were found to co-‐express GPR151. Via overexpression of GPR151 in human embryonic kidney cells (HEK293), we showed that GPR151 was located at the cell membrane. GPR151 mRNA expression was increased throughout development in the retrochiasmatic hypothalamus of female rats. In adult females, GPR151 mRNA expression was increased in preoptic area on proestrus vs diestrus day. Using a cellular clone expressing GPR151, current studies attempt to predict signaling pathways activated by possible endogenous ligands. In conclusion, early postnatal exposure to BPA altered the onset of puberty in female rats through disruption of the GnRH release. This effect could involve changes in expression of a potential new regulator of the GnRH network, GPR151.`

P20: Sex differences in basal hypothalamic anorectic and orexigenic gene expression after re-‐feeding Caughey S, Wilson PW, Mukhtar N, D’Eath RB, Dunn IC, Boswell T The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Scotland, UK; Scotland’s Rural College, Scotland, UK; School of Biology, Newcastle University, England, UK. We have previously demonstrated that AGRP and NPY gene expression in the basal hypothalamus of female meat-‐producing (broiler) chickens is sensitive to feeding history following food restriction and re-‐feeding whereas POMC and CART expression remain unchanged. To distinguish the effects of gut fullness from nutritional signal feedback on basal hypothalamic gene expression, we compared the effect in restricted-‐fed 12-‐week-‐old male and female birds of re-‐feeding either on a normal ad libitum diet for 2 days, or on a diet diluted with a non-‐nutritive bulking agent, ispaghula husk. Overall, expression of AGRP and NPY measured by real-‐time PCR was significantly decreased in birds re-‐fed ad libitum but was high and statistically indistinguishable between restricted-‐fed controls and birds re-‐fed on ispaghula husk. An identical pattern of group differences was observed for expression of POMC and CART, except that expression was significantly higher in the ad libitum re-‐fed group. Collectively, these results suggest that gut-‐fill alone is not sufficient to reduce orexigenic gene expression after re-‐feeding. We also observed pronounced sex differences in gene expression. AGRP and NPY mRNA levels were significantly higher in males compared to females in all groups, and the difference was more pronounced in the restricted control and ispaghula husk groups compared to birds re-‐fed ad libitum. For POMC and CART, mRNA levels were significantly higher in males in the ad libitum re-‐fed group compared to the other groups. However in females, there were no significant effects of treatment on gene expression, consistent with our results from previous experiments with females only. A change in CART gene expression in response to nutritional manipulation has not been observed previously in birds. The cause of differences in gene expression between males and females is unclear but may relate to sex differences in growth rate.

P21: Impact of repeated stress on caloric efficiency in the rat Rabasa Cristina; Askevik, Kaisa; Vogel, Heike; Dickson, Suzanne L Department of Physiology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Medicinaregatan 11, PO Box 434, SE-‐405 30 Gothenburg, Sweden

Chronic stress is believed to be an important factor driving the over-‐consumption of food and obesity. Here we explored the impact of acute versus repeated stress on caloric efficiency in the rat. Male rats fed chow or a combination high-‐fat/high-‐sucrose (HFS) were exposed to repeated forced swim stress (RFS-‐chow, n=20 and RFS-‐HFS, n=20) or handling (control-‐chow, n=16 and control-‐HFS, n=14) for 7 days. On day 8, all rats were exposed to 1 h of forced swim (FS) and blood sampled to study the HPA response. 24 h later (day 9) half of the rats were sacrificed and other half were assessed for caloric efficiency after the stress for 8 days. RFS reduced food intake in chow fed rats, but not in rats exposed to a HFS diet. However, both groups under chronic stress reduced their body weight (BWt) gain indicating that mechanisms other than food intake cause BW changes in RFS animals. During days 1 to 7, the non-‐stressed groups were the most caloric efficient. However, after the acute FS on day 8, chow fed rats with previous stress experience increased their caloric efficiency compared with acutely stressed rats. Thus, repeated stressed rats gained more weight although they continued to eat less food than rats after acute stress, suggesting that repeated stress causes metabolic adaptations, making the rats more able to transform the Kcal into BWt. Also, the RFS-‐chow rats sacrificed on day 9 had reduced white adipose tissue, leptin and thymus weight, while this was not seen in RFS-‐HFS rats, indicating that consuming a chow diet could attenuate some of the adipogenic effects of chronic stress. Our results demonstrate that repeated stress induces adaptations that increase caloric efficiency in rats. The mechanisms underpinning this metabolic adaptation are being explored as they may prove insight regarding the link between chronic stress and metabolic disorder. Supported by EC (Nudge-‐it, 607310)


P22: Long-‐term behavioural consequences of exposure to a high fat diet during the post-‐weaning period in rats Suzanne L. Dickson, Winsa Julia and Rabasa Cristina Department of Physiology/Endocrine, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg. Sweden.

It seems clear that early life diet can play a role in obesity development. What is much less explored is the the long-‐term consequences of dietary interventions during adolescence, a critical period for the consolidation of reward processing. Thus, in the present work we exposed adolescent rats (post natal day 21), to normal chow or high fat (HF) diet for 10 days (PN21-‐PN31). Right after PN31, one group of rats were moved to chow (n=82) or kept under HF + chow choice diet (n=42). Half of the rats were tested immediately after PN31 (adolescence), and the other half were tested for the long-‐term effects starting on PN60 (adult). We studied the food preferences in a three choice model (high fat, high carbohydrates and high proteins). The rewarding properties of chocolate in the conditioned place preference (CPP), the preference for saccharin and sucrose over water, the anxiety in the elevated plus maze (EPM), and the locomotor response to Quinpirole (that tests dopamine signalling) were studied at both time points. Strikingly, behaviour was unaltered in adult rats transiently fed HF diet post-‐weaning, suggesting that short exposure to HF diet does not induce long-‐term effects in food preferences, reward perception and value of palatable food, anxiety or locomotor activity. Nevertheless, the HF-‐continuous group ate less chocolate during the training in the CPP and consumed less saccharin and sucrose when they were tested during the young period. However, this effect was attenuated when they had access to HF diet until adulthood. On the other hand, the intake of HF over chow was reduced in the adult HF-‐continuous group, probably due to the lack of novelty of the treat. Our results demonstrate that longterm access to HF diet in adolescent rats causes a reduction in the consumption of sweet tastes (saccharin/sucrose) and that sucrose (but not saccharine consumption) recovers if the rats continue to have HF access until adult. Supported by Nudge-‐it, 607310.

P23: Palatability can drive feeding independent of AgRP neurons Raphaël G. P. Denis1,15, Aurélie Joly-‐Amado1,15, Julien Castel1, Céline Cansell1 , Emily Webber13,14, Stéphanie Padilla9, 10 Anne-‐Sophie Delbès1, Sarah Martinez1, Marie Schaeffer 2,3,4, Fanny Langlet 5,6, Bénédicte Dehouck 5,6 , Amélie Lacombe1, Claude Rouch1, Nadim Kassis1, Jean-‐Alain Fehrentz 7, Jean Martinez 7, Pascal Verdié 7, Thomas S. Hnasko 8, Richard. D Palmiter9,10, Christophe Magnan1, Michael Krashes13,14, Ali. D Güler9,10,11 and Serge Luquet1 1Univ Paris Diderot, Sorbonne Paris Cité, Unité de Biologie Fonctionnelle et Adaptative, CNRS UMR 8251, F-‐75205 Paris, France. 2CNRS, UMR-‐5203, Institut de Génomique Fonctionnelle, F-‐34000 Montpellier, France; 3INSERM, U661, F-‐34000 Montpellier, France and 4Universities of Montpellier 1 & 2, UMR-‐5203, F-‐34000 Montpellier, France. 5Inserm, Jean-‐Pierre Aubert Research Center, U837, F-‐59000 Lille, France, and 6Université droit et santé de Lille, Faculté de Médecine, F59000 Lille, France. 7CNRS, UMR 5247, Institut des Biomolécules Max Mousseron, Universities of Montpellier 1&2. 8 Department of Neurosciences, University of California, San Diego, La Jolla CA, USA. 9 Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, USA. 10Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA. 11 Department of Biology, University of Virginia, Charlottesville, VA 22904-‐4328, USA. 13Diabetes, Endocrinology, and Obesity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA. 14National Institute of Drug Abuse, Baltimore, MD 21224, USA.

Feeding behavior is exquisitely regulated by homeostatic and hedonic neural substrates that integrate energy demand as well as the reinforcing and rewarding aspects of food. Understanding the net contribution of homeostatic and reward-‐driven feeding has become critical due to the ubiquitous source of energy-‐dense foods and the consequent obesity epidemic. Hypothalamic, agouti-‐related protein-‐secreting neurons (AgRP neurons) represent primary orexigenic drives of homeostatic feeding. Using a models of neuronal inhibition or ablation we demonstrate that the feeding response to a fast, ghrelin or serotonin receptor agonist relies on AgRP neurons; however, when palatable food is provided, AgRP neurons are dispensable for an appropriate feeding response. In addition, AgRP-‐ablated mice present exacerbated stress-‐induced anorexia and palatable food intake—a hallmark of comfort feeding. These results demonstrate that hedonic circuitry can solely operate feeding and override the homeostatic circuitry especially in conditions where positive response to energy demands is chronically defective.

P24: Palatable food regulates the activity of magnocellular oxytocin neurons in the supraoptic nucleus of the hypothalamus. Hume Catherine1, Sabatier Nancy1, Menzies John1 and Leng Gareth1. 1. Centre for Integrative Physiology, The University of Edinburgh, Edinburgh, UK.

Oxytocin is now considered to be a key player in the homeostatic control of food intake. It’s anorexigenic effects may be mediated partly through peripheral satiety signals regulating oxytocin-‐releasing neurons in the supraoptic (SON) nuclei of the hypothalamus. We have developed an in vivo model to study the activity of oxytocin-‐releasing SON and PVN neurons in response to the oral gavage of palatable food using c-‐Fos-‐like immunoreactivity and in vivo electrophysiology. Fasted animals were anesthetised, a feeding tube inserted into the stomach and 5 ml of sweetened condensed milk (SCM) infused over 40 min. Animals were perfused-‐fixed 1 hr post gavage and the brains processed for c-‐Fos-‐ and oxytocin-‐like immunoreactivity. Sham gavage acted as a control. For electrophysiology, the ventral surface of the brain was exposed by transpharyngeal surgery and a microelectrode inserted into the SON. Oxytocin cells were identified by their excitatory response to i.v. cholecystokinin. Extracellular recordings were made continuously during gavage. In response to SCM gavage, c-‐Fos-‐like immunoreactivity was strongly increased in oxytocin-‐positive cells of the SON (p < 0.05, Mann Whitney test; n = 6). Nine identified oxytocin neurons were recorded from the SON, seven showed a progressive increase in firing rate beginning within 10 min after start of gavage (p < 0.001, two-‐tailed paired t-‐test). These findings indicate that the activity of SON oxytocin neurons is regulated by gut-‐brain signalling triggered by delivery of palatable food into the stomach, providing further evidence for a role of magnocellular oxytocin neurons in the homeostatic control of food intake. Whether this response is specific to high-‐sugar foods will be the focus of future experiments. This research has received Funding from the European Union's Seventh Framework programme for research, technological development and demonstration under grant agreements 607310 (Nudge-‐it) and 245009 (NeuroFAST)


P25: A new role for Monocyte chemoattractant protein 1/CCL2 in promoting weight loss through inhibition of melanin-‐concentrating hormone-‐expressing neurons Le Thuc Ophélia (1,2), Blondeau Nicolas (1,2), Guyon Alice (1,2), Cazareth Julie (1,2), Rostène William (3), Heurteaux Catherine (1,2), Nahon Jean-‐Louis (1,2) and Rovère Carole (1,2). (1) Université de Nice Sophia Antipolis, IPMC, Sophia Antipolis, F-‐06560, France ; (2) CNRS, IPMC, Sophia Antipolis, F-‐06560, France ; (3) Institut de la Vision, UMRS 968-‐Université Pierre et Marie Curie, 17 rue Moreau, 75012 Paris, France. Injuries or infections induce endocrine, autonomic and behavioural changes known as “sickness behaviour”. Among them, fever and weight loss appear driven by hypothalamic cytokines, although the exact mechanism remains elusive. Our goal was to investigate the neuroimmunological events driving appetite and weight loss in systemic high-‐grade inflammation. To study the role of the hypothalamic inflammation in the inflammation-‐driven weight loss, we performed intracerebroventricular (ICV) injections of lipopolysaccharide (LPS) in C57Bl6/J male mice. Blood and cerebral tissues were collected: mRNA and protein levels of cytokines/chemokines and hypothalamic peptides involved in the regulation of food intake were measured by qPCR and ELISA/EIA/FACS array, respectively. The effect of inflammatory factors on neuropeptidergic systems involved in food intake was investigated via perifusion and electrophysiology experiments. A central injection of LPS provokes a temporal sequence linking activation of pro-‐inflammatory cytokines and chemokines (notably CCL2) to down-‐regulation of the orexigenic neuropeptide Melanin-‐Concentrating Hormone (MCH). CCL2 particular activation kinetics lead us to investigate whether CCL2 could mediate LPS effects. ICV-‐injected CCL2 triggers neuroinflammation, downregulation of MCH and weight loss. Furthermore, CCL2 reduces KCl-‐induced MCH release from perifused hypothalamic explants and hyperpolarizes MCH neurons. These effects are reversed by the CCR2 antagonist INCB 3344 and in CCR2-‐deficient mice. Finally, the demonstration that MCH neurons expressed CCL2 receptor confirms that CCL2 could act directly on MCH-‐neurons promoting inflammation associated weight loss. In conclusion, CCL2 appears as a major intermediate between cytokine-‐producing cells and neurons in the cascade linking inflammation and eating disorders as LPS-‐induced weight loss is mediated by CCL2 up-‐regulation through modulation of the MCH neuronal network

P26: Using a single cell model to explain oxytocin neurons ability to reliably report absolute long term levels of gut peptides involved in satiety. Maicas Royo, Jorge; MacGregor, Duncan; Leng, Gareth. University of Edinburgh

In analysing the firing patterns of oxytocin cells in the supraoptic nucleus (SON) we noticed an unexpected feature: the mean firing rate at large binwidths is much less variable than expected from the variability at small binwidths, implying a structure in their activity that “smooths out” perturbations in activity. We have been using computational modelling in an attempt to determine whether this feature can be explained by the after-‐hyperpolarising potential (AHP) and if the AHP’s role in oxytocin cells is thus to help produce a relatively stable firing rate. However, a model of oxytocin neurons with an AHP and hyperpolarising after-‐potential (HAP) was not able to give a good match to both this behaviour and the interspike interval distribution. Our new model solves this by adding equations for a fast depolarising after potential (DAP) and increasing the magnitude of the AHP. To test the role of the AHP with this new model, we matched recordings of five oxytocin cells exposed to apamin, a blocker of the AHP, at two concentrations. With the new model we are able to obtain good matches for the five cells under all conditions – baseline, apamin 1 and apamin 2 -‐ by varying only the AHP amplitude and the synaptic input rate for each condition. Apart from now having a very accurate model for oxytocin cells, we have identified the membrane properties that enable these cells to be very sensitive to small changes in inputs while still having a stable firing rate. This work was supported by Nudge-‐it (http://www.nudge-‐it.eu/) a research program that aims to better understand decision-‐making in food choice and to build predictive models to contribute to improving public health policy. Nudge-‐it is a European Commission-‐funded FP7 project.

P27: A potential role in motivated feeding behaviour for the rat supramammillary nucleus Menzies John, Hume Catherine, Plaisier Fabrice, Sabatier Nancy, Leng Gareth Centre for Integrative Physiology, University of Edinburgh, George Square, Edinburgh, UK The supramammillary nucleus (SuM) is a posterior hypothalamic region with a potential role in goal-‐oriented behaviour (Pan et al., 2004. The supramammillary area. Prog Neurobiol. 74:127). However, the SuM is understudied in the context of food reward. We hypothesised that motivated eating behaviour would be associated with activation of the SuM. We fasted male rats to increase their motivation for food and used c-‐Fos to map neuronal activity after refeeding with standard laboratory diet. Compared to rats fed ad lib, re-‐fed rats showed an increase in c-‐Fos expression in the SuM. Next, to determine the effects of motivation for palatable food, we conditioned satiated rats to consume 5 ml sweetened condensed milk (SCM) daily. Compared to control animals, we saw increased Fos expression in the SuM of rats receiving daily SCM access. Levels of Fos were increased yet further in rats that received SCM at a different time of day to that used during conditioning. We also used in vivo electrophysiology to characterise SuM neurones in anaesthetised satiated male rats. The majority of SuM cells showed a short-‐burst firing pattern while the remainder showed a more regular pattern. The stomach-‐derived orexigenic hormone ghrelin is involved in food motivation (Menzies et al., 2013. Ghrelin, reward and motivation. Endocr Dev. 25:101). We tested six SuM cells with 10 µg ghrelin iv. Five cells responded with an increase in the mean firing rate. These data indicate that the SuM is activated by behaviours associated with food motivation and is sensitive to ghrelin, a key reward-‐related signal. This research has received Funding from the European Union's Seventh Framework programme for research, technological development and demonstration under grant agreements 607310 (Nudge-‐it), 266408 (Full4Health) and 245009 (NeuroFAST).


P28: Glucose excites hypothalamic neurons through the activation of Transient Receptor Potential Canonical (TRPC) channels Chrétien, Chloé; Fenech, Claire; Grall, Sylvie; Pénicaud, Luc; Leloup Corinne & Fioramonti, Xavier Centre des Sciences du Goût et de l’Alimentation, UMR 6265 CNRS, 1324 INRA, Université de Bourgogne-‐Franche Comté The mediobasal hypothalamus (MBH) houses specific glucose-‐sensitive neurons able to sense changes in glucose levels which are suggested to participate in the control of food intake and glucose homeostasis. Glucose-‐excited (GE) neurons increase their electrical activity in response to increased glucose level. The molecular mechanisms involved in GE neurons response to glucose are not totally understood. In view of 1/ the role of Mitochondrial Reactive Oxygen Species (mROS) in hypothalamic glucose detection and 2/ the ROS sensitivity of some transient receptor potential canonical (TRPC) channels, we hypothesized that GE neuron detect increased glucose level through a ROS-‐TRPC dependent signaling pathway. To test this hypothesis, dissociated rat MBH cells activity in response to increased glucose, was monitored using Fura-‐2 calcium imaging in presence of TRPC channel inhibitors or antioxidants. Hypothalamic detection of hyperglycemia was also evaluated in vivo in models of TRPC3 deficient mice. Quantification of the area under the curve of GE neurons responses to glucose shows that MBH GE neuron responses to 2.5-‐10 mM increased glucose are inhibited by antioxidants (trolox/gluthation or catalase) or the non-‐selective TRPC channel inhibitor SKF96365. Glucose responses are also partially inhibited by the TRPC3 inhibitor Pyr3 or mimicked by the TRPC3 activator O-‐Acyl Glycerol. In vivo, pharmacological inhibition of TRPC3 channel specifically into rat MBH significantly decreases insulin secretion in response to intra-‐carotid glucose injection. Hypothalamic detection of increased blood glucose level is also impaired in whole-‐body TRPC3 deficient mice in which MBH GE neurons response to glucose is impaired. The selective inhibition of MBH TRPC3 expression is currently under investigation. Altogether, these data highlight a new ROS-‐TRPC3 channel dependent pathway involved in GE neuron glucose response and the central control of glucose homeostasis.

P29: The hypothalamic neuropeptide 26RFa acts as an incretin to regulate glucose homeostasis Picot Marie 1,3,4 ; Prévost Gaëtan 1,2,3,4; Jeandel Lydie1,3,4; Arabo Arnaud 3,4; Coëffier Moïse 3,4,5,6; EL Ouahli Mariama 1,3,4,7; Alexandre David 1,3,4; Leprince Jérôme 1,3,4; Berrahmoune Hind 2,3,4; Déchelotte Pierre 3,4,5,6; Chigr Fatiha 7; Lefebvre Hervé 1,2,3,4; Anouar Youssef 1,3,4; Chartrel Nicolas 1,3,4 1, INSERM U982, Laboratory of Neuronal and Neuroendocrine Differentiation and Communication, Institute for Research and Innovation in Biomedecine (IRIB), Mont-‐Saint-‐ Aignan, France. 2, Department of Endocrinology, Diabetes and Metabolic Diseases, Institute for Research and Innovation in Biomedecine (IRIB), University Hospital of Rouen, Rouen, France. 3, Normandy University, Caen, France. 4, University of Rouen, Rouen, France. 5, INSERM U1073, Institute for Research and Innovation in Biomedecine (IRIB), Rouen, France. 6, Department of Nutrition, University Hospital of Rouen, Rouen, France. 7, Biological Engineering Laboratory, Life Sciences, Sultan Moulay Slimane University, Beni-‐ Mellal, Morocco.

26RFa, a hypothalamic neuropeptide discovered by our team, was identified as the endogenous ligand of an orphan human receptor, GPR103. It strongly stimulates food intake. 26RFa is up-‐regulated in obese animal models and its orexigenic activity is accentuated in rodent fed a high fat diet, suggesting that this neuropeptide might play a role in the development and maintenance of the obese status. Obesity and type II diabetes are frequently associated. Their significant and synchronous progression for 30 years so that the pandemic "diabesity" currently observed worldwide, is a real public health issue. Recent studies revealed that neuropeptides known to play a crucial role in the hypothalamic control of feeding behavior are also expressed in pancreatic islets, suggesting that hypothalamic neuropeptides could provide the link between energy and glucose homeostasis, and constitute potential therapeutic targets for the treatment of obesity associated with type II diabetes. Indeed, our human studies showed a moderate positive correlation between plasma 26RFa levels and plasma insulin in diabetic patients. Plasma 26RFa concentration also increases in response to an oral glucose tolerance test. In addition, we found that 26RFa and its receptor GPR103 are present in human pancreatic β cells as well as in the gut. In this context, we investigated whether 26RFa may be involved in the regulation of glucose homeostasis in mice. We found that 26RFa attenuates the hyperglycemia induced by a glucose load, potentiates insulin sensitivity and increases plasma insulin concentrations. Consistent with these data, 26RFa stimulates insulin production by MIN6 insulinoma cells. Finally, we show, using in vivo and in vitro approaches, that a glucose load induces a massive secretion of 26RFa by the small intestine. Altogether, the present data indicate that 26RFa acts as an incretin to regulate glucose homeostasis.

P30: 5-‐HT2CR agonist obesity medication increases the activity of appetitive brain stem neurons Teodora Georgescu, Giuseppe D’Agostino, Raffaella Chianese, Celine Cansell, David Lyons and Lora K Heisler Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen, UK The brain plays an essential role in the regulation of food intake and energy balance. This vital regulatory process is coordinated via the interaction of numerous brain regions. Amongst these is the nucleus of the solitary tract (NTS), a brain stem region that integrates satiety signals from the gastrointestinal tract and related digestive organs. 5-‐hydroxytryptamine (5-‐HT; serotonin) is a neurotransmitter involved in the regulation of energy homeostasis mainly via its action at the 5-‐HT2C receptor (5-‐HT2CR). This receptor is amenable to pharmacological manipulation for obesity treatment, as illustrated by the new obesity medication lorcaserin, which is a 5-‐HT2CR agonist. We utilised immunohistochemistry (IHC) in a reporter 5-‐HT2CR-‐yellow fluorescent protein (YFP) mouse line to map 5-‐HT2CR distribution and characterise its expression within the NTS. We report that 5-‐HT2CRs are most abundantly expressed in the medial-‐caudal NTS, a sub-‐region expressing energy balance regulating pro-‐opiomelanocortin (POMC) neurons. We observed that 5-‐HT2CRs are anatomically positioned to influence the activity of POMC cells as NTS POMC cells express 5-‐HT2CR mRNA. Furthermore, we observed that 5-‐HT2CR agonist obesity treatment lorcaserin increases c-‐fos immunoreactivity (a marker for neuronal activation) in NTS POMC neurons. These findings reveal that 5-‐HT2CR agonist obesity treatment lorcaserin influences the activity of appetitive NTS POMC neurons and this may be a mechanism through which its therapeutic effect is achieved. Work was supported by the Wellcome Trust (WT09801) and BBSRC (BB/K001418/1)


P31: Hedonic sensitivity to natural rewards is affected by prenatal stress in a sex-‐dependent manner Jenny Cigalotti1,7, Marie-‐Line Reynaert1,7, Jérôme Mairesse1,7, Luana Lionetto2, Maurizio Simmaco2, Lucie Deruyter1,7, Delphine Allorge3, Anna Moles4,5, Anna Pittaluga6, Stefania Maccari1,7, Sara Morley-‐Fletcher1,7, Gilles Van Camp1,7*, Ferdinando Nicoletti7,8* 1Glycobiology of Stress-‐related Diseases team, UMR 8576 University of Lille1/CNRS, Villeneuve d’Ascq, France 2Advanced Molecular Diagnostic, Sant’Andrea Hospital, Italy; 3EA4483, University Lille 2, France; 4Institute of Neuroscience, National Research Council (CNR), Italy; 5Genomnia, Italy; 6Department of Pharmacy, University of Genoa, Italy 7International Associated Laboratory “Prenatal Stress and Neurodegenerative Diseases” France/Italy (Glycobiology of Stress-‐related Diseases team, UMR 8576 University of Lille1/CNRS, Villeneuve d’Ascq, France; Neuromed, Pozzilli, Italy and Sapienza University of Rome, Rome, Italy) 8IRCCS Neuromed, 86077-‐Pozzilli, Italy

Palatable food is a strong activator of the reward circuitry and may cause addictive behavior leading to eating disorders. How early life events and sex interact in shaping hedonic sensitivity to palatable food is largely unknown. We used prenatally restraint stressed (PRS) rats, which show abnormalities in the reward system and anxious/depressive-‐like behavior. Some of the hallmarks of PRS rats are known to be sex-‐dependent.We report that PRS enhanced and reduced milk chocolate-‐induced conditioned place preference in males and females, respectively. Male PRS rats also show increases in plasma dihydrotestosterone (DHT) levels and dopamine (DA) levels in the nucleus accumbens (NAc), and reductions in 5-‐hydroxytryptamine (5-‐HT) levels in the NAc and prefrontal cortex (PFC). In male rats, systemic treatment with the DHT-‐lowering drug finasteride reduced both milk chocolate preference and NAc DA levels. Female PRS rats showed lower plasma estradiol (E2) levels and lower DA levels in the NAc, and 5-‐HT levels in the NAc and PFC. E2 supplementation reversed the reduction in milk chocolate preference and PFC 5-‐HT levels. In the hypothalamus, PRS increased ERα and ERβ estrogen receptor and CARTP (cocaine-‐and-‐amphetamine receptor transcript peptide) mRNA levels in males, and 5-‐HT2C receptor mRNA levels in females. Changes were corrected by treatments with finasteride and E2, respectively. These new findings show that early life stress has a profound impact on hedonic sensitivity to high-‐palatable food via long-‐lasting changes in gonadal hormones. This paves the way to the development of hormonal strategies aimed at correcting abnormalities in the response to natural rewards.

P32: Circulating triglycerides interaction with mesolimbic structures regulate the rewarding and motivational aspects of feeding Chloé Berland (1), Céline Cansell (1,2), Julien Castel (1), Raphaël G. P. Denis (1) PhD, Anne-‐Sophie Delbes (1), Thomas S. Hnasko (3) PhD, Matthias H. Tschöp (4,5) MD, PhD, Serge Luquet (1)PhD (1)Univ Paris Diderot, Sorbonne Paris Cité, Unité de Biologie Fonctionnelle et Adaptative, CNRS UMR 8251, F-‐75205 Paris, France (2)Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen AB21 9SB, United Kingdom (3) Department of Neurosciences, University of California, San Diego, La Jolla CA, USA (4) Helmholtz Diabetes Center, Helmholtz Zentrum München, German Research Center for Environmental Health, München/Neuherberg, Germany (5) Div. of Metabolic Diseases, Dept. of Medicine, Technische Universität München, Germany

Obesity results from a disruption of energy balance, where energy input (food intake) is greater than energy expenditure. Hyperphagia associated to obesity involves an uncontrolled craving for a calorie rich diet similar to the compulsive consumption seen in drugs addiction. Rewarding aspect of food intake is tightly associated with the release of dopamine in the mesocorticolimbic (MCL) system. Obesity has been associated with specific defect in dopamine signaling pathway and the ability to encode reward. Hence, obesity-‐associated hyperphagia could be the result of altered dopamine signaling in the MCL and the development of uncontrolled craving for food reward. Triglycerides (TG) increase after a meal and are chronically elevated in obese. Lowering plasma TG can resorbe cognitive deficits associated with obesity, suggesting that TG might act in the brain. Interestingly, the MCL expresses several genes involved in TG metabolism, suggesting that TG sensing in the MCL might be involved in DA signaling regulation. This suggests that circulating TG could serve as a satiety signal by modulating reward processes in the MCL. Using a model that allows brain-‐specific delivery of TG through intra carotid catheter, we showed that circulating TG act upon MCL structures to control the motivational and rewarding aspect of food intake. In order to precisely decipher if TG-‐per se-‐could act as a drug of abuse and be a positive reinforcer, we developed a conditioned place preference protocole (CPP) in which animals associate one environment to a TG delivery. 4 conditioning sessions are sufficient to learn a positive conditioning for the compartment associated with brain-‐specific delivery of TG (average time in associated compartment = 185 seconds on a 30 minutes test, n=6). This result strongly suggest that nutritional TG can act on the reward circuitry as a natural reinforcer.

P33: Humanization of hepatocyte results in metabolic and circadian change in rodent. Delbès AS.1, Hubert M1, Denis RG1, Berland C1, Castel J1, Philippe E1, Martinez S1, Parini P2, Vedin LL2, Bail J3, Wilson EM3, Steffensen KR2 & Luquet S1. 1Université Paris Diderot, Sorbonne Paris Cité, Unité de Biologie Fonctionnelle et Adaptative, CNRS UMR 8251, F-‐75205 Paris, France. 2Division of Clinical Chemistry, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden. 3Yecuris Corporation, Portland, Oregon, United States of America.

Despite being very useful to identify genes and pathways involved in basic biological mechanisms, their evolutionary distance from humans and rodent has been a major limitation in the overall understanding of the disease. To overcome these limitations, The European consortium "Health and the Understanding of Metabolism, Aging and Nutrition" (HUMAN) has generated mouse models highly repopulated with human hepatocytes. Here we described for the first time the metabolic and behavioural response of animal repopulated with either murine (FRG-‐KO-‐Mur) or human hepatocyte (FRG KO-‐Hum) to a high fat high sucrose (HFHS) challenge. FRG-‐Hum exhibited a strong shift toward lipid-‐substrate oxidative preference as assessed by respiratory quotient without changing food intake and overall energy expenditure. In addition, FRG-‐Hum mice exhibit increased insulin sensitivity associated with low hepatic gluconeogenesis compared to FRG KO-‐Mur and complete resistance to the deleterious action of HFHS diet on glucose metabolism and insulin sensitivity. Analysis of circulating lipoproteins confirms a "humanization" of the lipid profile of FRG-‐Hum mouse with the appearance of triglyceride-‐rich lipoprotein (LDL / IDL) and provide a potential mechanism by which enhanced muscle lipid availability could redirect oxidative change. Finally, we observed that circadian distribution of feeding, metabolic and activity profile displayed a ~2hrs shift in FRG-‐KO-‐Hum compared to FRG KO-‐Mur suggesting an alteration in circadian rhythm. Change in feeding pattern was also associated with altered expression of hypothalamic orexigenic neuropeptide. Exposure to dark further confirmed that liver-‐borne inputs was sufficient to drive endogenous clock. These data reveals an important new mechanism by which liver-‐born inputs-‐presumably to the brain-‐could act as a dominant signals in the control of feeding behaviour and metabolism


P34: Chemerin modulates the control of feeding and hypothalamic remodelling in seasonal animals Helfer, Gisela 1; Stoney, Patrick 2; Ross, Alexander W 1; McCaffery, Peter J 2; Morgan, Peter J 1 1 Rowett Institute of Nutrition and Health, University of Aberdeen, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, Scotland, UK; 2 Institute of Medical Sciences, School of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, UK

Long-‐term and reversible changes in growth and feeding are characteristic of seasonal animals. These changes are triggered by photoperiod through melatonin and involve altered thyroid and retinoic acid signalling within the ependymal cells and tanycytes of the hypothalamus. Here we examined signalling downstream of retinoic acid, and investigated how it links to the control of growth and feeding in photoperiod-‐sensitive F344 rats. Using microarray analysis, we identified the inflammatory chemokine chemerin as a physiological effector downstream of retinoic acid regulated transcription in the hypothalamus. Chemerin is a recently discovered adipokine that is involved in energy metabolism but a central role for chemerin is not known. We show that Chemerin and its receptors, Cmklr1 and Ccrl2, are expressed in the ependymal cells and tanycytes of the hypothalamus and are under strong photoperiod regulation. Acute intracerebroventricular injections of chemerin results in a decrease in body weight and food intake, accompanied by a change in hypothalamic expression of neuropeptides that play a pivotal role in growth and feeding, such as growth-‐hormone-‐releasing-‐hormone (GHRH) and pro-‐opiomelanocortin (POMC). In contrast, chronic infusion of chemerin into F344 rats causes a significant increase in food intake after two weeks, together with changes in POMC mRNA expression thereby mimicking long day conditions. Hypothalamic re-‐modelling plays an important role in the photoperiodic response and here we show that Chemerin is involved in this seasonal brain plasticity, monitored by hypothalamic changes in the intermediate filament protein vimentin. Thus, we show that inflammatory signalling downstream of thyroid hormone and retinoic acid is involved in seasonal brain plasticity and reveal a novel pathway within the hypothalamus that contributes to the neuroendocrine control of seasonal physiology.

P35: Chronic over-‐expression of VGF in the hypothalamus leads to decreased bodyweight despite increases in food intake in Siberian hamsters and mice Lewis J.E, Brameld J.M, Hill P.J, Barrett P, Ebling F.J.P, Jethwa P.H Division of Nutritional Sciences, School of Biosciences, University of Nottingham Sutton Bonington Campus, Loughborough, LE12 5RD. The catabolic phenotype of mice lacking the functional vgf (non-‐acronymic) gene has not been supported by subsequent functional studies using VGF-‐derived peptides in both mice and Siberian hamsters, particularly in terms of energy balance. One theory is that in the null mice the gene is ablated in all areas not just the hypothalamus. To further elucidate the role of VGF in the hypothalamic regulation of energy balance, we designed a recombinant adeno-‐associated viral vector (rAAV) utilizing the novel viral 2A sequence for dual expression of VGF mRNA and enhanced green fluorescent protein (eGFP), which would not only overexpress VGF mRNA, but also allow visualisation of transfected neurones. Bilateral infusion of the rAAV vector containing VGF, the viral 2A sequence and eGFP into the hypothalamus of Siberian hamsters resulted in a significant decrease in bodyweight from 5 weeks post infusion, an effect which lasted for the duration of the study (12 weeks). By 12 weeks the VGF treated hamsters weighed 12% less than eGFP controls (p<0.001), despite an 8% increase in food intake per gram of bodyweight (p<0.001). The comprehensive laboratory animal monitoring system (CLAMS) revealed a significant increase in oxygen consumption and carbon dioxide at 12 weeks (p<0.05), but no effect on locomotor activity. There was also a significant increase in food intake during the dark phase without affecting the frequency of meals. A similar effect was observed in mice. Mapping of GFP expression at the end of the two studies confirmed widespread expression of both the control and VGF-‐encoding constructs within the hypothalamus and in-‐situ hybridisation confirmed that the localisation of VGF mRNA expression within the brain was similar to the eGFP expression pattern. In conclusion the rAAV with viral 2A sequence allowed long-‐term dual expression of both VGF and GFP in mice and Siberian hamsters, and the over-‐expression of VGF results in a catabolic phenotype.

P36: Hypothalamic lipoprotein lipase plays a role in the adaptation to cold exposure. Laperrousaz Elise (1), Denis Raphaël G. (1), Luquet Serge (1), Contreras Cristina (2), Lopez Miguel (2), Magnan Christophe (1), Cruciani-‐Guglielmacci Céline (1) (1) University Paris Diderot , (2) University of Santiago de Compostela-‐Instituto de Investigación Sanitari The effect of the utilization of the adipose tissue during a cold exposure is well characterized in the literature. However, there is few data concerning brain lipid metabolism in this adaptation to cold exposure. We exposed C57Bl6/J mice at 4°C for 4 hours, compared to a group at room temperature, and analysed the gene expression of enzymes involved in lipids metabolism by PCR, in different areas of the brain. In a second set of experiments, we focused on lipoprotein lipase (LPL) role in response to cold. We found that cold exposure modulates the expression of some lipases in hypothalamus (HT), hippocampus (HP) and striatum, especially the LPL, the key enzyme of the hydrolysis of the triglycerides from circulating lipoproteins. This enzyme is mainly expressed in HP and HT and we measured that its expression and enzymatic activity are strongly decreased in HT during cold exposure. Then, we tested the response to cold exposure on mice deleted for the LPL in the ventro-‐medial HT (VMH). We used mice floxed for the LPL gene and C57Bl6/J as control. The 2 groups were injected bilaterally in the VMH with an adeno-‐associated-‐virus expressing the Cre recombinase. We also implanted an intraperitoneal probe to measure the body temperature every 15 minutes by telemetry. 10 days post-‐injection, during cold exposure, LPL VMH -‐/-‐ showed a decrease of locomotor activity and energy expenditure without any change in respiratory exchange ratio, compared to WT. Interestingly, WT mice decreased their body temperature whereas LPL VMH -‐/-‐ succeeded to maintain theirs. Thermogenic images showed that the brown adipose tissue temperature was also maintained in LPL VMH-‐/-‐ whereas is decreased in WT. We concluded that the hypothalamic LPL plays a role in the response to cold exposure, by a modulation of energy expenditure and thermogenesis.


P37: The role of prenatal leptin in the development of parasympathetic innervation of pancreatic b-‐cells Croizier Sophie, Bouret G Sebastien The Saban Research Institute, Neuroscience Program, Children’s Hospital Los Angeles, USC, Los Angeles CA 90027, USA -‐ Inserm, Jean-‐Pierre Aubert Research Center, U837, University of Lille 2, Lille 59045, France Epidemiological and experimental data have suggested that changes in the hormonal environment during perinatal life can play a significant role in the development of obesity and related diseases. Here, we investigated whether the adipocyte-‐derived hormone leptin acts, during embryonic life, in the hindbrain to influence the development of the parasympathetic fibers to pancreatic b-‐cells. Parasympathetic axons containing the vesicular acetylcholine transporter (VAchT) begin to innervate insulin-‐producing cells in the pancreas at embryonic day (E) E12. The density of cholinergic fibers innervating insulin-‐containing cells increases markedly between E12 and E18. High levels of leptin receptor mRNA are found in the hindbrain as early as at E12, suggesting that prenatal leptin might play a role in hindbrain development. Consistent with this idea, a single intracerebroventricular injection of leptin in leptin-‐deficient embryos at E12 causes a reduction in the parasympathetic innervation of pancreatic b-‐cells and induces lifelong glucose intolerance. Furthermore, direct exposure of dorsal vagal complex explants to leptin blunts VAchT+ axon growth. These data indicate that a single injection of leptin during an important period of fetal hindbrain development causes long-‐term alterations in glucose homeostasis that likely result from disruption parasympathetic control of b-‐cell function.

P38: Circadian and metabolic disruption caused by prenatal inflammation may be partly restored by scheduled feeding. Velarde, Elena; Biscaia, Jose Miguel, Llorente de Miguel, Ricardo; Marco, Eva European University of Madrid. School of Biomedical and Health Sciences. Department of Basic Biomedical Sciences; Complutense University of Madrid. School of Biology. Department of Animal Physiology II

Prenatal inflammation is commonly used for the investigation of developmental psychiatric disorders such as schizophrenia or autism. One of the most common approaches, LPS injection to the dams, might induce disruptions on the offspring circadian system. Therefore, feeding time arises as a potential strategy to counteract some of the long-‐term effects of prenatal LPS administration. We assessed metabolic and circadian parameters in the female offspring of Sprague Dawley rats administered with LPS (1mg/kg, sc) or saline (Sal) during the second half of pregnancy (gd 11 to 21). From weaning to sacrifice (adulthood), females were fed ad libitum (AL) or a scheduled feeding (SF) regime (restricted temporal access for 5 hours after lights off). AL animals consumed more food than SF during the first 3 weeks; thereafter no differences were found between AL and SF animals. Only on the last weeks (8 and 9) did LPS-‐AL animals decrease their food intake. Additionally, LPS-‐AL weighted less than Sal-‐AL but LPS-‐AL exhibited the higher percentage of subcutaneous adipose tissue. Notably, SF decreased body weight, although among SF animals no differences were found between treatments (Sal vs. LPS). Besides, expression of the clock genes Per1 and Per2 was assessed in the hypothalamus. Samples from each group were extracted after decapitation of the animals every six hours for 24 hours, and then analysed by RT-‐PCR. Following beheading, blood samples were extracted from the trunk to determine corticosterone levels and rhythmicity. Rhythmic parameters form both gene expression and serum corticosterone also points to significant effects of scheduled feeding on the LPS offspring, being similar to non-‐treated groups. Present results suggest that SF may counteract some of the disruptions described following prenatal LPS administration, and therefore could be a potential strategy to ameliorate some symptoms related to neuropsychiatric disorder associated with maternal immune activation.

P39: Identification of a selective glucocorticoid receptor modulator that prevents both diet-‐induced obesity and inflammation J.K. van den Heuvel1,2, M.R. Boon1,2, I. van Hengel1,2, E. Peschier-‐van der Put1,2, L. van Beek2,3, V. van Harmelen2,3, K. Willems van Dijk2,3, H. Hunt4, J.K. Belanoff4, P.C.N. Rensen1,2, O.C. Meijer1,2 1Dept of Medicine, Div. of Endocrinology, Leiden Univ. Medical Center, Leiden, The Netherlands 2Einthoven Laboratory for Experimental Vascular Medicine, Leiden, the Netherlands 3Dept of Human Genetics, Leiden Univ. Medical Center, Leiden, the Netherlands 4Corcept Therapeutics, CA, USA

High-‐fat diet consumption results in obesity and chronic low-‐grade inflammation in adipose tissue. Whereas glucocorticoid receptor (GR) antagonism reduces diet-‐induced obesity, GR agonism reduces inflammation, the combination of which would be desired in a strategy to combat the metabolic syndrome. The aim of this study was to assess the beneficial effects of the selective GR modulator C108297 on both diet-‐induced weight gain and inflammation in mice, and to elucidate underlying mechanisms. 10-‐week old C57Bl/6J mice were fed a high-‐fat diet (60% energy from fat) for 4 weeks while being treated with the selective GR modulator C108297, a full GR antagonist (RU486/mifepristone) or vehicle. C108297 and, to a lesser extent, mifepristone reduced body weight gain and fat mass. C108297 decreased caloric intake and increased lipolysis in WAT and free fatty acid levels in plasma, resulting in decreased fat cell size and increased fatty acid oxidation. Furthermore, C108297 reduced pro-‐inflammatory M1 macrophage infiltration in WAT and reduced inflammation in the hypothalamus as measured by immunostaining for the microglia marker CD45. On the other hand, mifepristone increased energy expenditure as measured by fully automatic metabolic cages and enhanced expression of thermogenic markers in energy-‐combusting brown adipose tissue (BAT), but did not affect inflammation. In conclusion, C108297 attenuates obesity by reducing caloric intake and increasing lipolysis and fat oxidation, and in addition attenuates inflammation. These data suggest that selective GR modulation is a viable strategy for the reduction of diet-‐induced obesity and inflammation


P40: Mice lacking TLR4 or CD14 are not protected against high-‐fat diet induced obesity Dalby, Matthew; Ross, Alexander; Morgan, Peter. The Rowett Institute of Nutrition and Health

The gut microbiota has been proposed to increase body weight and body fat in high-‐fat diet fed mice through increased systemic exposure to low levels of gut derived bacterial lipopolysaccharide (LPS), defined as metabolic endotoxemia. Chronic low-‐grade inflammation resulting from LPS activation of Toll like receptor-‐4 (TLR4) is implicated as a mechanism linking the gut microbiota to body weight. The hypothalamus is a region of the brain where inflammation has been implicated in disrupting energy homeostasis. Hypothalamic inflammation due to systemic LPS potentially links the microbiota to increased body weight. This study investigated whether mice lacking TLR4, or its co-‐receptor CD14, are protected from diet induced obesity and hypothalamic inflammation. Mice were fed high-‐fat or low-‐fat diets and body weight, body composition, and food intake measured. LPS exposure was assessed via serum lipopolysaccharide binding protein (LBP). Denaturing gradient gel electrophoresis (DGGE) was used to assess differences in caecal microbiota. Hypothalamic gene expression was investigated using microarray. Mice lacking TLR4 or CD14 were not protected against obesity. There was no difference in high-‐fat diet induced increases in body weight and body fat in TLR4 or CD14 null mice compared to wild-‐type mice. Food intake was not different in high-‐fat diet fed TLR4 or CD14 null mice compared to wild-‐type mice. This is despite increased serum LBP in high-‐fat diet mice indicating increased systemic exposure to LPS. Genotype influenced caecal microbiota composition but this did not influence obesity susceptibility. Hypothalamus gene expression did not indicate increased inflammatory gene expression in either wild-‐type mice, or mice lacking TLR4, or CD14. Mice lacking TLR4 signalling are not protected against high-‐fat diet induced obesity. This study does not support the role for gut-‐derived LPS or TLR4 mediated hypothalamic inflammation as causes of increased body weight or body fat.

P41: The effect of RFamides on food intake in two different photoperiodic conditions in male and female Siberian hamster. Cazarez-‐Márquez, Fernando; Laran-‐Chich, Marie-‐Pierre; Kalsbeek, Andries; Simonneaux, Valérie Neurobiology of Rhythms Department, Institute of Cellular and Integrative Neurosciences, Strasbourg, France. Hypothalamic Integration Mechanisms, Netherlands Institute for Neurosciences, Amsterdam, The Netherlands Kisspeptin and RFRP-‐3 are two peptides that control the activity of GnRH neurons and modify reproductive activity. It has been demonstrated that over the different seasons photoperiod synchronizes reproductive and metabolic activities via melatonin. The photoperiodic regulation of reproduction involves an action of kisspeptin and RFRP on GnRH neurons. Interestingly, some of the areas in the mediobasal hypothalams that show increased c-‐FOS expression after acute central injections of kisspeptin or RFRP3 (PVN, DMH, and ARC) are also involved in bodyweight regulation and feeding behavior. On the other hand metabolic factors, like leptin and glucose, are also strongly involved in the regulation of reproductive activity over the year and recent data suggest that both RF-‐amide peptides may be also be involved in the regulation of these metabolic processes. The aim of this project is to delineate how RFRP is involved in the metabolic seasonal programming of male and female siberian hamsters. We analyzed if RFRP-‐3 has a role in the food intake behavior in the two photoperiods that could reflect the changes in the body weight associated to the seasonal changes, and we will delineate the central mechanisms involved in the observed effect of RFRP. Preliminary results indicate photoperiodic and sex-‐dependent effect of central administration of RFRP on food intake.

P42: The RFRP system in the female Syrian hamsters: photoperiodic and oestral regulation of the reproductive axis Henningsen, Jo B; Poirel, Vincent-‐Joseph; Mikkelsen, Jens D; Gauer, François; Simonneaux, Valérie 1. Neurobiology of Rhythms, Institute of Cellular and Integrative Neuroscience, CNRS/University of Strasbourg, Strasbourg,France. 2. Neurobiology Research Unit, Rigshospitalet, Copenhagen University Hospital, Denmark.

Hypothalamic RF-‐(Arg-‐Phe) related peptides (RFRP-‐1 and -‐3) are considered to play a role in the (seasonal) regulation of reproduction and their expression is down-‐regulated in short photoperiod. RFRP-‐3 stimulates reproductive activity in male Syrian hamsters; however the effects of the peptides depend on species and gender. In females, the control of reproductive activity is more complex and depends on proper integration of environmental as well as cyclic changes. This study aimed at investigating the RFRP system in female Syrian hamsters by determining the photoperiodic and oestral changes in the RFRP system as well as the effects of intracerebroventricular administration of RFRP-‐3. The expression of RFRP neurons as well as GPR147 mRNA is strongly down-‐regulated in short photoperiod and interestingly, the number of RFRP-‐positive fibers in the MPN/AVPV is higher only in short photoperiod adjusted females. Chronic intracerebroventricular administration of RFRP-‐3 decreases the gonadal size of sexually active female hamsters, whereas in SP-‐adapted females, RFRP-‐3 potently stimulates gonadal size. In sexually active females, the number of c-‐Fos activated RFRP neurons are reduced at the time of the LH-‐surge, where the activity of kisspeptin neurons in the MPN/AVPV is at the highest. In the female Syrian hamster, the RFRP system is strongly regulated by photoperiod and central administration of RFRP-‐3 has opposite effects depending on season. Interestingly, RFRP-‐3 reactivates the reproductive axis despite photoinhibitory conditions. All together our results suggest that RFRP neurons are important for the seasonal control of female reproductive activity and might play a key role in the integration of the photoperiodic input onto the reproductive axis. Moreover, our results suggest that the RFRP system is important for proper female cycling and generation of the pre-‐ovulatory LH surge, possibly through a direct action onto the kisspeptin neurons located in the MPN/AVPV.


P43: The effect of RFRP-‐3 on the mouse reproductive axis Ancel Caroline, Kim Joon, Inglis Megan, Anderson Greg Centre for Neuroendocrinology and Department of Anatomy, Otago University School of Medical Sciences, Dunedin, New Zealand In 2000, gonadotrophin-‐inhibitory hormone (GnIH) was discovered in birds and shown to inhibit gonadotrophin secretion (Tsutsui et al. 2000). The mammalian ortholog was concurrently discovered in humans and rats and termed RFamide-‐related peptide-‐3 (RFRP-‐3) (Hinuma et al. 2000). Since then, a number of studies have aimed at determining the involvement of RFRP-‐3 in the regulation of reproduction in various mammalian species, including rats (Johnson et al. 2007, Murakami et al. 2008, Pineda et al. 2010), hamsters (Kriegsfeld et al. 2006, Ancel et al. 2012, Ubuka et al. 2012), sheep (Clarke et al. 2008, Sari et al. 2009, Caraty et al. 2012), and cattle (Kadokawa et al. 2009). This plethora of results has underlined the species-‐ and sex-‐specific effects of RFRP-‐3 on the reproductive axis, through the analysis of LH section in most cases. However, a comprehensive investigation of the role of RFRP-‐3 in the regulation of the mouse gonadotrophic axis remains to be carried out, in part because of the difficulty in taking repeated LH measurements from this species. In this study, we performed an extensive analysis of the effects of RFRP-‐3 on LH secretion in male C57BL/6 mice. Using a repeated tail tip blood sampling method combined with a highly sensitive ELISA, RFRP-‐3 dose-‐dependently stimulated LH secretion in mice when injected centrally (0.5-‐5 nmol/mouse), but had no effect when administered peripherally (5-‐50 nmol). In cultured HEK293 cells transfected with the human KISS1R, RFRP-‐3 showed some affinity to KISS1R and was able to potentiate the effects of kisspeptin, but did not show agonism alone. This potentiating effect may occur through allosteric modulation, although further studies are required. To conclude, our results show that RFRP-‐3 can stimulate the reproductive axis in male mice, as has been shown in hasmsters (Ancel et al. 2012, Ubuka et al. 2012). Thus, the idea that RFRP-‐3 and kisspeptin exert opposing effects is likely to be overly simplistic.

P44: Winter Is Coming: Linking Seasonal Cues to Reproduction in the Mouse Beymer, Matthew; Sáenz de Miera, Cristina; Simonneaux, Valerie University of Strasbourg, Strasbourg, France The Arginine-‐Phenylalanine-‐amide (RF-‐amide) family is a class of neuropeptides which is implicated in many physiological processes, including stress, metabolism, and reproduction. Two members of this family, in particular, regulate the hypothalamic-‐pituitary-‐gonadal (HPG) axis. Kisspeptin is expressed by neurons in the arcuate and medial preoptic nuclei. Release of kisspeptin can strongly activate the HPG axis. RF-‐amide related peptides (RFRP) are expressed in neurons located in and around the dorso-‐ and ventro-‐medial hypothalamic nuclei. The roles of RFRP seem to be species-‐, sex-‐, and photoperiod-‐dependent. The central pathways by which seasonal cues regulate reproduction are still unknown; however recent findings suggest that RFRP and kisspeptin may be involved. In order to elucidate the pathways involved in the transduction of seasonal cues to the HPG axis we are using three strains of mice with different melatonin profiles: C57BL/6, melatonin-‐deficient, CBA and MsM/MS, both melatonin-‐proficient. We placed males of all strains in either short day (SP) or long day (LP) conditions to determine the effects of melatonin on RF-‐amides in mice. Both CBA and C57 males showed no changes in body, paired testes, or seminal vesicle weight in response to different photoperiodic conditions. Interestingly, SP CBA show a decrease in Rfrp expression as compared to LP CBA, whereas there was no change in Rfrp expression in C57 in either photoperiod. This is in concordance to what is found in classical seasonal breeders adapted to SP where hypothalamic Rfrp expression is reduced. It is clear from these findings that the RFRP system in mice is still sensitive to seasonal cues in the form of melatonin. Furthermore, since we do not see a response of the testes in CBA due to changing photoperiods, it is tempting to conclude that another central factor, a likely candidate being kisspeptin, in non-‐seasonal breeders is blocking the full transduction of seasonal cues to the HPG axis. P45: Siberian hamster genome reveals novel mechanisms for neuroendocrine plasticity. Riyue Bao1, David G. Hazlerigg2, Perry Barrett3, Brian J. Prendergast4 & Tyler J. Stevenson5 1Center for Research Informatics, University of Chicago, 2Dept. Arctic and Marine Biology, University of Tromso, 3 Rowett Institute, University of Aberdeen, 4Institute for Mind and Biology, University of Chicago, 5Institute for Biological and Environmental Sciences, University of Aberdeen. Siberian hamsters have a strong tradition for studies that examine seasonal neuroendocrine plasticity due to the robust and naturally-‐occurring variation at multiple levels of analysis—from genomic to behavioural. Molecular and cellular advances have been stymied due to a lack of an available genome. Using next generation sequencing, we have sequenced the genome of the Siberian hamster (Phodopus sungorus). Genome comparisons with other high and low amplitude seasonal species reveal genome regions that may have been selected for robust circannual rhythms. Subsequent RNA sequencing of hypothalamic tissue from summer (LD) and winter (SD) phenotypes revealed extensive plasticity in both coding and non-‐coding transcripts. Remarkably, almost 80% of all transcripts that exhibited a significant change in expression were non-‐coding. mRNAs for several known ‘seasonal genes’ exhibited the anticipated photoperiodic changes in expression (e.g., increased dio3 mRNA in SD). Follow-‐up qPCR analyses were used to confirm photoperiod-‐ and melatonin-‐dependent regulation of the expression of select non-‐coding RNAs. The data suggest that non-‐coding RNAs may play a role in molecular mechanisms of neuroendocrine plasticity and afford novel insights into the complex nature of plasticity in non-‐coding RNA signaling in adult mammalian neuroendocrine systems.


P46: Ovine melatonin receptors (MT1 and MT2): Functional activity and potential role in the pineal gland Lépinay Julie, Gennetay Dominique, Delagrange Philippe*, Taragnat Catherine and Bozon Véronique UMR 7247 " Physiologie de la Reproduction et des Comportements" (INRA/CNRS/Université François-‐Rabelais Tours/IFCE); * Institut de Recherches Servier, 78290 Croissy-‐sur-‐Seine Melatonin (MLT) is synthetized in pineal gland and binds on MT1 and MT2 receptors. The aim of our work is to study functional properties of MT receptors and their role in ovine pineal gland. Transduction pathways induced by MLT were studied on primary culture of ovine pinealocytes. The role of MT receptors on MLT synthesis was analyzed with Luzindole (MT inverse agonist). The level of MT receptors expressed and binding affinity were studied with 2-‐[125I]–iodoMLT (MT agonist) and [125I]-‐S70254 (MT2 antagonist) according to the seasonality on pineal gland. The internalization mechanism was analyzed on pineal gland and pars tuberalis which doesn’t synthetize MLT. In pinealocytes, 10-‐7M MLT increases the amount of pErk1/2 which is inhibited by PTX (pertussis toxin). This result suggests an activation of this MAP kinase pathway by MLT through Gi-‐protein. During the estrus (September to February) and anoestrus (May to August), the number of MT receptors expressed (20±6.9 fmol/mg in estrus vs 23.7±4fmol/mg in anoestrus) and the binding affinity (2.24±1.11nM vs 1.7±0.4nM) don’t significantly vary. However, in the transition period (March-‐April), the number of MT receptors decreases (6.25±0,6fmol/mg) and the binding affinity is stronger (0.67±0,11nM). With the two radioligand assays, the proportion of MT1 and MT2 receptors could be estimated during the estrus, 70% MT1 and 30% MT2 receptors. MT receptors are able to be internalized rapidly (5min). The maximum level is reached after 10-‐15min of incubation. In transition period, internalization rate is similar between pineal gland and pars tuberalis (30-‐35%). This rate decreases in anoestrus (7%) for pinealocytes and increases in estrus for pars tuberalis (70-‐80%). Finally, Luzindole increases MLT secretion suggesting an inhibitory role of MT receptors on MLT synthesis in pinealocytes. In conclusion, MT receptors are functional in pineal gland, able to transduce an intracellular signal and regulate negatively MLT synthesis.

P47: Mu and delta opioid receptor autoradiographic binding in brains of a mouse model of autism Pantouli, Fani; Hourani, Susanna; Bailey, Alexis University of Tours

Autism Spectrum Disorders are a family of neurodevelopmental disorders that affect 1% of the general population. They are characterised by deficits in social communication and interaction, repetitive and restricted behaviour and difficulty in inferring others’ emotions and beliefs. There is no established pharmacological treatment for the core symptoms and the aetiology of the disorders remains largely unknown. Recent evidence has demonstrated an important involvement of the endogenous opioid system in the neurobiology of autistic like traits. In order to further investigate the involvement of the endogenous opioid system in autistic like disorder, we carried out Mu and delta opioid receptor autoradiographic binding in brain sections of a genetic mouse model of autistic-‐like behaviour, the Fmr1 KO mice. Mice lacking the so called Fmr1 gene (FMR1 protein expressed ubiquitously in the brain and appears to be involved in the development of synapses and the regulation of synaptic plasticity) is considered a good animal model for the human disorder Fragile X syndrome, a neurodevelopmental syndrome that includes autistic symptoms. Full autoradiographic mapping of Mu and delta opioid receptors in the brains of WT and Fmr1 KO mice showed no alterations of either of the receptors in any of the regions analysed. These findings show a lack of endogenous opioid dysregulation in this particular model of fragile X at least at the receptor level.

P48: Anti-‐opioid effects of RFamide related peptide-‐3 and reversal of morphine tolerance using a novel antagonist. Kim Joon, Brown Colin, Anderson Greg

Centre for neuroendocrinology, University of Otago Agonists of the neuropeptide FF receptors (NPFFR1 and NPFFR2) have been generically termed “anti-‐opioids” for their putative ability to block opioid function. However in vivo evidence for this has been limited to nociceptive tests, which are confounded by the pronociceptive effects of the NPFFR ligands. To elucidate the functions of the NPFFRs, we first identified and characterised a true, potent, and selective antagonist, called GJ14. Next, we used the vasopressin neurons of the supraoptic nucleus as a model to examine the anti-‐opioid function of the NPFFR ligand, RFamide related peptide-‐3 (RFRP-‐3). In extracellular single-‐unit recordings from urethane-‐anaethetised rats, the spontaneous firing rate of vasopressin neurons was significantly reduced by morphine (i.v. 30 ug/kg). This inhibition was virtually abolished by pretreatment with RFRP-‐3 (i.c.v. 12 nmol) and morphine sensitivity was recovered 10 min after RFRP-‐3 treatment. Control rats receiving 3 consecutive morphine treatments alone did not show any change in morphine sensitivity. RFRP-‐3 alone had no effect on vasopressin neuron firing rate. A challenging notion is that chronic opioid treatment triggers the upregulation of these anti-‐opioid systems, which in turn attenuates the effect of morphine, thereby producing tolerance. To test this hypothesis, rats were given a continuous dose of morphine (10 mg/kg/day) via osmotic mini pumps for 6 days. Vasopressin neuron reponses to morphine (i.v. 30 ug/kg) were virtually absent in morphine-‐infused rats, confirming morphine tolerance. Pretreatment with GJ14 (i.c.v. 50 nmol) increased the sensitivity to morphine in vasopressin neurons of tolerant rats. In summary, this is the first evidence demonstrating an anti-‐opioid function in vivo using electrophysiology. Furthermore using our novel antagonist, we report convincing evidence that the NPFFRs are an important part of a genuine anti-‐opioid system that regulates opioid sensitivity.


P49: Oxytocin efficacy in treating detoxified opioid dependent individuals. A randomised double blind placebo controlled pilot trial. Weber, Carol, Robinson Fiona, Koumtsidis Christos, Boyle Julia, Revell Vicky, Hourani Susanna, Bailey Alexis University of Tours The major problem for opioid dependent individuals who are recovering from their addiction is the maintenance of a drug-‐free state, with only 40% maintaining abstinence 1 month post detoxification. People dependent on opioids undergoing detoxification suffer from persistent emotional withdrawal symptoms which can serve as a motivational trigger to re-‐administer the drug and relapse. These symptoms associated with emotional distress and dysphoria such as anxiety, irritability, stress, depression may persist for months in recovering opioid addicts with 30%-‐50% comorbidity of depression/anxiety. Moreover, the adverse social consequences of drug withdrawal in recovering addicts has been recognised, especially in light of the benefits that psychosocial support has in maintaining abstinence in addicts. As opioid substitution pharmacotherapy, benzodiazepine based anxiolytics and antidepressants have limited benefit in term of relapse prevention and induce dependence in their own right, there is a need to develop more effective, safer treatment for emotional opioid withdrawal symptoms which would assist with relapse prevention. The study will test the efficacy of intranasal oxytocin (Syntocinon) administration and assess if a) Detoxified opioid dependent individuals subjected to intranasal oxytocin administration for 2 weeks will exhibit decreased rates of relapse and higher retention rates in an in-‐inpatient rehabilitation programme as well as following 30 days, compared to those subjected to intranasal placebo control. b) Intranasal oxytocin administered for 2 weeks will reduce anxiety, depression, social anxiety and sleep disturbances in these detoxified opioid dependent individuals compared to placebo control group. c) These post detoxified individuals subjected to intranasal oxytocin will have reduced cortisol levels compared to placebo control group.

P50: Adiponectin : a Key player in the antidepressant effects of enriched environment Nicolas Sarah, Veyssiere Julie, Gandin Carine, Zsürger Nicole, Pietri Marielle, Heurteaux Catherine, Glaichenaus Nicolas, Petit-‐Paitel Agnès, Chabry Joëlle Université de Nice Sophia antipolis, Institut de Pharmacologie moléculaire et cellulaire, CNRS

Major depression is a complex disorder characterized by cognitive impairments triggered by various factors including stress and environment. Many patients are resistant to current antidepressant therapy, thus alternative strategies are needed. "Positive" life experiences could help to remission of the disorder by alone or in association with antidepressant drugs. However, little is known about the molecular mechanisms involved during high levels of sensory, motor, social stimuli, such as those experienced by mice housed in an enriched environment (EE). The aim of the study was to fully characterize the antidepressant-‐like effects of EE in a well-‐known murine model of depression-‐like behavior induced by long-‐term administration of corticosterone. We showed that EE efficiently reverses the anxiety/depression-‐like state of mice assessed through behavioral tests requiring both neurogenesis-‐dependent (Novelty Suppressed Feeding, Learned Helplessness) and -‐independent (Open Field, Light and Dark, Forced Swimming test) mechanisms. The contribution of this latter pathway remains largely unexplored; however we shown here that the adipokine, adiponectin is an absolute requirement for its establishment. Indeed, EE prevented the anxiety/depression-‐like state of adiponectin knock out (adipo-‐/-‐) mice in some but not all behavioral tests. Our data suggest that the environmental conditions of life may favor the passage of the adiponectin from the blood to the brain rather than change the circulating levels of adiponectin. Our findings bring insight into the beneficial effects of "positive" life experiences in anxiety/depression-‐related behaviors and highlight the pivotal role of adiponectin

P51: Role of CD4+ T cells in beneficial effects of enriched environment on hippocampal plasticity in mice Zarif Hadi, Nicolas Sarah, Hosseiny Salma, Petit-‐Paitel, Heurteaux Catherine, Chabry Joëlle, Guyon Alice CNRS/IPMC -‐ Université de Nice Sophia antipolis

The importance of environment in the regulation of brain functioning, behavior and physiology has long been recognized in biological, social and medical sciences. Animals maintained under enriched housing conditions have been shown to have better learning abilities than those maintained under standard conditions. We have shown that raising mice 4 weeks in an enriched environment (EE), characterized by bigger cages with running wheels, toys and mazes and increased number of mice per cage to encourage social interactions, increases neurogenesis in the dentate gyrus and induces changes in neuronal morphology and synaptic plasticity in the CA1 region of the hippocampus, a structure of the brain with a major role in learning and memory. The immune system is primarily involved in the surveillance of body tissues and protection from infectious agents and various forms of injury but recent findings indicate that it can also be involved in normal neurobehavioral processes. In addition, immune system can be affected by EE. Other groups have already shown that CD4+ T cells depletion affects memory, learning, LTP and neurogenesis but nobody has ever investigated the role of CD4+ T cells on the effect of EE. In this study, we investigated whether CD4+ T cells and among them CD25+ T cells are involved in this reshaping of the hippocampus after EE. We show that deprivation of mature T cells (CD4+ or CD25+) in adult mice using selective depleting antibody reverses some but not all of the effects of EE on hippocampal plasticity. Indeed, while the increase in spontaneous postsynaptic excitatory currents and the decrease in long term potentiation (LTP) normally induced by EE at the CA3-‐CA1 synapse were reversed, the increase in neurogenesis in the dentate dyrus was unaffected. This suggests that T cells are necessary specifically to some of the effects of EE. The mechanisms by which T cells impact the hippocampus are currently under investigation.


P52: Neuronal and synaptic ultra-‐structural organization in layer 5 of the human Gyrus temporalis. Yakoubi R1,3, Mosbah R1,2, Benmouloud A1 , Rollenhagen A3, Lübke JHR3,4,5,6 1Department of Biology, Faculty of sciences-‐ University of Boumerdes; 2Laboratory of Animal Eco –Biology, ENS-‐Kouba-‐ Alger, Algeria; 3 Institute of Neuroscience and Medicine (INM-‐2), Research Center Jülich GmbH, Leo-‐Brand-‐Str, 52425 Jülich, Germany; 4 School of Biomedical Sciences, University of Ulster, Cromore Road, BT52 1SA, Co. Londonderry, UK; 5 Department of Psychiatry and Psychotherapy, Medical Faculty, RWTH/University Hospital Aachen, Pauwelstr. 30, 52074 Aachen, Germany; 6JARA Translational Brain Medicine, Germany.

Synapses are the key elements for signal transduction and plasticity in the brain. Despite a relatively large number of assumptions that have been made on the structure mature cortical synapses in many other animal species, little is known about these structures in human. Here, synapses in cortical layer 5, the main recipient layer of thalamo-‐cortical efferents, therefore representing the first station of cortical information processing, were investigated quantitatively using serial ultrathin sections and digital electron microscopic images and immunohistochemistry against glutamine synthetase. 3D-‐reconstruction is the only way to describe these structures in great details to provide adequate morphological and quantitative data for realistic structural models of cortical synapses which could be than used for numerical and/or Monte Carlo simulations of synaptic parameters still inaccessible to experiment. These structural parameters such as the size, number and distribution of active zones and the size and organization of the pools of synaptic vesicles are critical factors not only for the induction but also for the maintenance of synaptic transmission and plasticity in the neocortex. The quantitative 3D-‐reconstructions of cortical synapses will allow to directly comparing structural and functional aspects of synaptic transmission and plasticity thus leading to a better understanding of the function of cortical networks in the human neocortex. We observed large pyramidal neurons (85%); the remainder is GABAergic inter neurons and astrocytes. We found multiple innervations on either the same or different dendrites. 85% of the spines have a spine apparatus, a specialized form of endoplasmic reticulum. Perforations exist in the pre and post-‐synaptic densities; we also found multivesicular bodies as well as mitochondria (2-‐8) in the pre-‐synaptic element

Poster Session 2—Friday, September 25

P1: Multi-‐level monitoring of cellular responses of corticotrophs to secretagogue stimuli Romanò Nicola*, Walker Jamie#, Le Tissier Paul*, Shipston Mike*

* University of Edinburgh, # University of Exeter The hypothalamic–pituitary–adrenal axis is central for the regulation of stress responses in the organism. Corticotrophs in the pituitary gland control adrenal release of corticosteroids through ACTH release, in response to CRH/AVP hypothalamic stimuli. We are using primary dispersed pituitary cells and organotypic slice cultures to study the cellular and molecular correlates of these responses. Intracellular calcium levels are used as a proxy of cellular activity, and monitored by specifically expressing the calcium indicator GCaMP6s in corticotrophs using a lentiviral approach. This allows monitoring of the system for several hours, and determination of the cellular responses to different concentrations and patterns of hormonal stimulations. Furthermore, simultaneous activation of intracellular pathways through optogenetic tools (e.g. channelrhodopsin 2, the photosensitive adenylate cyclase bPAC or light activated GPCR Jellyop), allows precise activation of different pathways in the cell. The long-‐term and population responses of these stimulations can also be analysed by monitoring activation of CREB or by real-‐time monitoring of cAMP levels through a luminescent cAMP sensor. These data can then be fed to a mathematical model, which allows prediction of cellular responses that can be then further tested experimentally.

P2: A Luminescence-‐Based Toolkit To Bioassay Endocrine Cell Function Romanò, Nicola, Shipston, Mike and Le Tissier, Paul Centre For Integrative Physiology, University of Edinburgh, United Kingdom In all neuroendocrine axes, the precise amount of hormone produced and its specific pattern of release in response to stimulation are influenced by a complex interplay between multiple signals. Because these responses are dynamic over multiple time-‐scales, it is important to develop systems capable of quantifying these signals over extended periods with high time resolution. We are developing several luminescence-‐based biosensors which allow quantification of receptor activation (and thus bioactive hormone concentration), monitoring of the dynamics of resulting intracellular pathways activation and measurement of secreted hormone output. We are currently testing tools for measuring GHRH, SRIF, CRH and TSH, either through a split-‐luciferase approach, or using a luminescent cAMP probe. Tools for monitoring cellular pathways include the cAMP probe, CRE-‐luciferase to monitor CREB activation and secreted luciferase to monitor cell secretion. All of these constructs can be delivered either through transfection or lentiviral transduction of cell lines, tissue slices and cells in living animals. Since these tools are not based on the irreversible binding of hormones to antibodies, they offer the basis for dynamic measurements and future incorporation in flow cells or in vivo applications. Furthermore, being based on luminescence, they can be easily coupled with fluorescence markers, such as calcium indicators or optogenetic tools that can be used to precisely control the system.

P3: Disruption of the HPA axis by vitamin A is through direct action on the adrenal gland Stoney, Patrick, Bowman, Ellen, McCaffery, Peter

Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, UK The vitamin A-‐derived hormone retinoic acid (RA) is known to affect the activity of the hypothalamic-‐pituitary-‐adrenal (HPA) axis, but the sites of its action are not clear. Retinoic acid regulates transcription by binding to specific nuclear receptors (retinoic acid receptors, RARs). To investigate the mechanism by which RA affects the HPA axis, we examined the short-‐term effects of RA on transcription in the hippocampus, hypothalamus, pituitary and adrenal gland. 8-‐week-‐old rats were injected with 10mg/kg RA once a day for 3 days before sacrifice. Expression of genes important for HPA axis regulation was analysed by qPCR. 3 days of RA treatment did not significantly alter expression of the investigated genes in the hippocampus, hypothalamus or pituitary; expression of the RA target Rarb was upregulated in tissues from RA-‐treated rats, confirming the presence of functional RA. However, after 3 days of RA treatment, expression of Cyp11b2 (aldosterone synthase) was significantly decreased in the adrenal gland (P=0.003). Expression of Cyp11b1 (11-‐β-‐hydroxylase) was increased in the adrenals of RA-‐treated rats though not statistically significant (P=0.058). Decreased aldosterone synthase combined with elevated 11-‐β-‐hydroxylase may be expected to increase corticosterone synthesis. Cyp11b2 was also rapidly downregulated 5 hours after a single RA injection (P=0.018). This rapid effect of RA on Cyp11b2 was reproduced in short-‐term cultures of isolated adrenal glands treated with RA for 5 hours, suggesting a direct effect of RA in the adrenal gland itself. A 5-‐hour RA treatment, whether in vivo or in vitro, had no effect on expression of Cyp11b1. Rapid changes in gene expression, which may result in dysregulation of the HPA axis, suggest that the adrenal gland is a primary site of action for RA. Further studies are needed to investigate if the transcriptional effects of RA in the adrenal gland correlate with changes in corticosterone levels and behavioral changes


P4: The role and regulation of the adrenal cortex circadian clock Dumbell, Rebecca; Leliavski, Alexei; Oster, Henrik. Chronophysiology Group, Medical Department I, University of Lübeck, Lübeck, Germany The adrenal gland plays an integral role within the HPA axis, with glucocorticoid being secreted from the adrenal cortex with circadian and ultradian rhythmicity, important in the regulation of stress response. A further role of glucocorticoids is the proposed synchronisation of peripheral clocks in other tissues, downstream of SCN mediated signals, and thus the regulation of this rhythm is of particular interest in circadian physiology. The sensitivity of the adrenal cortex to ACTH stimulated glucocorticoid secretion is considered to be under the control of the local circadian clock, and mice lacking a functional circadian clock (Bmal1-‐/-‐) have severely dampened corticosterone rhythms and an impaired behavioural stress response, with adrenal explant cultures showing impaired response to ACTH. However the specific importance of the local circadian clock in the regulation of glucocorticoid rhythm is not entirely clear. We have characterised a cre-‐loxP-‐mediated conditional knockout of Bmal1 in the adrenal cortex, under the promoter Cyp11a1, confirming loss of BMAL1 immunoreactivity, and saw severely dampened or absent expression rhythms of core clock genes, clock output genes, ACTH receptor Mc2r and the key steroidogenesis limiting StAR, in the adrenal cortex of these mice. However the circadian rhythm of glucocorticoid, whether excreted or in circulation, does not appear altered and the behavioural stress response remains intact. In conditional knockout mice expressing a PER2::LUCIFERASE reporter we were able to measure circadian rhythmicity of explant adrenal tissue, revealing intact rhythms in whole adrenals, but a loss of rhythm in adrenal cortex alone. This suggests a role of the adrenal medulla to drive cortex rhythmicity upon loss of the local clock.

P5: IL-‐6 alters pSTAT3 signalling in the murine adrenal gland Tranter, Danielle E., Bunn, Stephen J. Department of Anatomy, University of Otago Immune challenge is associated with both the sympathomedullary and the hypothalamic adrenal (HPA) responses to stress. Previous research from our lab has shown that treatment with the pro-‐inflamatory cytokine interleukin-‐6 (IL-‐6) induces an upregulation of neuropeptides and enzymes involved in catecholamine synthesis in isolated adrenal medullary chromaffin cells. IL-‐6 has also been shown to modulate the HPA axis, altering glucocorticoid production from the adrenal cortex. The aim of the current study was to examine the murine adrenal response to IL-‐6 in vivo. Male mice were administered 1μg IL-‐6 by i.p injection, and were deeply anaesthetised and perfused with 4% paraformaldehyde 10, 30 or 60 minutes later. To investigate the effects of endogenous cytokines on adrenal function, an additional group of male mice were treated with 10 μg/kg lipopolysaccharide (LPS) for 3h prior to perfusion. The adrenal glands were removed, and 10μm sections cut and processed for immunohistochemistry. IL-‐6 treatment resulted in an increase in phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) immunoreactivity in the adrenal cortex compared to saline only controls after 10 min (p<0.05), with the response most apparent in the region adjacent to the medulla. An IL-‐6 response was also seen in the medulla, however dual-‐labelling showed that the majority of chromaffin cells were unaffected. In contrast, LPS treatment induced a dramatic increase in pSTAT3 in the adrenal medullary chromaffin cells (p<0.005). A pSTAT3 response could also be seen in the cortex (p<0.005), however the distribution of staining was distinctly different from the IL-‐6 treated tissue, with the bulk of immunoreactivity seen in the region bordering the outer capsule. Further experiments are underway using LPS alongside an IL-‐6 blocking antibody to determine whether this increase in pSTAT3 can be attributed to IL-‐6.

P6: Regulation of secretion in pheochromocytomas by microRNAs Quillet Aurélie, Tellier Marie-‐Capucine, Cartier Dorthe, Bérard Caroline, Vergne Nicolas, Courel Maïté, Yon Laurent, Caron Philippe, Tabarin Antoine, Anouar Youssef, Dubessy Christophe Inserm, France Pheochromocytomas are tumors of the adrenal medulla known for their hypersecretion of catecholamines which are responsible for numerous negative side effects (symptomatic pheochromocytomas, SP). In order to identify the molecular regulators of this hypersecretion, we study a specific form of pheochromocytoma that secretes physiological levels of catecholamines (Normotensive Incidental Pheochromocytomas, NIP). We are particularly interested by microRNAs (miRs) whose role has been shown in cancers. MiRs are non-‐coding small RNA that regulate transcription by fixing mRNA. We used molecular biology (qRT-‐PCR), statistics (Limma) and bioinformatics (miRabel, GeneCodis3) methods on 32 samples of pheochromocytoma (SP and NIP) to identify miRs and their putative mRNA targets. Experimental validations are done by qRT-‐PCR, western blot and luciferase reporter assay. Seven miRs have been shown to be differentially expressed (hsa-‐miR-‐7-‐1-‐3p, 7-‐2-‐3p, 497-‐3p, 32-‐5p, 190b-‐5p, 26a-‐1-‐3p et 550a-‐3p) between SP and NIP tumors. These miRs potentially regulate several hundreds of pathways within which 10 seems particularly affected (Wnt, MAPK, Ubiquitinated proteasome, Cell cycle, Axon guidance, Insulin, Cell-‐matrix adhesion, Cell-‐cell adhesion, Actin cytoskeleton regulation and SNAREs). The targets PAK3, MLCP, MLCK (Actin cytoskeleton), SNAP25 and STX1A (SNAREs) were selected to be experimentally validated on the basis of their selection score (according to miR’s expression, number and up or down-‐regulation) and their function in the regulation of catecholamines’ secretion. Functional tests for the selected interactions are still in progress. However, preliminary results indicate that miR-‐7-‐1 and miR-‐497 may exert an inhibitory effect on the expression of MLCP and MLCK mRNA respectively. Ultimately this study should allow to better understand the regulation of catecholamines hypersecretion that characterizes pheochromocytomas and neuroendocrine tumors.


P7: Cellular and Molecular mechanisms of hypersecretion in pheochromocytomas Croise Pauline 1, Houy Sebastien1, Lanoix Joel2, Calco valerie 1, Brunaud Laurent 3, Paramithiotis Eustache2, Chelsky Daniel 2, Tryoen Petra1, Ory Stephane1, Gasman Stephane1 1 Cellular and Integrative neuroscience institute (INCI), CNRS UPR 3212, Strasbourg, France 2 Caprion Proteome, Inc, Montreal, Canada 3 Hospital Nancy-‐Brabois (CHU), Chirurgie Digestive, Hépato-‐bilaire et Endocrinienne, Nancy, France

Neuroendocrine tumors (NETs) are neoplasms arising from hormone/peptide-‐secreting cells. Although NETs are heterogeneous, a common critical feature is the dysfunction of the secretory activity leading to hypersecretion. Pheochromocytomas (pheo) are NETs that arise from chromaffin cells of the adrenal medulla, which are characterized by an excess of catecholamine secretion, leading to hypertension, cardiomyopathy and high risk of stroke. Although this aspect is well known by clinicians, it has never been explored at the cellular and molecular level. Here, we have analyzed the aberrant secretion of catecholamine at a single cell level by applying carbon fiber amperometry technique on human pheo resection. We have observed a drastic increase of exocytotic events in tumoral cells comparing to non tumoral chromaffin cells from the same patient. These data demon-‐strate that hypersecretion is a direct consequence of a deregulation of the secretagogue induced secre¬tion and not simply a mass effect due to the proliferation of tumoral cells. According to their widely accepted involvement in tumorigenesis Rho GTPases and their regulator pathways appear as good candidates to be involved in secretion defect and/or development of pheo. Our results show a decrease of the GTPases Rac1 and Cdc42 activity in human pheo compared to non-‐tumoral tissu. Moreover, by investigating protein expression changes in tumor through a mass spectrometry approach, we have demonstrated that ARHGEF1 and FARP1, two guanine nucleotide exchange factors that activate Rho GTPases are down-‐regulated. We then confirmed by in vitro experiment that down-‐regulation of ARHGEF1 and FARP1 triggers the inactivation of Rac1 and Cdc42, respectively. Altogether, our results demonstrate a deregulation of the secretory activity at a cellular level, and an alteration of the Rho GTPase pathways in pheo. This work has been supported by a Ligue Contre le Cancer (CCIR-‐GE) and a Fondation ARC to SG.

P8: Involvement of the secretory pathway in the tumorigenesis of pheochromocytomas Denorme M. 1,2,3*, Croisé P. 4*, Montero-‐Hadjadje M. 1,2,3, Ory S. 5 , Carmont O. 1,2,3, Cartier D. 1,2,3, Grumolato L. 1,2,3, Courel M. 5, Yon L. 1,2,3, Anouar Y. 1,2,3, Gasman S. 4* , Dubessy C. 1,2,3* 1Institut National de la Santé et de la Recherche Médicale (INSERM), UMRS 982, Mont-‐Saint-‐Aignan, France 2Normandie Université, France 3Université de Rouen, Laboratoire Différenciation et Communication Neuronale et Neuroendocrine, Institut de Recherche et Innovation Biomédicale (IRIB), Mont-‐Saint-‐Aignan, France 4CNRS UPR 3212, Institut des Neurosciences Cellulaires et Intégratives, Université de Strasbourg, France 5Centre National de la Recherche Scientifique (CNRS) UMR7622, Laboratoire de Biologie du développement, Université Paris VI, Paris, France * = Equal contributions

Pheochromocytomas (PCCs) are rare neuroendocrine tumors (NETs) which arise from chromaffin cells of the adrenal medulla. Most PCCs secrete high levels of catecholamines that cause many deleterious effects in patients, including hypertension which is difficult to control and could be lethal for some patients. However, the impact of the secretory activity on the development of NETs has never been investigated before. We therefore evaluated the role of the secretory activity on tumor growth in a xenograft model of PCCs in nude mice, using two spontaneous variants of the rat PCC PC12 cell line, namely A35C and PC12-‐27, both defective for the secretory pathway. We have shown that the tumorigenic potential of these PCC cell variants is significantly lower than that of the parental PC12 cells, as shown by the tumor growth delay (TGD, 38 and 14 days, respectively) and the increase in the tumor doubling time (TDT, 10 and 9.2 vs 4.8 days ; P <0.001) of the xenografts. To assess more specifically the involvement of the secretory pathway in the development of PCC tumors, we performed a lentiviral delivery of specific shRNA to inhibit the expression of chromogranin A (CgA), a key regulator of the formation of secretory vesicles (SV) in PC12 cells. Unlike parental PC12 cells, the PC12-‐shCgA cell line showed a strong decrease (92%) in CgA expression along with that of other granins, associated to a lack of SV and dopamine secretion. PC12-‐shCgA xenografts exhibited a high TGD (24 days) and an increased TDT (8.2 vs 2.6 days, P <0.001) compared to controls. Altogether, these results unveil a causal link between the secretory activity and PCC tumor growth. Moreover, our data suggest that the secretory pathway could be targeted to develop new treatments for NETs. This work was supported by INSERM, University of Rouen and Région Haute-‐Normandie.

P9: The combined treatment cyclosporine A ( CsA) (20mg/kg) and ketoconazol (KTCZ) (25mg/kg) induced a repair in all levels in testicular , adrenal , Thymic , hormonal and behavior disorders in wistar rats. 1-‐Aziez Chettoum, 2-‐Guedri Kamilia ,3_ Frih Hacene ¹Department of Animal Biology, University Mentouri, Constantine, Algeria; 2-‐3 Department of Biology, University BadjiMokhtar, Annaba,Algeria

Purpose: Cyclosporine (CsA ) , is a neutral lipophilic decapeptide cyclic and he was successfully used in transplant medicine and in the treatment of autoimmune diseases such as uveitis, psoriasis and rheumatoid arthritis. However, it has been reported that the administration of CsA (20mg/kg) causes a dysfunction at both gonadotropic that neurobehavioral. The objective of our study is to propose a therapeutic strategy based on the combined CsA treatment while relying on the assumption of the central role of corticosterone – proinflammatory cytokines in the development of disorders induced by the administration of CsA and Ketoconazole ( KTCZ), ( antidepressant and anxiolyique) and physiological (anti-‐ gonadotropic and corticoblocking). Methods: In our study, we performed the combined CsA administration (IP) and KTCZ (oral) for 14 consecutive days in male Wistar rats subsequently the forced swim test (FST). The forced swimming test in rats is a preclinical behavioral model has good predictive validity and is widely used to determine the effectiveness of antidepressant drugs (ADS) In addition ,conventional histopathological investigations in target organs (testes , thymus, adrenal ) neuropharmacological behavioral tests were used in this study, including the measure of the anxiety degree of the animal by the elevated place maze test, measure of the locomotor activity by the open fields test the and the use of Morris water maze test to assess the capacity of rat memory. Results: Our results indicated that the intraperitoneal injection of CsA(20mg/kg) cause testicular damage associated with a decrease in testosterone , adrenal cortical hyperplasia and lobular thymic degeneration. At the behavioral level , we recorded a significant improvement in learning and memory that an antidepressant and anxiolytic effect . Similar results were found following administration of KTCZ . However, we reported thymic hypertrophy, adrenal medulla hyperplasia and memory disorders.


P10: The orexin type 1 receptor is overexpressed in advanced prostate cancer with a neuroendocrine differentiation, and mediates apoptosis Alexandre David1, Hautot Coralie1, Mehio Marwa1, Jeandel Lydie1, Courel Maïté1, Voisin Thierry2, Couvineau Alain2, Leprince Jérôme1, Pfister Christian3, Anouar Youssef1, Chartrel Nicolas1 1, INSERM U982, Laboratory of Neuronal and Neuroendocrine Differentiation and Communication, Biomedical Research and Innovation Institute (IRIB), University of Rouen, Mont-‐Saint-‐Aignan, France. 2, University Paris-‐Diderot, Sorbonne Paris Cité, CRB3, Centre de Recherche Biomédicale Bichat Beaujon (CRB3), UMR773, INSERM, France. 3, Department of Urology, University Hospital of Rouen, Institute for Biomedical Research, University of Rouen, Rouen, France Here, we have examined the presence of orexins and their receptors in prostate cancer (CaP) and investigated their effects on the apoptosis of prostate cancer cells. First, we have localised the orexin type 1 and 2 receptors (OX1R and OX2R) and orexin A (OxA) in CaP sections of various grades. OX1R is strongly expressed in carcinomatous foci exhibiting a neuroendocrine differentiation, and the number of OX1R-‐stained cancer cells increases with the grade of the CaP. In contrast, OX2R is only detected in scattered malignant cells in high grade CaP. Then, expression of OX1R was also evaluated in the androgeno-‐dependent LNCaP and the androgeno-‐independent DU145 prostate cancer cells submitted or not to a neuroendocrine differentiation. OX1R is expressed in the AI DU145 cells but is undetectable in the LNCaP cells. Acquisition of a neuroendocrine phenotype by the DU145 cells is associated with an overexpression of OX1R. Finally, we showed that orexins induce the apoptosis of DU145 cells submitted to a neuroendocrine differentiation. The present data indicate that OX1R-‐driven apoptosis is overexpressed in androgeno-‐independent CaP exhibiting a neuroendocrine differentiation opening a gate for novel therapies for these aggressive cancers which are incurable until now.

P11: Developmental chemical exposure alters key steroidogenic enzymes in the neonatal sheep testis Bellingham, Michelle; Nikolatou Konstantina; Evans Neil

Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland

The hypothalamo-‐pituitary-‐gonadal axis (HPG) axis is tightly regulated by sensitive inputs. This system is a possible target for environmental endocrine disrupting chemicals (EDCs) whereby developmental EDC exposure can affect reproductive function in later life. Biosolids (sewage sludge) contains a mixture of EDCs and its application to pasture is a relevant model to examine effects of EDC mixtures on the HPG axis. Our previous studies have shown that maternal exposure to biosolids can affect expression of regulatory genes in the hypothalamus and pituitary gland and can alter fetal and adult testicular structure in offspring exposed in-‐utero. However, the effects of this exposure on the normal patterns of gene expression in the developing neonatal testis are still unknown. The present study examined whether developmental exposure to EDCs via maternal grazing on pastures fertilised using biosolids, affects mRNA expression of key genes which regulate testicular function in neonatal male lambs. Testes were collected from 1 day old male lambs from mothers who had been pastured on fields fertilised with inorganic fertiliser (control, n=9) or Biosolids (n=8). Testis were immersed in RNAlater® solution for 24 hours before storage at -‐80°C until RNA was extracted (25 mg) and reverse transcribed into cDNA. Using qRT-‐PCR, mRNA expression of genes of interest were quantified relative to 2 housekeeping genes (ß2-‐microglobulin and mitogen-‐activated protein kinase 14). Data were normalised to testis weight, log transformed and analysed using Student’s T-‐test (P<0.05 considered significant). Testicular expression of STAR and CYP17A1 was significantly reduced whereas expression of HSD3B and LHR was significantly increased in biosolid exposed compared to control lambs. These data add to an increasing body of evidence that developmental EDC exposure can have long term effects on the male.

P12: Ameliorating role of Fertimax in treatment of male infertility Mosbah Rachid 1,2*, Kourta Lamia2, Habel Mohamed Amine3. 1Laboratory of Animal Eco-‐Physiology, ENS-‐ Kouba-‐Alger, 2Departement of Biology, University of Boumerdes, 3Clinic El-‐Bordj for Assisted Reproductive Technology, Bordj El kifan Alger, ALGERIA. Over recent decades, there is a growing concern around the infertility problem in Algeria. In the latest census of the health and populations ministry, the infertility affects approximately 10-‐12% of the couples and in 30% of these cases, the male is the main associated origin by a reduction in semen quality under environmental and lifestyle factors. Many studies have incriminated sperm oxidative stress in the pathogenesis of men infertility through sperm DNA damage and membrane peroxidation. In this research, we attempt to determine the etiology of reproductive failure in infertile men and to assess the effectiveness therapy of Fertimax™ treatment as combination of a specific micronutrients and antioxidants on semen quality. Therefore, thirty six men consulting for infertility spousal in clinic of assisted reproductive technology "El Bordj"-‐ Algiers were interviewed, examined for clinical signs and their sperm was analyzed, then after, some of them were subjected to Fertimax™ treatment for six months and their sperm was reanalyzed. The obtained results revealed that Fertimax™ intake for six months can improve significantly all semen parameters including seminal volume, viscosity, spermatozoa number, motility, viability and morphology in these patients. Besides, in 33.33% of cases, these treated patients with Fertimax™ have fertilized their partners without recourse to in vitro fertilization process. This wonderful ameliorating role of Fertimax™ may be related to the particularly potent antioxidant properties of its components and thus we recommend this treatment as remedy for patients suffered from subfertility in both sexes.


P13: Selenoprotein T, a novel thioredoxin reductase, is involved in maintenance of ER homeostasis Hamieh A1, Cartier D1, Abid H1, Calas A2, Anouar Y1 and Lihrmann I1 1Inserm U982, Neuronal and Neuroendocrine Differentiation and Communication Laboratory, (IRIB), Rouen University, 76821 Mont-‐Saint-‐Aignan, France ²CNRS UMR 5297, Interdisplinary Institute of Neurosciences, Bordeaux, France Selenoprotein T (SelT) is a thioredoxin-‐like protein which was discovered during a transcriptomic analysis of the PACAP-‐induced neurodifferentiation process. This protein contains a thioredoxin fold structural pattern, which suggests that it participates to the regulation of redox homeostasis. SelT gene is silenced in adulthood, except in pituitary and numerous endocrine organs. Supporting a role in hormone secretion, SelT was reported to be a crucial intermediate in the calcium-‐dependent regulated secretory pathway in the rat pheochromocytoma PC12 cells. This study aimed at elucidating the role of SelT in endocrine tissue, using the pituitary as model. We found that SelT is a novel thioredoxin reductase and this enzymatic activity is dependent on the presence of Sec or Cys residues in its active site. We next characterized SelT expression in mouse pituitary. Double immunostaining and confocal microscopy analysis showed that SelT is expressed in some corticotroph, gonadotroph, somatotroph, lactotroph and thyreotroph cells. SelT expression is induced by ER stress and can protect pituitary cells against ER and oxidative stresses. This activity requires anchoring of SelT in the ER membrane, online with results obtained by electronic microscopy, as well as the integrity of the CXXU/C redox domain. Finally, SelT interacts with a member of the N-‐glycosylation complex OST, and is involved in POMC N-‐glycosylation and ACTH secretion. In conclusion, we have shown that SelT is a new member of the OST complex, which could be critical in tissues with high secretory activity. Its thioredoxin reductase activity and its specific presence in cells producing specific hormones (ACTH, prolactin, TSH, LH, FSH, insulin, somatostatin) suggested that it may play a specific role in the N-‐glycosylation of proteins containing disulfide bridges, as it has been shown for other OST subunits.

P14: Neat1 a long non-‐coding RNA involved in pituitary circadian rhythms: characterization of its interactome Torres Manon , Becquet Denis, Guillen Séverine, Boyer Bénédicte, Moreno Mathias, Blanchard Marie-‐Pierre, Franc Jean-‐Louis and François-‐Bellan Anne-‐Marie Aix Marseille Université, CNRS, CRN2M-‐UMR7286, Faculté de Médecine Nord, 51 Bd Pierre Dramard CS 80011, 13344 Marseille cedex 15, France Post-‐transcriptional regulation appears increasingly essential to the circadian system functioning. Paraspeckles are nuclear bodies that control gene expression through a post-‐transcriptional mechanism based on nuclear retention of RNA containing in their 3’UTR region inverted repeats of Alu sequences (IRAlu). Paraspeckles are form around the long non-‐coding RNA, NEAT1, together with numerous RNA-‐binding proteins including NONO, SFPQ, PSPC1 and RBM14. We found that all these key components of paraspeckles including Neat1 displayed a circadian expression pattern in pituitary cells. In this study, we asked whether IRAlu elements inserted in 3’-‐UTR of a reporter mRNA may allow for its circadian retention within the nucleus by paraspeckles. The single antisense AluSp, or the IRAluSp element cloned from the 3’-‐UTR of Nicolin1 gene were inserted each between the 3’-‐end of EGFP cDNA and a polyadenylation signal in the expression vector pEGFP-‐C1. These constructs were stably transfected into pituitary GH4C1 cells. Then we monitored by real-‐time video-‐microscopy EGFP expression and determined by RTqPCR the nuclear and cytoplasmic distribution of EGFP mRNA over a circadian period. We also aimed to identify the endogenous RNA targets of paraspeckles in GH4C1 cells. We developed an approach used to analyze RNA targets of the main component of paraspeckles, Neat1.With this approach which employed capture antisens oligonucleotides designed to specifically hybridize to Neat1, Neat1 is enriched together with its targets that are identified by RNA sequencing. Analysis of the 3’-‐UTR sequences of these RNAs shows that others structural elements than IRAlus can mediate the binding of RNA to the paraspeckles.

P15: Stem cells in the hypothalamo-‐pituitary axis Sam Goldsmith, Christophe Galichet , Robin Lovell-‐Badge and Karine Rizzoti The Crick Institute, London, UK Organ-‐specific cell renewal can be achieved by cell division and/or by differentiation from stem cells (SC). Recently, populations of adult hypothalamic and pituitary SCs have been characterized. In the pituitary, these express the transcription factors SOX2 and SOX9 (Fauquier et al., 2008). Using cell-‐lineage tracing we showed that SOX2;SOX9+ve progenitors self-‐renew and give rise to endocrine cells, from embryogenesis to adulthood. In the embryo SOX2 is required for proliferation and the protein is also required later, for melanotrophs differentiation (Goldsmith et al, in prep.). In the adult, SCs mostly maintain their identity, so we explored their regenerative potential. Pituitary target organ ablation induces a mitotic wave in the gland followed by generation of new hormonal cells; cell division is observed in non-‐endocrine cells so SCs may be activated (Nolan et al, 2006). Combining this ablation model with lineage tracing, we showed that pituitary SCs proliferate and differentiate in the appropriate cell type (Rizzoti et al., 2013). To understand the mechanisms of SC mobilization, we used the somatostatin analogue Pasireotide as it affects the pituitary response to adrenalectomy (Nolan et al., 2007). Pasireotide administration after adrenalectomy affects proliferation but not differentiation of SCs, suggesting that these events are regulated independently. In the hypothalamus, SCs express SOX2 and SOX3. Sox3-‐/-‐ mutants are affected by hypopituitarism (Rizzoti et al., 2004) but the deficiencies only appear after weaning. We show that Sox3 loss affects proliferation and differentiation of hypothalamic progenitors but only after weaning. In Sox3-‐/-‐ median eminence, using lineage tracing, we observed a reduction in oligodendrocyte differentiation, as pituitary deficiencies develop. This suggests that SCs give rise post-‐natally to oligodendrocytes, required in the median eminence for a functional hypothalamo-‐pituitary connection (Galichet et al, in prep).


P16: Pituitary lactotroph cell ultrastructure in normal and reproductive mutant male mice Paul, Nadine; Abel, Margaret; Christian, Helen Department of Physiology, Anatomy and Genetics, University of Oxford Testosterone exerts genomic and non-‐genomic rapid effects in rats to stimulate prolactin secretion. However, there is relatively little known about the effects of testosterone on the structure and function of lactotrophs in male mice. Castration of mice results in reduced prolactin content in the pituitary and decreased circulating prolactin. In the present study we have compared the effects of mutations that perturb the hypothalamic-‐pituitary-‐gonadal axis on lactotroph ultrastructure in male mice. Testicular feminised (tfm, naturally occurring deletion of androgen receptor), luteinising hormone (LH) receptor knockout (LuRKO), and hypogonadal (hpg, lack GnRH) mice were compared with control wild-‐type mice. Pituitary glands (8 week old mice) were collected (n=4 per group) and prepared for quantitative electron microscopy. In control mice type I lactotrophs were identified by irregular shaped secretory granules (10-‐200nm diameter) and type II lactotrophs were identified by regular-‐shaped secretory granules, as in the rat although the secretory granule size was smaller. In hpg, tfm and LuRKO mice type I lactotrophs were smaller in size than controls (P<0.05) and the amounts of dilated rough endoplasmic reticulum were reduced (P<0.05) in hpg and LuRKO mice consistent with lack of testosterone stimulation, but increased in tfm mice. Despite androgen insensitivity testosterone is present in tfm mice and may act on lactotrophs indirectly via conversion to estradiol. Secretory granule density was not significantly different between control and mutant mice in type I lactotrophs but was increased in type II lactotrophs. Fewer (P<0.01) secretory granules were observed adjacent to the plasma membrane in hpg and LuRKO type II lactotrophs than in control. These findings indicate that lactotroph ultrastructure is altered in different gonadal steroid environments and are consistent with a lack of stimulation by testosterone

P17: The gonadotroph-‐vascular unit and build-‐up of LH pulses HOA Ombeline, LAFONT Chrystel, GUILLOU Anne, SAMPER Patrick, FONTANAUD Pierre, MOLLARD Patrice

CNRS UMR5203, INSERM U1191, University of Montpellier The hypothalamic-‐pituitary-‐gonadal (HPG) axis controls the reproductive function. The pituitary gland was considered as a patchwork of randomly distributed cells which simply respond to hypothalamic regulation, but it turns out that this organ is highly organized into structural and functional cell networks, and that dynamics of pituitary cell networks are essential for the build-‐up of hormone pulses. Recent preliminary data suggested the existence of a Gonadotroph-‐Vasculature Unit (GVU) in which crosstalk between the network of pituitary gonadotrophs and vasculature (pericytes and fenestrated endothelial cells) may be prerequisites for regulation of LH release into the bloodstream. Indeed, the gonadotroph network displays at proestrus a highly dynamic spatial reorganization which is correlated with changes in vascularisation/pericyte coverage. Our working hypothesis is that GVU plasticity would allow pericytes to play a role in blood flow regulation which would occur during LH pulses/pre-‐ovulatory surge. To do so, we use optogenetic tools expression in transgenic mice models to modulate the activity of pericytes during LH surges following i.v. GnRH or Kisspeptin injections. We study GnRH and Kisspeptin effects in pituitary blood flow and their correlation with LH secretion. LH secretion will be determined by tail vein blood sampling and ultra-‐sensitive hormone ELISA. Moreover we are currently adapting the use of GRIN lenses to image and manipulate blood flow during LH pulses in conscious head-‐fixed mice. Finally we are setting up pituitary gland infection with AAV-‐GCAMP6s in order to monitor calcium signals in GnRH stimulated gonadotrophs in conscious animals. These experiments are expected to help understand the role of GVU remodelling in the HPG function.

P18: The gonadotrope lock : silencing of miR-‐125 necessary for GnRH activation of gonadotropins expression Lannes J, L’hôte D, Garrel G, Laverrière JN, Cohen-‐Tannoudji J and Quérat B Université Paris-‐Diderot, Paris-‐7 ; CNRS UMR 8251; INSERM U1133

Gonadotropin-‐releasing hormone (GnRH) plays a key role in the vertebrate reproductive system by stimulating biosynthesis and secretion of pituitary gonadotropins. Although the transcriptional control of gonadotropin subunit genes has been largely studied, the involvement of microRNAs (miRNAs) still needs to be deciphered. We recently showed that GnRH activates a miR-‐132/212 pathway, involving a lowered level of Sirt1 deacetylase and an increase in the acetylated form of FoxO1, releasing its transcriptional inhibitory action on Fshb subunit gene. In this study, we investigated the role of miR-‐125, an miRNA that showed an acute decline in response to a GnRH treatment in the murine gonadotrope cell line, LβT2. We first demonstrated that GnRH was not able to stimulate gonadotropins subunit expression when miR-‐125 was overexpressed in rat pituitary gondadotrope cells. Overexpression of miR-‐125 in LβT2 cells induced a marked decline in mRNA steady-‐state level and/or in protein level of a number of known mediators of GnRH activation: Gq/11 alpha, IP3R, CamK, MKK7 and c-‐JUN, Elk-‐1 and c-‐Fos, thus affecting both calcium and MAPK signalling pathways. Blocking miR-‐125 action had an inverse effect on all these actors. In contrast, the unmodified phosphorylation state of CREB indicated that the cAMP signalling pathway is not altered by modulating miR-‐125 levels. Interestingly, miR-‐132 expression which has been shown to be CREB-‐dependant, exhibited an opposite dynamic to that of miR-‐125. In addition, GnRH treatment stimulated the recruitment of miR-‐132 into the RNA-‐induced Silencing complex, whereas it lowered that of miR-‐125. The mechanisms involved in the down-‐regulation of miR-‐125 are still under investigation. In conclusion, we show that an acute decline in miR-‐125 is necessary for GnRH activation of both calcium and MAPK signaling pathways and for the resulting activation of gonadotropin subunit genes expression.


P19: MicroRNAs flip the switch for the production of hypothalamic GnRH before puberty Messina Andrea1,2, Langlet Fanny 3, Chlachaki Konstantina 1,2 , Roa Juan 4,5 , Rasika S 1, Jouy Nathalie 1,2, Gallet Sarah 1,2, Parkash Jyoti 1,2, Accili Domenico 3, Tena-‐Sempere Manuel 4,5, Giacobini Paolo 1,2,3, Prevot Vincent 1,2,3 1 Inserm, Laboratory of Development and Plasticity of the Neuroendocrine Brain, Jean-‐Pierre Aubert Research Centre, UMR1172, F-‐59000 Lille, France 2 University of Lille, School of Medicine, Lille, F-‐59000, France 3 Columbia University, New York, USA 4 Department of Cell Biology, Physiology and Immunology, University of Cordoba & Instituto Maimonides de Investigación Biomédica de Cordoba (IMIBIC/HURS), 14004 Cordoba, Spain 5 CIBER Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Carlos III, 14004 Cordoba, Spain A sparse population of a few hundred primarily hypothalamic neurons forms the hub of a complex neuroglial network that controls reproduction in mammals by secreting the "master molecule", gonadotropin-‐releasing hormone (GnRH). The mechanisms underlying the timely changes in GnRH expression necessary for the onset of puberty are unknown. Here, we report that a dramatic inversion in microRNA species expressed by postnatal GnRH neurons acts as an epigenetic switch that triggers the prepubertal increase in GnRH expression and the correct initiation of puberty. The disabling of this microRNA-‐mediated switch leads to hypogonadotropic hypogonadism and infertility in mice. The underlying mechanism involves a complex and multilayered network of GnRH transcriptional activators and repressors reciprocally controlled by several microRNA species tuning the balance between inductive and repressive signals and triggering a rise in hypothalamic GnRH expression. Anomalies in this microRNA-‐mediated switch, which appears essential for the neuroendocrine control of reproduction, could thus underlie dysfunctions of human puberty and fertility when a genetic cause is not apparent.

P20: Dmxl2 haploinsufficiency impedes the postnatal maturation and activation of GnRH neurons in mice. Tata Brooke K , Csaba Zsolt , Jacquier Sandrine , de Roux Nicolas

INSERM U1141, Paris Diderot University, Robert Debré Hospital Puberty involves the maturation of neuronal circuits where synaptic inputs modify GnRH neuron activity. We recently described a new neurodevelopmental disorder in humans associated with pubertal and fertility defects due to haploinsufficiency of DMXL2. DMXL2 encodes rabconnectin-‐3α (rbcn3-‐α), a scaffolding protein for Rab3-‐GAP and RAB3-‐GEP proteins and V-‐ATPase, which are important for synaptic plasticity and neurotransmitter release. We previously found that neuronal loss of Dmxl2 in mice (Nes-‐Cre+/-‐;Dmxl2-‐/wt) causes infertility associated to a loss of GnRH neurons in the OVLT. Because the GnRH deficiency in Nes-‐Cre+/-‐;Dmxl2-‐/wt mice cannot explain the complete infertility, we suspect an additional functional defect of the GnRH neuron system. As such, we pursued to characterize the GnRH neuron phenotype in Nes-‐Cre+/-‐;Dmxl2-‐/wt mice. Loss of neuronal Dmxl2 impedes morphological maturation of OVLT GnRH neurons which could not respond to Kisspeptin-‐10 nor Estradiol. Female Nes-‐Cre+/-‐;Dmxl2-‐/wt mice had a blunted circadian-‐timed LH surge. Nes-‐Cre+/-‐;Dmxl2-‐/wt male mice showed a feminized profile of kisspeptin and tyrosine hydroxylase in the AVPV, suggesting that sexual differentiation is perturbed. We found evidence indicative that Nes-‐Cre+/-‐;Dmxl2-‐/wt mice also have a glutamatergic defect. Conditional cre-‐dependent viral filling of OVLT GnRH neurons in GnRH-‐Cre+/-‐;Dmxl2wt/wt mice showed that rbcn3-‐α-‐containing terminals contact OVLT GnRH dendritic spines, associate with VgluT2-‐positive terminals, wrap between juxtaposed GnRH dendrites, and is in the GnRH soma. Collectively, Nes-‐Cre+/-‐;Dmxl2-‐/wt mice display a defect in the maturation of GnRH neurons that is probably related to a decrease in glutamatergic inputs on GnRH neurons during the juvenile period. This study opens new insights in the characterization of the postnatal neurodevelopment of the GnRH neuronal network for puberty and complete gonadotropic function in adulthood.

P21: Nitric oxide says “NO” to promote and maintain reproductive capacity Chachlaki Konstantina 1 Messina Andrea 1 Garthwaite John 2, Prevot Vincent 1 1 Inserm, U1172, University of Lille, France 2 The Wolfson Institute for Biomedical Research, University College London, London, UK The regulation of the reproductive axis is governed by a delicate balance of elaborate positive and negative signals received from hypothalamic neurons. This balance is of pivotal importance for the initiation of puberty and normal fertility. Nitric oxide (NO) and its downstream signaling cascades are critical to various cellular functions in the brain, including the neuroendocrine control of reproduction. NO is produced by neuronal NOS-‐expressing neurons (nNOS) found in the vicinity of GnRH-‐containing perikarya in the preoptic region of the hypothalamus. The fact that nNOS cells surround GnRH cell bodies, creates an anatomical relationship between the two neuronal populations, which is complemented by the capacity of NO to modulate GnRH electrical activity, enabling the peak release of GnRH (Clasadonte et al.,2008). Thus, nNOS-‐expressing cells are considered an integral part of the neuronal network controlling ovarian cyclicity and ovulation, provoking the peak release of LH (Bellefontaine et al.,2014;Hanchade et al.,2012). Additionally, recent results from our lab demonstrate a different role of NO, proposing its implication in the transcriptional regulation of GnRH during the crucial infantile period (Messina et al.,subm). Since the biological actions of NO depend critically on its concentration, which is difficult to measure, we use a novel cGMP biosensor in combination with an ultrasensitive detector cell line in order to quantify the active concentration of NO being released in a mouse hypothalamic slice using live imaging techniques, under physiological conditions (Bhargava et al.,2013;Wood et al.,2011). Our results suggest that nNOS neurons within and in direct proximity to the OVLT, a site devoid of the blood brain barrier and to which GnRH neurons extend dendrites, relay necessary signals to GnRH cells, acting as mediators of repressive cues necessary for the establishment of the reproductive balance needed to promote and maintain reproductive capacity.


P22: Essential role of α-‐MSH signaling in mediating the permissive effects of leptin on puberty onset and its interplay with kisspeptin pathways Manfredi-‐Lozano, M1,2; Roa, J1,2; Ruiz-‐Pino, F1,2; Piet, R3, Garcia-‐Galiano, D1,2; Zamora, A1,2; Leon, S1,2; Sanchez-‐Garrido, M.A.1,2; Romero-‐Ruiz, A1,2; Dieguez, C.4; Vazquez, M.J.1,2; Pineda, R.4; Herbison, A.E. 3; Pinilla, L.1,2; Tena-‐Sempere, M1,2. 1Department of Cell Biology, Physiology and Immunology, University of Córdoba; 2Instituto Maimónides de Investigaciones Biomédicas de Córdoba (IMIBIC)/Hospital Universitario Reina Sofía, 14004 Córdoba, Spain; 3Centre for Neuroendocrinology, Department of Physiology, University of Otago School of Medical Sciences, Dunedin 9054, New Zealand; 4University of Santiago de Compostela, 15705 Santiago de Compostela, Spain; and 4Centre for Integrative Physiology, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK.

POMC neurons in the hypothalamic arcuate nucleus (ARC) play a key role in energy homeostasis, mainly via the release of α-‐MSH, which acts through melanocortin 3 and 4 receptors (MC3-‐R & MC4-‐R) to suppress food intake and mediate the effects of leptin. Melanocortin pathways are likely involved also in the central control of reproduction, and bidirectional interactions with kisspeptins (Kp), ligands of Gpr54 and indispensable elements of the reproductive brain, have been suggested. Yet, the role of α-‐MSH in the regulation of puberty onset remains ill defined. We report herein that central activation of α-‐MSH signaling, mainly via MC4-‐R, elicited robust luteinizing hormone (LH) responses, as surrogate marker of reproductive axis activity, in pubertal, but not infantile rats, even against unfavorable metabolic conditions. In addition, chronic central blockade of MC3/4-‐R during the pubertal transition delayed the timing of puberty and prevented the permissive effect of leptin on puberty onset. Central blockade of MC3/4-‐R or selective elimination of Kp receptors from POMC neurons did not affect LH responses to Kp. Conversely, Gpr54 null mice displayed markedly attenuated net LH responses to MC receptor activation, whereas blockade of α-‐MSH signaling suppressed Kiss1 expression in the ARC of pubertal female rats. Neuroanatomical studies documented close appositions between POMC and Kp neurons in the ARC of pubertal female rats. Yet, Kiss1 electrical activity was not overtly modified by direct application of α-‐MSH agonist, suggesting either indirect or non-‐action potential-‐mediated mode of action. In sum, our data document the essential role of α-‐MSH pathways in the physiological control of puberty and in transmitting the permissive effects of leptin. While α-‐MSH signaling seems dispensable for the reproductive effects of Kp, the reproductive/pubertal actions of α-‐MSH are, at least partially, mediated via direct or indirect modulation of Kp pathways.

P23: A link between GnRH neuronal migration and central precocious puberty: the role of Semaphorin3A/Neuropilin-‐1 signaling in GnRH neurons Charlotte Vanacker 1, , Filippo Casoni1, Andrea Messina1, Sophie Croizier2, Naresh Kumar Hanchate1, Sébastien G Bouret1,2, Paolo Giacobini1, Vincent Prevot1 1 Inserm U1172, University of Lille, CHRU of Lille, France 2 Developmental Neuroscience Program, The Saban Research Institute, Children’s Hospital Los Angeles, University of Southern California, Los Angeles, California, United States of America

Semaphorin3A (Sema3A) and its receptor, neuropilin 1 (Nrp1), are involved in the guidance of GnRH neuron migration during embryogenesis. Disruption of Sema3A gene expression or of neuropilin 1 signaling indeed causes marked GnRH migratory defects in mice, and Sema3A has recently been described as a Kallmann gene. However, the specific role of Nrp1 expression in GnRH neurons remains to be elucidated. Here, we analyze the phenotype of mice in which Nrp1 expression was selectively knocked out in GnRH neurons. Animals harboring the conditional Nrp1 allele were crossed with a mouse line expressing the Cre recombinase under the control of the endogenous GnRH gene promoter. The efficacy of our genetic strategy was shown by the loss of Nrp1 expression in GnRH neurons of mutant (GnRH-‐Cre; Nrp1lox/lox) but not wild-‐type (Nrp1lox/lox) littermates. Unexpectedly, mutant mice that did not show any alteration of adult reproductive function, exhibited precocious puberty. While wild-‐type mice reach puberty (first ovulation) at postnatal day 50 (P50), mutant mice first ovulate at P45. Interestingly mutant mice also have impaired energy balance, as they exhibit higher body weight and fat mass than control littermates. Our preliminary neuroanatomical data suggest that while there is no overt difference in the total number of GnRH neurons in the brain, their distribution differ between mutant mice and wild-‐type littermates. This phenomenon is probably due to differential migration of the cells during embryogenesis, since we have already noted differences in GnRH neuronal distribution from nose to brain at embryonic age 14,5. Even though further analyses are required to determine the causes of this precocious puberty, the present study report a role for Nrp1 expression in GnRH neurons in the correct development/physiology of the reproductive axis.

P24: A novel role for anti-‐müllerian hormone on the development of the GnRH system Malone, SA, Cimino, I, Jamin, SP, Prevot, V, Giacobini, P INSERM U1172 & University of Lille, France

Gonadotropin-‐releasing hormone (GnRH) neurons originate in the olfactory placode and must migrate during embryogenesis to reach the basal forebrain where in postnatal life they function as the master regulators of reproductive function. Disruption of the GnRH migratory process results in hypogonadotrophic hypogonadism and infertility. A number of factors have been identified that regulate this migration and here we present novel evidence that anti-‐Müllerian hormone (AMH) acts as a putative regulator of this developmental process. Recent immunohistochemical data from our lab have demonstated that the AMH receptor (AMH-‐R2) is expressed by GnRH neurons both during development and postnatal life and that AMH is expressed in the olfactory epithelium and along the olfactory fibers. Further, Amhr2 null mice display a 30% reduction in the total number of GnRH neurons located in the brain, coupled with an altered spatial distribution. Taken together these data suggest that AMH may be important in regulating GnRH cell survival during migration or to facilitate correct migratory behavior. We first validated that the immortalized GnRH cell lines Gn11 and GT1-‐7 express the receptors required to transduce the AMH signal. We then assessed the effect of AMH on the survival of both Gn11 and GT1-‐7 cell lines by MTT and flow cytometry; neither approach suggesting that AMH is able to increase cell survival in either cell line when challenged with pro-‐apoptotic factors. AMH was, however, able to significantly increase cell motility in Gn11 cells – a model of migrating GnRH neurons. Together this data identify AMH as a novel putative regulator of the GnRH migratory process


P25: Beta-‐NGF regulates GnRH neurones activity Pinet-‐Charvet Caroline, Duittoz Anne PRC, INRA U85, CNRS UMR727, Université de Tours, IFCE, Tours France. Université de Poitiers, Poitiers France GnRH is an important hormone for adequate regulation of reproduction. The frequency of its pulsatile release is the major condition to the secretion of two pituitary hormones, FSH which is involved in the ovarian follicle maturation, and LH whose peak is responsible for ovulation. In animals such as llamas, alpacas and camels, an ovulation inducing factor (OIF) triggers ovulation at the time of mating. Mass spectrometry studies have shown this OIF to be beta-‐NGF, present in 80% of the seminal plasma in these species. Several studies have demonstrated that intramuscular injection of beta-‐NGF resulted in the release of LH induced by secretion of GnRH in alpaca but also in species with spontaneous ovulation such as ewes. The purpose of the present study was to investigate the effects of recombinant beta-‐NGF on GnRH neurones. Olfactory placodes explants were dissected from mouse embryos (E11.5). Loose-‐patch electrophysiology and calcium imaging experiments were performed on cultured GnRH neurons (10 DIV). Here we found that beta-‐NGF increased the frequency of action potentials of GnRH neurons in a dose-‐dependent manner. Calcium events frequency and synchronization were also affected at single cell and network levels. In addition, immunostainings revealed rearangements between surrounding glial cells and GnRH neurones, suggesting a mechanism involving a glial-‐neuronal cross-‐talk. Thus our results bring new insights into the understanding of beta-‐NGF effect on GnRH neurons. As beta-‐NGF targets two different receptors p75 and TrkA, which are differentially expressed in neurons or glial cells, more experiments are necessary to determine if beta-‐NGF acts directly on neuron or through glial cells.

P26: Investigating sexual dimorphism and morphological plasticity in GABAergic input to gonadotropin-‐releasing hormone neurons Moore, Aleisha M.; Yip, Siew Hoong; Campbell, Rebecca E. University of Otago, Centre for Neuroendocrinology and Department of Physiology, Dunedin, New Zealand 9013

The density of GABA terminals to gonadotropin-‐releasing hormone (GnRH) neurons is enhanced in prenatally androgenised female mice that display impaired steroid hormone negative feedback. GABA neurons, in the arcuate nucleus in particular, have recently been implicated in mediating steroid hormone feedback directly to GnRH neurons. The present study aimed to determine whether GABAergic input to GnRH neurons differs between males and females, and exhibits morphological plasticity over the female estrous cycle. Tissue collected from GnRH-‐green fluorescent protein (GFP) male (n= 5) and female mice in diestrus (n= 5) underwent immunofluorescent labelling of GFP and the vesicular GABA transporter (vGaT). The density of vGaT-‐immunoreactive (-‐ir) puncta apposed to GnRH neuron somata and proximal dendrites was not different between males and females. To assess putative GABAergic innervation to the full extent of GnRH neurons and over the female estrous cycle, GnRH-‐Cre mice were stereotaxically injected with a cre-‐dependent adenovirus expressing farnesylated enhanced green fluorescent protein into the preoptic area. Tissue was collected at least one week later from mice in the diestrous, proestrous or estrous stage of the cycle (n= 4/ group) and sagittal brain sections were labelled for GFP and vGaT. Putative GABAergic inputs were greatest to the proximal dendrites of GnRH neurons in the preoptic area. The density of vGaT-‐ir puncta to GnRH neurons was not significantly different in animals in different estrous cycle stages, indicating that GABAergic input to GnRH neurons does not exhibit significant morphological plasticity across the estrous cycle. Together, these data suggest that prenatal androgen induced re-‐wiring of GABA inputs to GnRH neurons do not reflect masculinization of GABA innervation and that cyclic fluctuations in steroid hormone feedback over the female estrous cycle is unlikely to result in plastic changes in GABA inputs to GnRH neurons

P27: Design of a selective kisspeptin analog capable of synchronizing ovulation Beltramo Massimiliano1, Robert Vincent1, Lomet Didier1, Anger Karine1, Galibert Mathieu2, Madinier Jean-‐Baptiste2, Marceau Philippe2, Delmas Agnès2, Caraty Alain1, Aucagne Vincent 2, Decourt Caroline1. 1UMR Physiologie de la Reproduction et des Comportements (INRA, UMR85; CNRS, UMR7247; Université François Rabelais Tours; IFCE) F-‐37380 Nouzilly, France; 2Centre de Biophysique Moléculaire (CNRS UPR 4301) F-‐45071 Orléans cedex 2, France. GnRH secretion is central to reproduction control and the neuropeptide kisspeptin (Kp) is the most potent GnRH secretagogue. Hence, Kp system is an appealing target to develop new methods to manage reproduction and heal related pathologies. Endogenous Kp isoforms have short in vivo half-‐life and continuous administration is required to obtain the wanted effects. However, in livestock management and in human therapy a single injection is preferable to continuous administration. To meet this need we designed analogs of the 10 amino acid isoform of Kp with improved pharmacokinetics and pharmacodynamics. To this aim we combined various modifications improving resistance to degradation and reducing renal clearance. Our effort produced analogs that compared to Kp10 have equal efficacy, similar or better potency, and a prolonged half-‐life in blood serum. Several compounds were active by intramuscular injection at very low doses (5 to 15 nmoles/ewe) and LH level was still higher than basal nine hours after analog injection. When injected during the breeding season, in ewes pretreated with flugestone acetate (FA) for 14 days, the best Kp analog produced a superior synchronization of LH surge compared to available treatment (pregnant mare serum gonadotropin). In presence of a ram treated ewes showed all behavioral signs of estrus. Ovulations triggered by Kp analog were fertile as indicated by the rate of pregnancy (7 out of 10) obtained after servicing. Experiments are ongoing to assess if analogs could also induce ovulation during the non-‐breeding season. In conclusion we generated a Kp analog with improved pharmacokinetics and pharmacodynamics capable of inducing, after a single intramuscular injection, synchronized fertile ovulations in ewes pretreated with FA. This molecule holds a strong potential to improve management of livestock reproduction and possibly to treat human reproductive disorders due to reduced GnRH secretion. Supported by Région Centre (Reprokiss grant).


P28: Neuromedins U and S differentially regulate pulsatile LH and prolactin secretion and are expressed in the supraoptic and paraventricular nuclei in ewes. Grachev, Pasha; Valent, Miroslav; Goodman, Robert L. Department of Physiology & Pharmacology, West Virginia University, Morgantown, WV, USA Prolactin (PRL) secretion is elevated annually during long days and is stimulated by estradiol, while pulsatile LH secretion is subject to negative feedback effects of gonadal steroids and photoperiodic suppression. We have recently demonstrated the biphasic effect of central administration of NMU-‐8, a synthetic peptide the sequence of which resembles the receptor-‐binding portion of endogenous ovine neuromedins U and S, on LH pulse amplitude in ewes – stimulatory in ovary-‐intact and inhibitory in ovariectomized sheep. In light of the differential regulation of prolactin and LH, we proposed that NMU-‐8 would have converse effects on PRL pulse amplitude. To test this hypothesis, we examined the effects of intracerebroventricular administration of NMU-‐8 on PRL levels in frequently sampled blood from anestrous or ovariectomized ewes. Immunohistochemistry, utilizing a polyclonal antibody raised in rabbit against NMU-‐8, was performed as a first step in exploring the localization of endogenous ovine neuronal populations expressing neuromedins U and S. Contrary to our hypothesis, NMU-‐8 failed to affect PRL pulse amplitude in either anestrous or ovariectomized ewes, but did exert significant biphasic influence over PRL pulse frequency: inhibitory in ovary-‐intact anestrous and stimulatory in ovariectomized ewes. Discrete populations of neuromedin U/S-‐immunoreactive soma were localized to the supraoptic (including auxiliary supraoptic) and paraventricular nuclei. These are the first data in sheep that detail effects of central neuromedin U/S signaling on pulsatile PRL secretion and the hypothalamic expression of endogenous ovine neuromedins U and/or S. Source of research support: NIH RO1 HD039916 (RLG)

P29: Anatomical and phenotypical evidences for early prenatal maturation of the kisspeptin-‐GnRH system

Alfaïa Caroline, Poissenot Kevin, Faure Mélanie, Robert Vincent and Franceschini Isabelle UMR INRA-‐CNRS-‐Université de Tours-‐IFCE, Physiologie de la Reproduction et des Comportements. 37380 Nouzilly

Kisspeptin (Kp), encoded by Kiss1, is a potent secretagogue of GnRH playing key roles in sexual differentiation and regulation of reproductive cycles. Kp is synthesized by neurons with specific estrogen sensitivity, located in the periventricular region of the preoptic area (POA) and in the arcuate nucleus (Arc) where these also express neurokinin B (NKB). Close anatomical interconnections between POA Kp neurons, Arc kp neurons and GnRH neurons exist in adults. Recently, we discovered that Kp is already produced and regulated prenatally in the Arc which prompted us to refine the prenatal development of Kp neurons. Here, we used a knock-‐in mouse expressing cre-‐GFP under control of the Kiss-‐1 promoter to define by immunohistochemistry the spatiotemporal pattern of Kiss-‐GFP expression in the fetal brain, in relation to Kp, NKB, GnRH, sox2 and Era. GFP-‐immunoreactive (ir) cells increased with age, were first detected at embryonic day (E) 13.5 in the mantle layer of the tuberal hypothalamus, were clustered on either side of the infundibular recess up to the mammillary recess at E14.5 and accumulated around the infundibulum at E16.5. Phenotypic differences were further noted: between E14.5 and E16.5 the proportion of GFP/ERa-‐ir increased while that of GFP/sox2-‐ir decreased and cells of the posterior Arc displayed a more immature profile than the anterior one. Preliminary data suggests an increase in Kp-‐ir between E14.5 and E16.5 and in NKB-‐ir between E16.5 and E18.5 in females. Kp-‐ir at E16.5, and NKB-‐ir at E18.5 were detected around many GFP-‐ir cell nuclei and along fibers in the Arc, median eminence and POA including in its periventricular part. No evidence for GFP or Kp-‐ir cell bodies were found outside the Arc at any stage analyzed. GnRH fibers were seen close to GFP/kp cell bodies and reciprocally. Taken together, these results show an early prenatal maturation of Arc Kp neurons. Potential sex differences in this prenatal maturation are currently being investigated.

P30: Somatostatin (SOM) contacts on kisspeptin (KP) neurons in the arcuate nucleus both in ewes and rats: a possible involvement of SOM on KP-‐induced LH secretion. Dufourny Laurence, Delmas Oona, Decourt Caroline, Martinet Stéphanie

UMR Physiologie de la Reproduction et des Comportements (PRC), Centre INRA de Tours, 37380 Nouzilly. Kisspeptin (KP) is the most potent secretagogue of GnRH secretion known to date in mammals. Its secretion is under the control of several endogenous hormones and neuromodulators among which some are involved in the control of both reproduction and metabolic homeostasis. A former study performed by Pillon et al. (2004) in the ewe pinpointed an inhibitory action of somatostatin (SOM) on LH pulsatility within 30 min following intracerebroventricular (icv) delivery suggesting that SOM acting directly or through interneurons was able to inhibit GnRH neuron activity. Our hypothesis is that SOM inhibits KP neuron activity directly in order to modulate GnRH release. We performed a triple immunofluorescent approach to detect simultaneously KP, SOM and synapsin, a marker for synaptic vesicles, on sections from ewes killed during the breeding season having therefore an activated gonadotrope axis and high numbers of KP neurons in the arcuate nucleus (ARC). The same approach was also performed on sections from male rats killed following a colchicine icv treatment. Sections from the preoptic area (POA) and from the mediobasal hypothalamus were counterstained with DAPI and examined under a confocal microscope. KP neurons were randomly chosen in the POA and ARC and, SOM neurons were observed in these 2 regions and in the ventromedial hypothalamus (VMH). Both in ewes and rats, ARC KP neurons were densely surrounded by SOM terminals while in the POA few SOM terminals were found on KP neurons. SOM neurons were scarcely contacted by KP terminals in the POA and VMH of both species while more SOM neurons with KP terminals were observed in the ovine ARC than in the rat ARC. Quantitative estimations are currently underway to strengthen these observations. Our data suggests a strong inhibitory action of SOM on KP neurons found in the ARC both in males and females of ovine and rodent species. This remains to be ascertained using other approaches such as electrophysiology.


P31: Inputs to arcuate kisspeptin neurons revealed by conditional viral transneuronal tracing Shel-‐Hwa Yeo, Victoria Kyle and William H. Colledge Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK Kisspeptin neurons are well established as the crucial regulator of reproduction and fertility in mammals. However, the circuitry controlling kisspeptin neurons remained uncharacterised. We therefore investigated the inputs onto ARN kisspeptin neurons using genetically restricted transsynaptic retrograde tracing strategy. We injected the conditional pseudorabies virus (Ba2001) into the ARN that can replicate only in Cre-‐expressing kisspeptin neurons. Since Ba2001 transports across synapses exclusively in a retrograde manner, we were able to identify a subset of the neurons upstream of the Cre-‐expressing ARN kisspeptin neurons. Expression of green fluorescent protein (GFP), the marker for both viral infection and replication, was observed in kisspeptin neurons within the injection site 32 hours post-‐injection (females, n=3; males, n=3). Two to three days post-‐injection, we identified forebrain inputs from the subfornical organ, ventral tuberomammillary, ventrolateral part of the ventromedial nucleus, paraventricular and periventricular nuclei. We also observed midbrain input from the interpeduncular nucleus. Interestingly, we observed a small subset of proopiomelanocortin (POMC)-‐expressing neurons in the ARN labelled with GFP 48-‐72 hours post-‐innoculation (n=3, females; n=2, males), indicating synaptic connectivity between POMC neurons and ARN kisspeptin neurons. Five days after Ba2001 injection, GFP-‐expressing cells were found in additional brain regions, the bed nucleus of stria terminalis, suprachiasmatic nuclei, posterodorsal part of the medial amygdala, amygdalohippocampal region and the medial habenula; which represented the secondary or tertiary afferents of the ARN kisspeptin neurons. Our observations demonstrate that ARN kisspeptin neurons receive direct projections from brain regions that are involved in appetite and metabolic regulation, sleep/wake cycle, stress response and sexual behaviour. This work is supported by BBSRC grant (BB/K003178/1).

P32: Vasopressin effect on intracellular calcium in pre-‐optic kisspeptin neurons Richard Piet1, Antoine Fraissenon1, Ulrich Boehm, 2, Allan E. Herbison, 1 1 Centre for Neuroendocrinology and Department of Physiology, Otago School of Medical Sciences, University of Otago, 9054 Dunedin, New Zealand. 2 Department of Pharmacology and Toxicology, University of Saarland School of Medicine, D-‐66421 Homburg, Germany The Luteinizing hormone (LH) surge driven by gonadotropin-‐releasing hormone (GnRH) neurons is crucial for ovulation in mammals. Kisspeptin neurons located in the rostral periventricular area of the third ventricle (RP3V) are thought to integrate ovarian steroids feedbacks and circadian inputs controlling LH surge timing. Recently, RP3V kisspeptin neurons were shown to express the arginine Vasopressin Receptor 1a (V1a) and to receive Arginine Vasopressin (AVP) projections from the Supra-‐Chiasmatic Nucleus (SCN), the biological clock driving circadian rhythms in mammalian. Previous works in the lab demonstrated that AVP increases RP3V kisspeptin neurons firing rate in an estrogen dependent manner. Here, we firstly explored the AVP effect on the intracellular calcium concentration ([Ca!!]!) in RP3V kisspeptin neurons using a transgenic mice expressing a genetically encoded calcium indicator called GCaMP3 in kisspeptin neurons. Following AVP administration, a significant and reversible [Ca!!]! increase was observed in RP3V kisspeptin neurons. Moreover, in presence of sodium channel and fast amino acid synaptic blockers, a second AVP application also induced [Ca!!]! increase in RP3V kisspeptin neurons. Secondly, we performed AVP administration on RP3V kisspeptin neurons from ovariectomized mice and demonstrated that ovarian steroid deprivation suppressed [Ca!!]! rise evoked by AVP. Altogether, we showed that AVP induces a reversible, reproducible and directly-‐mediated [Ca!!]! rise in RP3V kisspeptin neurons. We also demonstrated that ovarian mediators are required for [Ca!!]! increase evoked by AVP administration in RP3V kisspeptin neurons. Therefore, these data support that ovarian steroids influence how RP3V kisspeptin neurons integrate the vasopressinergic circadian input controlling the LH surge timing.

P33: Increased kisspeptin fibre innervation in the pregnant mouse paraventricular and supraoptic nuclei Rachael Augustine, Gregory Bouwer and Colin Brown Centre for Neuroendocrinology and Department of Physiology, University of Otago, New Zealand

Oxytocin secretion is required for normal birth. An increase in action potential firing of oxytocin neurons of the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON), releases oxytocin from the posterior pituitary gland into the circulation. Oxytocin causes uterine contractions, which aids in the delivery of the newborn. The trigger for increased oxytocin cell firing is largely unknown. However, we have shown that intracerebroventricular injection of the neuropeptide, kisspeptin, increases the firing rate of SON oxytocin neurons in vivo in late-‐pregnant rat but not in non-‐pregnant rats. Coincident with the emergence of kisspeptin excitation of oxytocin cells, is an increase in kisspeptin fibre density around the SON at the end of pregnancy. Here, we tested whether this plasticity in kisspeptin fibre density and oxytocin neuron response to kisspeptin is evident in pregnant mice. Kisspeptin and oxytocin double-‐label immunofluorescent staining was carried out on perfused brain slices from non-‐pregnant (n = 7), day 7, 14 and 19 (n = 8/group) pregnant and day 7 lactating (n = 7) mice. Sections were photographed on a confocal microscope and the average voxels/section (one section/animal) in the PVN and SON were analysed using FIJI as a measure of kisspeptin fibre density in the PVN and SON. Kisspeptin fibre density within the PVN of day 19 pregnant mice (337.0 ± 107.8 voxels/section) was higher than in non-‐pregnant mice (92.9 ± 25.01 voxels/section; P < 0.05). Similarly, kisspeptin fibre density within the SON of day 19 pregnant mice (68.3 ± 12.1 voxels/section) was higher than in non-‐pregnant mice (19.0 voxels/section ± 4.1; P < 0.05). We are also investigating whether oxytocin neurons in the PVN and SON of mice are activated after ICV kisspeptin administration using cFos as a marker of activation in these neurons. Preliminary results suggest ICV kisspeptin induces Fos in the SON and PVN but Fos is not co-‐expressed in oxytocin neurons and appears to be co-‐expressed in vasopressin neurons. Kisspeptin fibre innervation to the SON and PVN increases over pregnancy and might contribute to oxytocin neuron excitation at parturition but an acute kisspeptin injection at the end of pregnancy does not increase Fos expression in oxytocin neurons at this time.


P34: Kisspeptin does not affect excitatory postsynaptic current frequency or amplitude in magnocellular neurosecretory cells from virgin or pregnant rats Seymour, Alexander; Piet, Richard; Campbell, Rebecca; Brown, Colin Centre for Neuroendocrinology and Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand Oxytocin magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) and paraventricular nucleus secrete oxytocin from the posterior pituitary gland in response to action potential firing to cause contraction of the uterus during parturition and mammary gland ducts during lactation. The mechanisms that underpin oxytocin neuronal activity at these times remain poorly understood. Using in vivo electrophysiology in anaesthetised rats, we have shown that intracerebroventricular kisspeptin administration excites oxytocin neurons in late pregnant rats but not in virgin rats. To determine the mechanisms of this excitation, we performed whole-‐cell recordings of MNCs in hypothalamic slices from virgin and late-‐pregnant rats (gestation day 18 – 21). We measured miniature excitatory postsynaptic current (mEPSC) frequency and amplitude in response to kisspeptin at a holding potential of -‐60 mV under constant perfusion of 100 μM picrotoxin to block GABA-‐mediated events and 0.5 μM tetrodotoxin to block action potential-‐mediated events. While spontaneous mEPSC frequency was significantly lower in late-‐pregnant rats compared to virgin rats (P = 0.02, t-‐test), 1 μM kisspeptin did not affect the mEPSC frequency (P = 0.70, two way repeated measures ANOVA) or amplitude (P = 0.77) in MNCs from virgin or late-‐pregnant rats. The mean baseline current was also unchanged by kisspeptin (P = 0.82), which might indicate that kisspeptin has no direct effect on MNCs. These results suggest that the oxytocin MNC excitation by kisspeptin in late-‐pregnant rats is not a local effect on excitatory inputs. We are currently testing whether kisspeptin alters inhibitory postsynaptic currents in MNCs from virgin and late pregnant rats.

P35 : Sex-‐dependent effects of prenatal stress on social memory in rats Grundwald, Natalia; Brunton Paula J Division of Neurobiology, The Roslin Institute, University of Edinburgh, UK

Prenatal stress (PNS) affects a number of traits in the offspring including neuroendocrine stress axis regulation, cognition and emotionality. Here we investigated the effect of PNS on social recognition in the adult offspring of dams exposed to social stress during pregnancy. We first tested social preference using a modified 3 chamber sociability test: containing either a same sex conspecific, an object or a neutral chamber. Next using a social discrimination task, we assessed social memory with and without prior acute stress exposure (30 min restraint). Rats were initially exposed to 1, and after a lag time, 2 juveniles (1 known and 1 novel). Memory was assessed by comparing investigation times. Finally we assessed olfaction in female rats for social and non-‐social odours using wooden beads. Oxytocin (OT) and vasopressin play key roles in regulating social memory, thus central OT and vasopressin-‐1a (V1a) receptor mRNA expression was quantified using in situ hybridization. PNS has no effect on sociability in rats, however it impaired social memory under basal conditions in PNS females, but not PNS males. In accordance, V1a mRNA expression in the rostral part of the lateral septum and the bed nucleus of stria terminalis was significantly lower in PNS females, compared with controls. Acute stress impaired social memory in both sexes in control rats, however PNS males were not affected. Moreover, in PNS females social memory was significantly enhanced by prior stress exposure. Social memory deficits in PNS females persisted when social odours were used, however these social memory deficits do not appear to be linked to impaired olfaction as memory for non-‐social odours was similar in control and PNS females. PNS has a negative effect on social memory in females under basal conditions. However our data support the environmental mismatch hypothesis of prenatal programming, as the PNS females perform markedly better after acute stress than controls. Support:BBSRC/BSN

P36: Suppression of maternal HPA axis activity may be mediated indirectly by prolactin. Gustafson, Papillon, Bunn, Stephen, Grattan, Dave. Centre for Neuroendocrinology, University of Otago, Dunedin.

Stress during pregnancy and lactation is associated with increased postpartum maternal anxiety, impaired lactation, and the development of an anxiety phenotype in the offspring. To avoid these outcomes, the activity of the hypothalamic-‐pituitary-‐adrenal (HPA) axis is attenuated at these times. The mechanism generating this change is not known; however, the anterior pituitary hormone prolactin may play a significant role. This project aimed to investigate basal HPA axis activity during pregnancy and lactation in the mouse, focussing upon the corticotropin-‐releasing hormone (CRH) neurons. In situ hybridisation was used to characterise CRH mRNA expression in the hypothalamic paraventricular nucleus (PVN) of pregnant (Day 18; n=7), lactating (Day 7; n=7) and pup-‐deprived (24 h) lactating (Day 7; n=7) mice in comparison to virgin controls (n=6). Quantification of the number of CRH mRNA-‐expressing neurons in the PVN revealed significant reductions in both pregnant (49.6 ± 9.3) and lactating (53.6 ± 4.9) mice in comparison to controls (158.2 ± 21.5, P<0.0001), while removal of the pups, and thus the associated suckling-‐induced prolactin secretion, partially restored CRH neuron number (107.6 ± 9.4, P<0.05 versus lactation). Evidence of prolactin receptor expression and activation within the PVN suggested prolactin may be acting directly upon the CRH neurons. Using dual label immunohistochemistry for pSTAT5 (a marker of prolactin action) and green fluorescent protein expression (a marker of CRH neurons in a CRH-‐Cre reporter mouse line), no co-‐expression of pSTAT5 and GFP was found, suggesting prolactin does not directly regulate CRH neurons. These data show that basal HPA axis activity is suppressed in late pregnant and lactating mice and suggest a potential role for prolactin in mediating this suppression through an indirect mechanism. Future studies aim to determine the cellular target and precise role of prolactin in mediating maternal HPA axis suppression.


P37: Novel prolactin signalling in the median eminence of stressed male mice Kirk, Siobhan, Grattan, Dave, Bunn, Stephen Department of Anatomy and Centre for Neuroendocrinology, University of Otago. Specific stressors have been shown to elevate prolactin early in the stress response. Serum prolactin is regulated by dopaminergic inhibition from hypothalamic neurons. These neurons project to the median eminence (ME) where they release dopamine into the hypothalamic-‐pituitary portal vasculature. Elevated prolactin activates Signal Transducers and Activators of Transcription (STAT)5 in these and other cells. We have described a prolactin dependent activation of STAT5 in the ME. Unlike phospho-‐STAT5 in hypothalamic cells, which is nuclear located, the ME shows a distinct non-‐nuclear distribution, as shown by colabelling for phospho-‐STAT5 and DAPI. Male mice (n=15) were treated with either bromocriptine (10μg) or saline for 24h prior to the experiment. Mice were exposed to either restraint stress (+/-‐ prior treatment of bromocriptine), or home cage environment for 15 minutes prior to euthanasia using pentobarbitone, and transcardiac perfusion with 4% paraformaldehyde. Brains were excised and immunohistochemical preparations made, identifying phospho-‐STAT5 in ME sections. Analysis of nuclear labelling in the arcuate nucleus showed significant phospho-‐STAT5 in ventral regions when exposed to restraint stress (p<0.05). This response was abolished by bromocriptine, indicating it is prolactin dependent (p<0.05). Densitometric measures of granular labelling in the ME showed a similar response, with restraint stress increasing phospho-‐STAT5 from control levels (p<0.05), and bromocriptine treatment reducing the effect. Immunolabelling of prolactin-‐induced phospho-‐STAT5 with either vimentin or β-‐III tubulin (markers for tanycytes or neurons) identified colocalisation of phospho-‐STAT5 within neuronal processes, however the specific neuronal type has not been identified. These results show a novel response of the ME to prolactin during stress, suggesting that neurons in the ME are sensory to circulating prolactin.

P38: Prolactin receptor-‐mediated enkephalin expression in TIDA neurons of late pregnant and lactating mice Yip Siew Hoong, Williams Eloise, Grattan David, Bunn Stephen Centre for Neuroendocrinology, University of Otago, New Zealand

During late pregnancy and lactation, the tuberoinfundibular dopaminergic (TIDA) neurons, located in the arcuate nucleus exhibit a reduction in dopamine synthesis and induction of enkephalin expression. Evidence suggests that elevated prolactin may contribute to this phenomenon. The current study aimed to test the hypothesis that this induction of enkephalin expression in TIDA neurons is prolactin-‐dependent. Quantitative PCR analysis revealed that there was a significant reduction in the mRNA level of TH (a dopaminergic marker) of about 50%, while pro-‐enkephalin A mRNA level significantly increased by 10-‐ and 4-‐fold in the arcuate nucleus of late pregnant and lactating mice respectively compared to diestrous mice (n=5-‐6). While the TH mRNA level decreased during lactation, immunohistochemical analysis showed that the number of TH expressing cells was not significantly altered (92.3±8.2 lactating vs. 68.3±5.7 diestrous mice) (n=3). Consistent with the rise in enkephalin mRNA levels, the number of enkephalin-‐positive cells was significantly higher in lactating compared to diestrous mice (39.0±3.5 vs. 1.0±0.6 respectively; p<0.001) (n=3). Dual-‐labeling revealed approximately 80% of the enkephalin-‐positive cells in lactating mice were also TH-‐positive. The suppression of prolactin in lactating mice by bromocriptine administration significantly reduced the number of enkephalin-‐positive cells by approximately 50% (n=5-‐6), suggesting that enkephalin expression in TIDA neurons requires prolactin. This was further supported by deletion of prolactin receptor specifically in forebrain neurons, which abolished the increased number of enkephalin-‐positive cells seen in the late pregnant mice (n=5-‐6). This study therefore indicates that the induction and maintenance of enkephalin expression in TIDA neurons during late pregnancy and lactation is prolactin-‐dependent.

P39: Evaluating Prolactin-‐sensitive Somatostatin Neurones in the Rat Paraventricular Nucleus of the Hypothalamus I.C. Kokay, T.J. Sapsford, D.R. Grattan Centre for Neuroendocrinology, Department of Anatomy, University of Otago, Dunedin, New Zealand During lactation when circulating prolactin levels are high, prolactin has important actions in the brain. The paraventricular nucleus (PVN) contains a number of populations of neuroendocrine neurones that are defined based on the hormones they synthesise. We have identified prolactin-‐responsive cells in the PVN. Previously, we have shown that oxytocin neurones express prolactin receptors and using phosphorylated signal transducer and activator of transcription 5 (pStat5) as a marker of prolactin receptor activation, we have shown that oxytocin cells rapidly respond to administration of ovine prolactin (oPRL). In addition, in the PVN, we also have observed a population of non-‐oxytocin neurones that express prolactin receptors. As a substantial population of somatostatin (SST) neurones is present in the periventricular region of the PVN, we investigated whether these SST neurones respond to prolactin. We performed immunohistochemistry to determine whether SST neurones co-‐localised with prolactin–induced pStat5 following icv injection of oPRL. We also conducted in situ hybridization to verify the presence of prolactin receptor mRNA in SST neurones. We found that SST neurones responded strongly to administration of oPRL with around 80% of SST neurones co-‐localising with nuclear pStat5, strongly suggesting that SST neurones are prolactin–sensitive. Surprisingly, however, we found that very few SST neurones expressed prolactin receptor mRNA. It is possible that prolactin receptors are expressed in very low abundance on SST neurones and in situ hybridization methodology lacks the sensitivity to detect the mRNA. Alternatively, oPRL may activate non-‐prolactin receptors such as growth hormone receptors, that are present on SST cells. We are currently investigating this latter possibility by comparing the pattern of pStat5 activation following oPLR administration with that activated by rat prolactin.


P40: Microglia are not activated in the hypothalamic supraoptic or paraventricular nuclei at the onset of angiotensin II-‐dependent hypertension Korpal, Aaron; Schwenke, Daryl; Brown, Colin Centre for Neuroendocrinology and Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand Arginine vasopressin (AVP) magnocellular neurosecretory cells are located in the paraventricular nucleus (PVN) and supraoptic (SON) nucleus of the hypothalamus. AVP signalling causes systemic vasoconstriction as well as renal reabsorption of water at the site of the collecting duct, ultimately increasing blood pressure. Normally, AVP release is suppressed by tonic input arising from arterial baroreceptors, which detect acute changes in blood pressure. Decreased blood pressure attenuates this inhibitory input to allow vasopressin release, helping return blood pressure back towards normal. Paradoxically, some patients with essential hypertension exhibit an increased plasma concentration of AVP. We have found that AVP cell activity is increased early during the induction of moderate angiotensin (ANG) II-‐dependent hypertension. Microglia are activated in the PVN in severe ANG II-‐dependent hypertension, so we aimed to investigate whether microglia are activated in the SON and PVN during the onset of moderate hypertension. We used the Cyp1a1-‐Ren2 transgenic rat model, which allows for the controlled onset of ANG II-‐dependent hypertension by administering a diet containing 0.225% indole-‐3-‐carbinol over 7 days. We carried out immunohistochemistry for the brain microglia marker, ionized calcium-‐binding adapter molecule 1 (Iba1), to quantify microglial activation in the SON and PVN. A region of interest (ROI) was selected within the SON or PVN, and microglial activation was measured as percentage area above a set threshold of the ROI immunolabelled for Iba1. No increase in Iba1 thresholded area was observed in the SON (control = 9.2% vs. hypertensive = 11.3%; P = 0.19 unpaired t-‐test; n = 5 -‐ 7). Similarly, no change in Iba1 staining was observed in the PVN. In conclusion, we can eliminate microglia activation as a trigger for AVP cell activity in hypertension and, thus, refine the scope for identifying key targets in AVP cell activation.

P41: Electrophysiological properties of lateral septum neurons are modulated by oxytocin and vasopressin, two neuro-‐hormones involved in social behavior regulation Borie, A.M. (1)(2), Guillon G. (1)(2), Muscatelli, F. (3) and Desarménien, M.G. (1)(2) (1) Institut de Génomique Fonctionnelle, CNRS UMR5203, Inserm U1191, Montpellier, France (2) Université de Montpellier, Montpellier, France (3) INMED, Inserm U910, Marseille, France Central oxytocin (OT) and vasopressin (VP), have almost opposite roles in mammal behavior: OT facilitates social interactions while VP promotes aggressivity and anxiety, but their mechanism of action is unknown. In the lateral septum (LS), both OT and VP increase social interactions and social memory. This structure highly expresses OT and VP receptors but electrophysiological consequences of their activation have not been studied in detail so far. Using patch clamp recordings from acute brain slices, we characterized electrophysiological responses of mouse LS neurons to TGOT (a specific OT receptor agonist) and VP. Strikingly, the firing frequency of almost all recorded neurons was significantly modulated by at least one of these peptides. Accordingly, we classify LS neurons into three populations: i) activated by TGOT; ii) activated by VP; iii) inhibited by both. Since the septo-‐hippocampal network can modulate theta rhythm, we analyzed the effect of TGOT and VP on spike patterning. We show that modulation of electrical activity in the LS results from a modification of inter-‐burst intervals (1.5-‐5 s range) rather than intra-‐burst frequency (close to the theta rhythm, 3-‐5 Hz range). To decipher a local microcircuitery involving the three above-‐mentioned neuronal categories, we studied spontaneous synaptic events and demonstrate that they are mostly GABAergics. Their frequency is increased by TGOT and VP, opening the possibility that these peptides act locally by regulating interactions between interneurons. We observe similar effects of OT and VP, consistent with their behavioral action and demonstrate that OT and VP set the tone of electrical activity in the LS. Disruption of OT or VP systems in mouse leads to social deficits and this is broadly used to define animal models of social diseases. We will now compare LS modulation by TGOT and VP in these models and wild type animals.

P42: Intrinsic osmotic regulation of vasopressin neurons in virgin and lactating rats in vivo Brown, Colin; Jaquiery, Zoë; Bouwer, Gregory: Augustine, Rachael Centre for Neuroendocrinology and Department of Physiology, University of Otago, New Zealand

Body fluid balance is regulated in large part by the anti-‐diuretic actions of vasopressin in the kidney. Vasopressin secretion is stimulated by increased plasma osmotic pressure. During pregnancy and lactation, plasma osmotic pressure is reduced and plasma volume is increased. Despite the decreased plasma osmotic pressure, circulating vasopressin is paradoxically increased during lactation. Vasopressin release from the posterior pituitary gland is triggered by action potential firing in magnocellular neurosecretory neurons that are mainly located in the hypothalamic supraoptic nucleus and paraventricular nucleus. Vasopressin neuron activity is increased by osmosensitive TRPV1 channels that are activated by increasing extracellular osmolality. Here, we tested whether TRPV1 channels contribute to the spontaneous activity of vasopressin neurons in virgin and lactating rats by microdialysis administration of ruthenium red into the supraoptic nucleus to block TRPV1 channels during extracellular single unit recording in vivo. Ruthenium red reduced the firing rate of vasopressin neurons (P < 0.01, two-‐way repeated measures ANOVA) and the degree of inhibition was similar (P = 0.97) in virgin rats (n = 9) and lactating rats (n = 7). By contrast to its inhibition of vasopressin neurons, ruthenium red did not affect the firing rate of supraoptic nucleus oxytocin magnocellular neurosecretory neurons in virgin rats (P = 0.65, n = 5). Hence, it appears that the spontaneous activity of vasopressin neurons, but not oxytocin neurons, is driven, in part, by TRPV1 activity in vivo and that this intrinsic osmotic drive remains during lactation despite the decreased plasma osmotic pressure evident during lactation. We are currently completing western blotting for TRPV1 and dual-‐label immunohistochemistry for TRPV1 and vasopressin to determine the abundance and location of TRPV1 in the supraoptic nucleus of virgin and lactating rats.


P43: A vasopressin neuron network model – using dendritic communication to regulate heterogeneous population response MacGregor, Duncan; Leng, Gareth Centre for Integrative Physiology, University of Edinburgh The magnocellular vasopressin neurons form part of the homeostatic systems that maintain osmotic pressure. Experiments show a robust linear relationship between osmotic pressure and vasopressin hormone secretion despite very non-‐linear properties of spike generation and stimulus-‐secretion coupling in the neurons. Spiking activity across the population is also highly heterogeneous, but we have previously shown, in our spiking and secretion model that this heterogeneity actually helps to produce a linear population response, despite the non-‐linear response in individual neurons. We now consider is how to maintain this response during sustained osmotic challenge. In an unconnected heterogeneous population, the most active neurons rapidly deplete their vasopressin stores. Increased activity upregulates the rate of synthesis but not sufficiently to match the high rates of secretion. A role for dendritic communication may be to coordinate the neurons’ response, taking turns at being the most active, and allowing stores to recover. To test this we added synthesis to our model, based on a previous model using spike activity dependent upregulation of mRNA stores; and dendritic communication, turning the unconnected population model into a coordinated network. Dendritic vasopressin secretion acts to suppress the activity of neighbouring neurons, allowing the more active neurons to maintain their dominance until they deplete their stores. Assuming that dendritic stores deplete in parallel with the stores in the pituitary terminals, this can successfully cycle activity between neurons, dynamically controlling the heterogeneity to maintain the population response to sustained challenge. The network works most effectively when we connect the neurons in bundles, using a ‘small world’ network structure. MacGregor D, Leng G. (2013) Spike triggered hormone secretion in vasopressin cells; a model investigation of mechanism and heterogeneous population function. PLoS Comp Bio.

P44: Activation of presynaptic oxytocin receptors enhances glutamate release in the ventral hippocampus of prenatally restraint stressed rats Alexandre Beuttin1,6, Jérôme Mairesse1,6, Eleonora Gatta1,6, Marie-‐Line Reynaert1,6, Sara Morley-‐Fletcher1,6, Marion Soichot2, Lucie Deruyter1,6, Gilles Van Camp1,6, Hammou Bouwalerh1,6, Francesca Fagioli3, Anna Pittaluga4, Delphine Allorge2, Ferdinando Nicoletti5,6*, Stefania Maccari1,6*. 1Glycobiology of Stress-‐related Diseases team, UMR 8576 University of Lille1/CNRS, Villeneuve d’Ascq, France 2Faculté de Médecine de Lille, UDSL, Lille, France; 3Azienda Sanitaria Locale, RM.E. Unità Operativa Complessa Adolescent, 00100-‐Rome, Italy 4Dept. of Pharmacy, Univ. of Genoa, Genoa, Italy 5IRCCS Neuromed, Pozzilli, Italy 6International Associated Laboratory “Prenatal Stress and Neurodegenerative Diseases” France/Italy (Glycobiology of Stress-‐related Diseases team, UMR 8576 University of Lille1/CNRS, Villeneuve d’Ascq, France; Neuromed, Pozzilli, Italy and Sapienza University of Rome, Rome, Italy) Oxytocin receptors are known to modulate synaptic transmission and network activity in the hippocampus, but their precise function has been only partially elucidated. Here, we have found that activation of presynaptic oxytocin receptor with the potent agonist, carbetocin, enhanced depolarization-‐evoked glutamate release in the ventral hippocampus with no effect on GABA release. This evidence paved the way for examining the effect of carbetocin treatment in “prenatally restraint stressed” (PRS) rats, i.e. the offspring of dams exposed to repeated episodes of restraint stress during pregnancy. Adult PRS rats exhibit an anxious/depressive-‐like phenotype associated with an abnormal glucocorticoid feedback regulation of the hypothalamus-‐pituitary-‐adrenal (HPA) axis, and, remarkably, with a reduced depolarization-‐evoked glutamate release in the ventral hippocampus. Chronic systemic treatment with carbetocin (1 mg/kg, i.p., once a day for 2-‐3 weeks) in PRS rats corrected the defect in glutamate release, anxiety-‐ and depressive-‐like behavior, and abnormalities in social behavior, in the HPA response to stress, and in the expression of stress-‐related genes in the hippocampus and amygdala. Of note, carbetocin treatment had no effect on these behavioral and neuroendocrine parameters in prenatally unstressed (control) rats, with the exception of a reduced expression of the oxytocin receptor gene in the amygdala. These findings disclose a novel function of oxytocin receptors in the hippocampus, and encourage the use of oxytocin receptor agonists in the treatment of stress-‐related psychiatric disorders in adult life.

P45: Neuroglobine protects astroglial cells against oxidative stress-‐induced apoptosis AMRI Fatma universite aix marseille (France) -‐ universite tunis el manar (Tunisia) Oxidative stress resulting from the accumulation of reactive oxygen species (ROS) and / or reactive nitrogen species (RNS) plays an essential role in astroglial cell death associated with neurodegenerative diseases. An excess of a hydrogen peroxide (H2O2) causes an imbalance in production of ROS and an alteration of cellular antioxidant defense, leading to oxidative damage and cell death by apoptosis. Neuroglobin (Ngb) is a family member involved in vertebrate globin cellular homeostasis of oxygen. Ngb is mainly expressed in the nervous system, and acts as an important scavenger of toxic reactive species, since it may bind directly to the nitric oxide (NO). Ngb exerts a potent neuroprotective effects in ischemic or hypoxic conditions, and many neurodegenerative diseases, but little is known regarding its potential protective effect in astroglial cells. In this study, we propose to evaluate the potential role of Ngb to protect astrocytes against oxidative stress induced by H2O2. In fact, incubation of cultured astrocytes with Ngb inhibited a cell death and prevented the morphological modifications induced by H2O2. Thereby, Ngb reduces significantly the overproduction of ROS induced by H2O2.


P46: TLDA-‐based mRNA profiling of laser capture microdissected spinal cord white matter astrocytes reveals high estrogen/phenol sulfotransferase expression during neuroinflammation Guillot Flora (1,2), Garcia Alexandra (1-‐3), Salou Marion (1,2), Brouard Sophie (1-‐3), Lapaud David A (1-‐4), Nicot Arnaud B (1,2) (1) INSERM UMR1064/CRTI, Nantes, France ; (2) Université de Nantes, Faculté de Médecine, Nantes, France ; (3) ITUN, CHU de Nantes, France ; (4) Service de Neurologie, CHU de Nantes, France White matter (WM) reactive astrocytes are important players in amplifying neuroinflammation. However, their mRNA profile has not been specifically defined because they are difficult to purify, compared to gray matter astrocytes. Here, we isolated WM astrocytes by laser capture microdissection (LCM) in a murine model of multiple sclerosis to better define their molecular profile focusing on selected genes related to inflammation. Based on previous data indicating anti-‐inflammatory effects of 17beta estradiol only at high nanomolar doses, we also examined mRNA expression for enzymes potentially involved in steroid hormone inactivation. Experimental autoimmune encephalomyelitis (EAE) was induced in female C57BL6 mice with MOG35-‐55 immunization. FACS analysis of a portion of individual spinal cords at peak disease confirmed the infiltration of immune cells, composed mostly of CD4 T cells and macrophages. GFAP immunolabeled WM astrocytes were microdissected from spinal cord sections of control and EAE mice. TLDA (Taqman-‐low-‐density-‐array)-‐based mRNA profiling of WM astrocytes confirmed EAE-‐induced gene expression of proinflammatory cytokines or chemokines. Strikingly, among phase II metabolism enzymes, SULT1A1 was expressed in control WM spinal cord astrocytes. Moreover, its expression was further increased in EAE. Immunohistochemistry on spinal cord tissues confirmed preferential expression of this enzyme in WM astrocytic processes. In conclusion, we evidenced, in WM astrocytes, relative high astrocytic expression of SULT1A1, an enzyme having -‐ among several phenolic compounds -‐ estrogens as substrates. This high astrocytic expression may account for the relative resistance of this cell population to the anti-‐neuroinflammatory effects of estradiol. Supported by the Region Pays de Loire (AN).

P47: The impact of glucocorticoid hyporesponse to stress on memory function de Medeiros, G.F., Cerpa J.C., Helbling, J.C. , Ferreira GF, Lafenêtre P., Moisan, M.P. INRA, Université de Bordeaux, Nutrition et neurobiologie intégrée, Bordeaux, France

Glucocorticoids (GCs) are the adrenal hormones secreted in response to the HPA axis activation. GCs are known to influence memory processes, and their effects follow an inverted-‐U-‐shape dose–response relationship. Corticosteroid binding globulin (CBG), a plasma glycoprotein that binds GCs with high affinity, endorses a major role in regulating free GCs levels, especially under stress conditions. Our team has developed the Cbg ko mice which presents a blunted rise of GCs after stress and thus constitutes a model of GC hyporesponse. A study from our lab used the spontaneous alternation paradigm to demonstrate that the Cbg ko mice are insensitive to the memory retrieval impairments induced by stress due to a lower rise of GC levels in the brain, notably in the hippocampus. In this context, we were interested in investigating the impact of CBG deficiency , and thus GC hyporesponse, on hippocampus-‐dependent memory paradigms. For that, adult male Cbg ko and wild-‐type (wt) mice were submitted to the fear conditioning (FC) and to the novel object recognition test (NORT). The NORT was also performed with variations of the standard protocol, including habituation sessions between the familiarization and the test and a stress delivery right after the familiarization session. Cbg ko mice displayed an impaired association between the context and the footshock in the FC when compared to wt mice. In addition, only wt animals showed increased exploration of the novel object in the NORT, while Cbg ko mice’s exploration levels did not differ from chance level. Habituation sessions in the NORT were able to enhance the performance of Cbg ko mice, while the stress delivery right after the familiarization session not only enhanced the performance of the Cbg ko mice, but also impaired the performance of wt animals. Altogether, these results suggest that the lower GC levels in the hippocampus due to CBG deficiency might underlie the memory impairment observed in Cbg ko mice.

P48: Interactions of corticosteroids and estrogens that influence aggression in male mice Rainville, Jennifer., Clark, Sara., Kemp, Brekel., and Vasudevan, Nandini

1: Cell and Molecular Biology Department, Tulane University, New Orleans, LA 70118. 2: Neuroscience Program, Tulane University, New Orleans, LA 70118.

Steroid hormones such as 17β-‐estradiol (17β-‐E) and corticosterone (cort) are necessary for the display of aggression in male rodents. Both hormones slowly regulate genomic transcription by binding classical nuclear receptors but also rapidly and non-‐genomically regulate kinases or calcium via a putative receptor on the plasma membrane. Though it has been implicitly assumed that aggression is dependent on genomic signaling, rapid signaling by 17β-‐E or cort increased aggression in rodents within 20 minutes. Though this short time-‐scale is indicative of non-‐genomic signaling, the relative contributions of genomic vs. non-‐genomic signaling by either hormone to aggression are not well understood. Our aim was to elucidate the control of hormone concentrations and the signaling mechanisms that mediate aggression. Since previous experimental approaches do not allow one to distinguish the contribution of non-‐genomic signaling, we developed a novel paradigm where an aromatase inhibitor, letrozole, given in drinking water for two weeks, reduced 17β-‐E levels and aggressive behavior in male mice, compared to vehicle treated mice. Injection of cyclodextrin conjugated 17β-‐E (cE2) rapidly increased aggressive behavior in male mice within 20 minutes, suggesting that non-‐genomic signaling is sufficient to driven aggression. cE2 also restored cort levels that were decreased in letrozole treated mice, within 20 minutes of injection. These data suggest that 17β-‐E rapidly increases cort which could in turn increase aggressive behavior. Surprisingly, a 48-‐hour treatment of hypothalamic cultures with the GR agonist, dexamethasone increased neuroestrogen concentration, possibly via aromatase transcription. Hence, intermale aggression could be controlled by a “feed-‐forward loop” of cort and 17β-‐E that signal via both non-‐genomic and genomic mechanisms to maintain the behavior. Supported by NSF CAREER IOS-‐ IOS-‐1053716 and Tulane startup funds to N.V.


P49: Effect of early life stress on synaptic plasticity in the developing hippocampus of male and female rats N.A.V. Derks1, R.A. Sarabdjitsingh1, H.J. Krugers2, C.C. Hoogenraad3, M. Joëls1 1) Dept Translational Neuroscience, Brain Center Rudolf Magnus, Univ. Medical Center Utrecht. 2) Swammerdam Institute for Life Sciences, Univ. of Amsterdam. 3) Dept Cell Biology, Faculty of Science, Utrecht University.

Early life stress (ELS) increases the risk for developing psychopathology in adulthood. How brain development is affected by ELS before symptoms emerge in adults is largely unknown. An established rodent model for ELS is 24h maternal deprivation (MD). Earlier studies showed that MD reduces hippocampal synaptic plasticity and impairs spatial memory functioning in adult rats. The aim of the present study is to determine how MD affects the development of hippocampal synaptic plasticity from early life up to adulthood. Male and female Wistar rats were maternally deprived for 24h on postnatal day (PND)3 or left undisturbed with their mother (control). On PND4, 8, 22 and 90, plasma corticosterone (CORT) levels, body weight and thymus and adrenal weights were determined to validate stress effects of MD. Furthermore, field potentials in the CA1 hippocampus were recorded in vitro before and after high frequency stimulation. Brain slices were incubated with 100nM CORT, to mimic high-‐stress conditions in vitro, or vehicle. Preliminary results: At PND4 and 8, rats showed a trend towards increased basal CORT levels after MD, which was normalized again in adults. Reduced body weight and increased adrenal weight were seen in PND90 MD females compared to controls, indicating a partial long-‐term effect of MD on the stress system. In line with previous studies, we found a CORT-‐induced suppression of LTP in control PND90 males; this was not seen in females. PND90 females did show a trend towards more stable LTP after MD, which was reversed by CORT. Furthermore, age in itself affected field potential size by yielding smaller maximum slopes on PND4 and 8 while requiring a larger stimulus intensity than adults. Future recordings will indicate how MD affects synaptic plasticity in early life and during adolescence. Together, this information will enhance our understanding of the mechanisms behind the development of psychopathology and indicate possible windows for intervention after ELS.

P50: Sex-‐dependent trans-‐generational effects of prenatal stress on the neuroendocrine stress axis and anxiety-‐like behaviour in rats Grundwald, Natalia J. and Brunton, Paula J. Division of Neurobiology, The Roslin Institute, University of Edinburgh, Easter Bush Campus, Midlothian, EH25 9RG, UK

Exposure to social stress during pregnancy results in hyperactive hypothalamo-‐pituitary-‐adrenal (HPA) axis stress responses in the adult male and female offspring and heightened anxiety behaviour in the males, but not the females. Here we tested whether the effects of prenatal stress (PNS) are transmitted to the second filial generation (F2) via the maternal line. F1 control and PNS female rats were mated with control males and housed under non-‐stress conditions throughout pregnancy. HPA axis responses to stress and anxiety-‐like behaviour were assessed in the adult F2 offspring. ACTH and corticosterone responses to acute stress were markedly enhanced/prolonged in F2 PNS females. This was associated with greater levels of corticotropin releasing hormone (Crh) mRNA in the paraventricular nucleus and reduced glucocorticoid (Gr) and mineralocorticoid receptor (Mr) mRNA expression in the hippocampus. Whereas in the F2 PNS males, HPA axis responses to acute stress were attenuated and hippocampal Gr and Mr mRNA expression was greater compared with controls. F2 PNS males exhibited greater anxiety-‐like behaviour (light-‐dark box and elevated plus maze) compared with control F2 males. Anxiety-‐like behaviour did not differ between F2 control and PNS females during metestrus/diestrus, however at proestrus/estrus, F2 control females displayed a reduction in anxiety-‐like behaviour, but this effect was not observed in the F2 PNS females. Crh mRNA expression was significantly greater in the central nucleus of the amygdala in F2 PNS males compared with controls, but there was no difference in the F2 females. Moreover, CRH receptor-‐1 (Crhr1) mRNA expression was significantly increased, whereas Crhr2 was significantly decreased in discrete regions of the amygdala in F2 PNS males compared with controls, with no differences in the F2 females. In conclusion, some of the effects of PNS are transmitted to subsequent generations and this occurs in a sex-‐dependent manner. [Support: BBSRC]

P51: Altered circadian locomotor activity in the males and females rats prenatally restraint stressed, a model of depression. Remy Verhaeghe1,4, G. Van Camp1,4, M. L Reynaert1,4, E Gatta1,4, S. Morley-‐Fletcher1,4, R Ngomba2, A. Tramutola3, P. Navarra3, S. Maccari1,4, F. Nicoletti2,4 and J. Mairesse1,4 1Glycobiology of Stress-‐related Diseases team, UMR 8576 University of Lille1/CNRS, Villeneuve d’Ascq, France 2IRCCS, NEUROMED, Pozzilli, Italy 3Instituto di Farmacologia, Univ. Catt. Sacro Cuore, Rome, Italy 4International Associated Laboratory “Prenatal Stress and Neurodegenerative Diseases” France/Italy (Glycobiology of Stress-‐related Diseases team, UMR 8576 University of Lille1/CNRS, Villeneuve d’Ascq, France; Neuromed, Pozzilli, Italy and Sapienza University of Rome, Rome, Italy)

Prenatal restraint stress (PRS) in rats is a well-‐documented model of early stress known to induce depression-‐like behaviour. Recently, we have shown that in male PRS rats the decrease of glutamate release in the ventral hippocampus is associated with reduction of the expression of synaptic-‐vesicle-‐ associated proteins. Conversely, the pharmacological enhancement of glutamate release of the animals in vivo and the chronic antidepressant treatment is able to reverse anxiety-‐like behavior. Interestingly, most the enduring changes induced by PRS are sex-‐dependent, with a depression-‐like phenotype being present in both males and females, and an anxiety-‐like phenotype predominating in males. Because PRS also results in changes in circadian rhythms and sex dependence is not defined, we studied whether changes in circadian patterns caused by PRS are also sex-‐dependent. Here, we examined the relationships between PRS, gender and the circadian system by monitoring the running wheel behaviour in male and female adult PRS rats, first under a regular light-‐dark (LD) cycle, and then after an abrupt 6h advance shift in the LD cycle. Furthermore, to investigate whether hypothalamic modifications are associated with these circadian behavioural activities, we also measured the hypothalamic CRH content in males and females at 08h00 and at 20h00. Among biological substrates, we studied if hormones such as cortisol and also a driving circadian hormone, melatonin are involved in these circadian differences. Finally, measured gene expression of biological clock parameters (clock, Bmal, Per and cry) in suprachiasmatic nucleus. Our results showed a pattern of locomotor activity in PRS rats that was erratic and more fragmented, particularly in female PRS rats. PRS increased and decreased total locomotor activity in males and females, respectively, and induced a significant phase advance in the rhythm of circadian activity only in males.


P52: Evidence for an imbalance between tau O-‐GlcNAcylation and phosphorylation in the hippocampus of a mouse model of Alzheimer’s disease Eleonora Gatta1,7, Tony Lefebvre2, Silvana Gaetani3, Marc Dos Santos4, Tommaso Cassano5, Ferdinando Nicoletti6,7, Stefania Maccari1,7, Jérôme Mairesse1,7 1Glycobiology of Stress-‐related Diseases team, UMR 8576 University of Lille1/CNRS, Villeneuve d’Ascq, France 3Dept of Physiology and Pharmacology, Sapienza University of Rome, 00185-‐Rome, Italy 4INSERM, UMR-‐S 1130, Neuroscience Paris Seine, 75005-‐Paris, France 5Dept of Clinical and Experimental Medicine, University of Foggia, 71100-‐Foggia, Italy 6IRCCS Neuromed, 86077-‐Pozzilli, Italy 7International Associated Laboratory “Prenatal Stress and Neurodegenerative Diseases” France/Italy (Glycobiology of Stress-‐related Diseases team, UMR 8576 University of Lille1/CNRS, Villeneuve d’Ascq, France; Neuromed, Pozzilli, Italy and Sapienza University of Rome, Rome, Italy) Intracellular accumulation of hyperphosphorylated tau protein is linked to neuronal degeneration in Alzheimer’s disease (AD). Mounting evidence suggests that tau phosphorylation and O-‐N-‐acetylglucosamine glycosylation (O-‐GlcNAcylation) are mutually exclusive post-‐translational modifications. O-‐GlcNAcylation depends on 3-‐5% of intracellular glucose that enters the hexosamine biosynthetic pathway. To our knowledge, the existence of an imbalance between tau phosphorylation and O-‐GlcNAcylation has not been reported in animal models of AD, as yet. Here, we used triple transgenic (3xTg-‐AD) mice at 12 months, an age at which hyperphosphorylated tau is already detected and associated with cognitive decline. In these mice, tau was hyperphosphorylated on both Ser396 and Thr205 in the hippocampus, and to a lower extent and exclusively on Thr205 in the frontal cortex. Tau O-‐GlcNAcylation, assessed in tau immunoprecipitates, was substantially reduced in the hippocampus of 3xTg-‐AD mice, with no changes in the frontal cortex or in the cerebellum. No changes in the expression of the three major enzymes involved in O-‐GlcNAcylation, i.e., glutamine fructose-‐6-‐phosphate amidotransferase, O-‐linked β-‐N-‐acetylglucosamine transferase, and O-‐GlcNAc hydrolase were found in the hippocampus of 3xTg-‐AD mice, raising the interesting possibility that the amount of glucose entering the hexosamine pathway is reduced in the hippocampus of 3xTg-‐AD mice. These data demonstrate that an imbalance between tau phosphorylation and O-‐GlcNAcylation exists in AD mice, and strengthens the hypothesis that O-‐GlcNAcylation might be targeted by disease modifying drugs in AD.

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ABSTRACT BOOK - hypothalamus.euhypothalamus.eu/wp-content/uploads/BSN-SNE2015_Program_low.pdf · LocalOrganizingCommittee !! Vincent Prévot, Chair Bénédicte Dehouck, Deputy Chair

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