11th world congress on neurohypophysial...
TRANSCRIPT
PROGRAMME and
ABSTRACTS
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Programme-at-a-glance
Saturday 29 August
12.00 pm Registration opens
12.00 pm-2.00 pm Poster Mounting
2.00 pm-2.30 pm Opening: Helen Nicholson, University of Otago
2.30 pm-3.30 pm Plenary Lecture 1: Larry Young, Emory University
3.30 pm-4.00 pm Afternoon Coffee
4.00 pm-6.00 pm Symposium 1: Molecular and cellular aspects of magnocellular neurons
6.00 pm Welcome Reception
Sunday 30 August
9.00 am-10.00 am Plenary Lecture 2: Andries Kalsbeek, Netherlands Institute for Neuroscience
10.00 am-10.30 am Morning Tea
10.30 am-12.30 pm Symposium 2: Oxytocin and behaviour
12.30 pm-1.30 pm Lunch
1.30 pm-3.30 pm Young Investigator Prize Presentations and Datablitz Presentations 3.30 pm-4.00 pm Afternoon Coffee
4.00 pm-7.00 pm Poster Presentations Odd numbered posters @ 4.00pm-5.30pm Even numbered posters @ 5.30pm-7.00pm
8.00 pm Speakers’ Dinner: Sasso Young Investigators’ Dinner: Flame Bar and Grill
Monday 31 August
9.00 am-10.00 am Plenary Lecture 3: Mike Ludwig, University of Edinburgh
10.00 am-10.30 am Morning Tea
10.30 am-12.30 pm Symposium 3: Vasopressin secretion in health and disease
12.30 pm-1.30 pm Lunch
1.30 pm-3.30 pm Young Investigator Presentations
3.30 pm-4.00 pm Afternoon Coffee and Poster Removal
4.00 pm-6.00 pm Symposium 4: Neurohypophysial hormones acting at the hippocampal CA2 regulate social behaviours
8.00 pm Congress Dinner: Jervois Steak House
Tuesday 1 September
9.00 am-10.00 am Plenary Lecture 4: Paul Alewood, University of Queensland
10.00 am-10.30 am Morning Tea
10.30 am-12.30 pm Symposium 5: Integration of central and peripheral systems
12.30 pm-1.30 pm Plenary Lecture 5: Joe Verbalis, Georgetown University
1.30 pm-2.00 pm Closing: Colin Brown, University of Otago, and Maria José Alves da Rocha, Universidade de São Paulo, Brazil
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General information
Venue Please contact for the venue for services and facilities available. Millennium Hotel Queenstown 32 Frankton Road Queenstown Phone: +64 3 450 0150 Fax: +64 3 441 8779 E-mail : [email protected] Website: http://www.millenniumhotels.co.nz/millenniumqueenstown/ Queenstown Queenstown is the Southern Hemisphere’s premier four season lake and alpine resort. See more at: http://www.queenstownnz.co.nz/. Weather WCNH 2015 is a winter meeting. Please dress appropriately. Queenstown weather information can be found here: http://www.metservice.com/towns-cities/queenstown. Conference Catering Catering is provided for the meeting. In addition to the welcome reception, Congress, Speakers’ and Young Investigators’ Dinners, morning and afternoon tea/coffee and lunches will be provided. Please advise the team from Dinamics (http://www.dinamics.co.nz/) if you have any special dietary requirements. Dining out Queenstown has many restaurants and bars to suit most tastes and budgets. When dining out, please note that tips are not normal, but will not cause offence. Queenstown Research Week WCNH 2015 is being held in association with Queenstown Research Week. There are many meetings and events that you may attend in addition to the WCNH schedule; see http://www.queenstownresearchweek.org/. Speakers’ Instructions A PC will be available for PowerPoint presentations. Please load and check your presentation during one of the breaks. If you are unable to use the PC provided, you may use your own laptop but please note that the technical support are volunteers and might not be able to assist troubleshooting any problems that you have with connecting to the projector. Poster Presentation Instructions Please mount your poster on the numbered board that corresponds to your abstract number. Posters should be mounted during registration on Saturday afternoon and removed on Tuesday morning. Poster boards are 1.2 m wide x 2.3 m high. We recommend A0 portrait format. Odd numbered posters should be staffed at 4.00pm-5.30pm, and even numbered posters at 5.30pm-7.00pm on Sunday.
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Venues
Conference Scientific Sessions: Millennium Hotel, Frankton Road Conference Dinner: Jervois Steak House, Brecon Street Speakers’ Dinner: Sasso Italian Dining, Church Street Young Investigators’ Dinner: Flame Bar & Grill, Beach Street Nearest Bar: Pig & Whistle, Ballarat Street Late-opening Bar: Winnies, Mall Street
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Organisers
Local Organising Committee Colin Brown, University of Otago, New Zealand Dave Grattan, University of Otago, New Zealand Iain McGregor, University of Sydney, Australia Karl Iremonger, University of Otago, New Zealand Pawel Olszewski, University of Waikato, New Zealand International Advisory Committee Alastair Ferguson, Queen's University, Canada Ben Nephew, Tufts University, USA Bice Chini, University of Milan, Italy Celia Sladek, University of Colorado, USA Charles Bourque, McGill University, Canada David Murphy, University of Bristol, UK Gareth Leng, University of Edinburgh, UK Gil Levkowitz, Weizmann Institute of Science, Israel Gina Yosten, Saint Louis University, USA Inga Neumann, University of Regensburg, Germany Maria José Alves da Rocha, Universidade de São Paulo, Brazil Maurice Manning, University of Toledo, USA Mike Ludwig, University of Edinburgh, UK Oliver Bosch, University of Regensburg, Germany Paula Brunton, University of Edinburgh, UK Ryoichi Teruyama, Louisiana State University, USA Scott Young, National Institute of Mental Health, USA Simone Meddle, University of Edinburgh, UK Tatsu Onaka, Jichi Medical University, Japan Valery Grinevich, Max Planck Institute for Medical Research, Germany William Armstrong, University of Tennessee, USA Yoichi Ueta, University of Occupational and Environmental Health, Japan Zouxin Wang, Florida State University, USA Conference Management Dinamics PO Box 558 Queenstown Phone: +64 3 441 1515 Fax: +64 3 441 1575 E-mail: [email protected] Website: http://www.dinamics.co.nz/
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Programme
Saturday 29 August
12.00 pm Registration opens
12.00 pm-2.00 pm Poster Mounting
Opening Address Chaired by Colin Brown (University of Otago, NZ)
2.00 pm-2.30 pm Helen Nicholson Deputy Vice-Chancellor (External Engagement), University of Otago, NZ
Plenary Lecture 1 Chaired by
Geert de Vries (Georgia State University, USA)
2.30 pm-3.30 pm Larry Young Emory University, USA Oxytocin and the neural mechanisms of social cognition
3.30 pm-4.00 pm Afternoon Coffee
Symposium 1: Molecular and cellular aspects of magnocellular neurons Chaired by
Gil Levkowitz (Weizmann Institute of Science, Israel) Hitoshi Ozwawa (Nippon Medical School, Japan)
4.00 pm-4.30 pm Katsuhiko Nishimori Tohoku University, Japan Molecular genetic study for the regulatory mechanism of social behavior regulated by GABAergic neurons, expressing oxytocin receptor
4.30 pm-5.00 pm Valery Grinevich University of Heidelberg, Germany Central axonal oxytocin pathways: pain, fear and sociality
5.00 pm-5.30 pm Yoichi Ueta University of Occupational and Environmental Health, Kitakyushu, Japan Electrophysiological study combined with fluorescent imaging and optogenetic approaches in rat vasopressin neuron
5.30 pm-6.00 pm Mingkwan Greenwood University of Bristol Transcription factor CREB3L1 mediates the cAMP regulation of the vasopressin
6.00 pm Welcome Reception
Sunday 30 August
Plenary Lecture 2 Chaired by
Alex Tups (University of Otago, NZ)
9.00 am-10.00 am Andries Kalsbeek Netherlands Institute for Neuroscience Vasopressin neurons in the suprachiasmatic nuclei (SCN): Critical signaling inside and outside the biological clock
10.00 am-10.30 am Morning Tea
Symposium 2: Oxytocin and behaviour Chaired by
Pawel Olszewski (University of Waikato, NZ)
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Yannis Paloyelis (King’s College London, UK)
10.30 am-11.00 am Pawel Olszewski University of Waikato, NZ Oxytocin as a secretagogue of behaviours: Where does feeding regulation fit in the puzzle?
11.00 am-11.30 am Adam Guastella University of Sydney, Australia Oxytocin therapeutics for psychiatric disorders: Applications and mechanisms
11.30 am-12.00 pm Oliver Bosch University of Regensburg, Germany When she’s gone: Emotional consequence of partner loss due to suppression of oxytocin signaling in the NAc shell in male prairie voles
12.00 pm-12.30 pm Michael Bowen University of Sydney, Australia Oxytocin inhibits ethanol consumption and intoxication in rats: interactions with dopamine and extrasynaptic GABAA receptors
13.30 pm-1.30 pm Lunch
Young Investigator Prize Presentations and Datablitz Presentations Chaired by
Mike Ludwig (University of Edinburgh, UK)
1.30 pm-1.50 pm Alison Douglas Young Investigator Award Winner Katrina Choe UCLA, USA, and McGill University, Canada Diverse morphology and structural plasticity of glial cells in the supraoptic nucleus
1.50 pm-2.10 pm Glenn Hatton Postdoctoral Award Winner Adam Smith National Institute of Mental Health, USA Vasopressin enhances social memory: Illuminating a PVN to hippocampal CA2 circuit
2.10 pm-2.30 pm Glenn Hatton Student Award Winner Joon S. Kim University of Otago, NZ Blockade of the anti-opioid effects of the Neuropeptide FF System using a novel antagonist reverses morphine tolerance in vasopressin neurons
2.30 pm-3.30 pm Datablitz Presentations
3.30 pm-4.00 pm Afternoon Coffee
4.00 pm-7.00 pm Poster Presentations Odd numbered posters @ 4.00pm-5.30pm Even numbered posters @ 5.30pm-7.00pm
8.00 pm-late Dinner Speakers’ Dinner: Sasso, 14-16 Church St, Queenstown http://sasso.co.nz/ Young Investigators’ Dinner: Flame Bar and Grill, 61 Beach St, Queenstown http://flamegrill.co.nz/
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Monday 31 August
Plenary Lecture 3 Chaired by
Jeff Tasker (Tulane University, USA)
9.00 am-10.00 am Mike Ludwig University of Edinburgh, UK There is more to vasopressin than meets the eye
10.00 am-10.30 am Morning Tea
Symposium 3: Vasopressin secretion in health and disease Chaired by
José Lemos (University of Massachusetts, USA) André de Souza Mecawi (Federal Rural University of Rio de Janeiro, Brazil)
10.30 am-11.00 am Mike McKinley Florey Institute, Melbourne, Australia Inhibitory influence of median preoptic/medial septal/diagonal band region on suppression of vasopressin secretion and rapid excretion of a water load
11.00 am-11.30 am Song Yao Florey Institute, Melbourne, Australia Blood-brain barrier disruption activates vasopressinergic PVN neurons
11.30 am-12.00 pm Evelin Cappellari do Cárnio Universidade de São Paulo, Brazil Regulation of vasopressin secretion during septic shock
12.00 pm-12.30 pm Maria José Alves da Rocha Universidade de São Paulo, Brazil Oxidative stress in vasopressinergic neurons during sepsis
12.30 pm-1.30 pm Lunch
Young Investigator Presentations Chaired by
Marta Busnelli (Institute of Neuroscience, Milan, Italy) Mike Greenwood (University of Bristol, UK)
1.30 pm-1.50 pm Thomas Grund University of Regensburg, Germany The anxiolytic effect of neuropeptide S is mediated via the oxytocin system
1.50 pm-2.10 pm Benjamin Jurek University of Regensburg, Germany Molecular effects of oxytocin on stress: Oxytocin delays CRF gene expression through CRTC3
2.10 pm-2.30 pm Arthur Lefevre CNRS and Université Claude Bernard Lyon 1, France Neural mechanisms of oxytocin and serotonin interaction in non-human primates
2.30 pm-2.50 pm Natalie Mills Louisiana State University, USA Aldosterone activated mineralocorticoid receptor regulation of epithelial sodium channels (ENaC) in rat hypothalamic vasopressin-synthesizing neurons
2.50 pm-3.10 pm Luis Paiva University of Edinburgh, UK Central activation of oxytocin neurons by Melanotan-II
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3.10 pm-3.30 pm Xander Seymour University of Otago, NZ Emergence of a kisspeptin projection from the periventricular nucleus to the perinuclear zone of the supraoptic nucleus during pregnancy
3.30 pm-4.00 pm Afternoon Coffee
Symposium 4: Neurohypophysial hormones acting at the hippocampal CA2 regulate social behaviours Chaired by
Dave Grattan (University of Otago) Simone Meddle (University of Edinburgh, UK)
4.00 pm-4.30 pm Limei Zhang Universidad Nacional Autónoma de México, Mexico Hypothalamic vasopressin system innervation of the hippocampus: Characteristics and functional implications
4.30 pm-5.00 pm Heather Caldwell Kent State University, USA A role for the Avpr1b in social behaviours: what we have learned from Avpr1b knockout mice and lesion studies
5.00 pm-5.30 pm Serena Dudek National Institute of Environmental Health Sciences, USA Oxytocin and vasopressin induce synaptic potentiation in CA2 pyramidal neurons
5.30 pm-6.00 pm Scott Young National Institute of Mental Health, Bethesda, USA Vasopressin innervation of the hippocampal CA2 area enhances social memory
8.00 pm-late Congress Dinner Jervois Stake House, Brecon Street, Lower Steps, Queenstown http://www.jervoisqueenstown.co.nz/
Tuesday 1 September
Plenary Lecture 4 Chaired by
Iain McGregor (University of Sydney, Australia)
9.00 am-10.00 am Paul Alewood University of Queensland, Australia Selenoether oxytocin analogues are analgesic in a mouse model of chronic abdominal pain
10.00 am-10.30 am Morning Tea
Symposium 5: Integration of central and peripheral systems Chaired by
Karl Iremonger (University of Otago, NZ) Ryoichi Teruyama (Louisiana State University, USA)
10.30 am-11.00 am Charles Bourque McGill University, Montreal, Canada Salt loading increases blood pressure via BDNF-mediated downregulation of KCC2 and impaired baroreceptor inhibition of vasopressin neurons
11.00 am-11.30 am Joanna Dabrowska Rosalind Franklin University, Chicago, USA Stress induced oxytocin release – modulation by corticotropin releasing factor peptide family
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11.30 am-12.00 pm Richard Piet University of Otago Mouse preoptic kisspeptin neurons integrate estrogen feedback and vasopressin signaling in female mice: implications for the generation of the preovulatory gonadotropin releasing hormone surge
12.00 pm-12.30 pm Tom Cunningham University of North Texas, USA Regulation of vasopressin neurons and fluid balance in a model of dilutional hyponatremia
Plenary Lecture 5 Chaired by
Allan Herbison (University of Otago, NZ)
12.30 pm-1.30 pm Joe Verbalis Georgetown University, USA Clinical and translational applications of vasopressin and oxytocin: Groundbreaking past, exciting future
Closing Address
1.30 pm-2.00 pm Colin Brown University of Otago, NZ Maria José Alves da Rocha Universidade de São Paulo, Brazil
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Poster Presentation Summary *Poster prize candidate, prizes sponsored by the Otago Centre for Neuroendocrinology
No. Title Presenter Affiliation 1 Induction of angiotensin II-dependent
hypertension does not activate microglia in the hypothalamic supraoptic and paraventricular nuclei
*Aaron Korpal University of Otago, NZ
2 Love drug to treat abdominal pain Aline Dantas de Araujo University of Queensland, Australia
3 Effects of arginine vasopressin (AVP) in the long term regulation of arterial blood pressure in the rat
Andres Quintanar-Stephano
Universidad Autónoma de Aguascalientes, México
4 Effects of arginine vasopressin on innate immunity in the rat
Andres Quintanar-Stephano
Universidad Autónoma de Aguascalientes, México
5 Oxytocin and familiarity: preferences for conspecifics and objects in male mice
Anica Klockars University of Waikato, NZ
6 Study of combined action of vasopressin receptor agonists and GLP-1 mimetic on renal sodium transport
Anna Kutina Russian Academy of Sciences, Russia
7 Visualization of vasopressin neurons in hypothalamic organotypic cultures using vasopressin-Venus transgenic mice
Daisuke Hagiwara Nagoya University Graduate School of Medicine, Nagoya, Japan
8 Vasopressin receptors and renal effects of arginine-vasopressin and arginine-vasotocin in rat kidney
*Darya Golosova Russian Academy of Sciences, Russia
9 Whole body pharmacokinetic distribution of intranasally delivered oxytocin in rats
Dean Carson Stanford University, USA
10 A network model of vasopressin neurons – using dendritic communication to regulate heterogeneous population response
Duncan MacGregor University of Edinburgh, UK
11 Oxytocin affects feeding for energy and reward via the nucleus accumbens core
*Florence Herisson University of Waikato, NZ
12 Insect neuropeptides as novel ligands for human oxytocin and vasopressin receptors
Christian Gruber Medical University of Vienna, Austria
13 Contrasting the organizational vs. activational roles of the oxytocin system in the regulation of zebrafish social behavior
Rita Nunes Weizmann Institute of Science, Israel
14 Osmotic responsiveness of supraoptic nucleus neurons in virgin and lactating rats
Greg Bouwer University of Otago, NZ
15 The effect of P2X7 receptor blockers on ATP release from rat hypothalamic slices
Hana Zemkova Czech Academy of Sciences, Czech Republic
16 Kiss1r expression in the rat hypothalamic oxytocin neurons
Hitoshi Ozawa Nippon Medical School, Japan
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17 Social deficits in dysbindin-1 knockout mice are ameliorated by intranasal oxytocin
Huiping Huang Istituto Italiano di Tecnologia, Italy,
18 Can acute intranasal oxytocin modulate neural responses to socio-emotional cues in a neurodegenerative population?
Izelle Labuschagne Australian Catholic University, Australia
19 Discovery of an oxytocin-like plant peptide as template for oxytocin and vasopressin receptor ligand design
Johannes Koehbach Medical University of Vienna, Austria
20 Modulatory role of zinc on neuropeptide release from neurohypophysis
José Lemos Massachusetts Medical School, USA
21 Re-examining the importance of vasopressin receptors in myometrial contraction
Markus Muttenthaler University of Queensland, Australia
22 Unveiling the mechanism of action and superagonism of oxytocin receptors bivalent ligands
Marta Busnelli Institute of Neuroscience, Italy
23 Upregulation of gonadotrophin inducible transcription factor 1 mRNA in the PVN and SON by cAMP pathways
Mike Greenwood University of Bristol, Bristol, UK
24 Systemic vasopressin V1a receptor antagonism blocks oxytocin's attenuation of reinstatement of methamphetamine-seeking behaviour in rats
*Nick Everett Macquarie University, Australia
25 Synthesis of [lys8]-oxytocin dendrimers that maintain potency and structure and inhibit visceral nociceptive responses
Paul Alewood University of Queensland, Australia
26 Kisspeptin regulation of oxytocin neurons in pregnant mice
Rachael Augustine University of Otago, NZ
27 Myocardial infarction increases Fos protein expression in the paraventricular and supraoptic nuclei
*Ranjan Roy University of Otago, NZ
28 Genetic evidence for people’s attitudes towards time
Richard Ebstein National University of Singapore, Singapore
29 Selective localization of oxytocin receptors and vasopressin 1a receptors in human brain tissue
Sara Freeman University of California-Davis, USA
30 Chronic methamphetamine self-administration dysregulates oxytocin plasma levels and oxytocin receptor expression in the nucleus accumbens core and subthalamic nucleus of the rat
Sarah Baracz Macquarie University, Australia
31 Manipulation of Avpr1b-expressing neuronal activity: Effects on social behavior
Sarah Williams National Institutes of Health, USA
32 Tonic endocannabinoid signaling in hypothalamic magnocellular neuroendocrine cells
Shi Di Tulane University, USA
33 Social behaviour and nonapeptide binding in passerine species of the Tibetan plateau
Simone Meddle University of Edinburgh, UK
34 The role of vasopressin expressing retinal ganglion cells projecting to the hypothalamic suprachiasmatic nucleus
Takahiro Tsuji University of Edinburgh, UK
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35 Radioimmunological assessment of OXT in human saliva during running, sexual self-stimulation and the Trier Social Stress Test: an OXT challenge study
Thomas Grund University of Regensburg, Germany
36 Juxtacellular recording and labelling uncovers hypertonicity-sensitive theta oscillations in hypothalamic periventricular nucleus and intracerebral axonal projections from individual magnocellular vasopressinergic neurons
Vito Hernandez UNAM, Mexico
37 Effects of maternal oxytocin administration on fetal vole pups
William Kenkel Indiana University, USA
38 The analgesic effect of oxytocin in humans: a double-blinded placebo controlled cross-over study using laser-evoked potentials
Yannis Paloyelis King’s College London, UK
39 Investigating the mechanisms through which intranasal oxytocin affects brain function in humans
Yannis Paloyelis King’s College London, UK
40 Oxytocin-like neuropeptide signalling in ants
Zita Liutkeviciute Medical University of Vienna, Austria
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Abstracts
No part of the conference proceedings, including the abstracts, may be referenced. Please contact the authors directly for permission to cite their work.
Saturday afternoon
Plenary Lecture 1
Oxytocin and the neural mechanisms of social cognition
Young L.J.
Silvio O. Conte Center for Oxytocin and Social Cognition, Center for Translational Social Neuroscience, Department of Psychiatry and Behavioral Sciences, Yerkes National Primate Research Center, Emory University, Atlanta GA, USA
Oxytocin (OXT) modulates several social behaviors including parental care, social recognition, and social bonding. Single nucleotide polymorphisms (SNPs) in the human OXT receptor gene (OXTR) are associated with variation in social behavior and autism. OT is thought to enhance the salience and reinforcing value of social stimuli. Intranasal OT improves some aspects of social functioning in autism and clinical trials are underway to determine the efficacy of chronic OT for treating social deficits in autism. We use the socially monogamous prairie vole to explore the neural mechanisms by which OT modulates social behavior. OXTR signaling in the prefrontal cortex (PFC) and nucleus accumbens (NAcc) promotes mating-induced pair bonds in male and female prairie voles. OXT coordinates the activity of a network of brain regions involved in processing social information and in reward. Using Fos immunoreactivity to assess neural activity during mating in males, we find strongly correlated activity within individuals across several nodes of the social behavioral neural network, including olfactory nuclei, amygdala, PVN, NAcc and PFC. Blocking central OXTR during mating disrupts this correlated activity. There is remarkable individual variation in NAcc OXTR density in voles that contributes to variation in social behavior. We have identified SNPs in the non-coding region of the prairie vole Oxtr gene that explain up to 80% of the variance in NAcc OXTR. This variation in NAcc OXTR has an important influence on the development of social behavior. In a model of neglect, we show that daily 3 hour social isolations for the first 2 weeks of life in female prairie voles impairs pairs bonding in adulthood (1). However, females with naturally high NAcc OXTR density are resilient to early social isolation, suggesting that neonatal NAcc OXT signaling evoked by parental nurturing has life-long effects on the capacity for form social relationships. Pharmacologically evoking central OXT release during the social isolations using a melanocortin agonist (MT II) prevents the disruption in social bonding. In adults, MT II potentiates the release of OXT in the NAcc and facilitates pair bonding that endures well after the drug is cleared, suggesting that the melanocortin system may be a viable drug target to enhance central OXT activity, and thus to improve social cognition in psychiatric disorders (2, 3). In addition to pair bonding, we recently found that OXT enhances consoling behavior in male prairie voles toward a stressed partner or familiar individual. Blocking OXTR in the anterior cingulate cortex, but not in the PFC, prevents this empathy related behavior. In rodents, OXT primarily affects behaviors mediated by olfaction. In humans we have found that polymorphisms in the OXTR gene predict face recognition abilities in families with an autistic child. In primates, OXTR are concentrated in brain regions involved in visual attention and auditory processing, including the nucleus basalis of Meynert and superior colliculus.
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Thus OXT plays a conserved role in modulating social information processing in rodents and man across sensory modalities.
1. C. E. Barrett, S. E. Arambula, L. J. Young, The oxytocin system promotes resilience to the effects of neonatal isolation on adult social attachment in female prairie voles. Transl. Psychiatr. In press, (2015).
2. M. E. Modi et al., Melanocortin Receptor Agonists Facilitate Oxytocin-Dependent Partner Preference Formation in the Prairie Vole. Neuropsychopharmacology, Online Ahead of Print (2015).
3. L. J. Young, C. E. Barrett, Neuroscience. Can oxytocin treat autism? Science 347, 825 (2015).
Symposium 1: Molecular and cellular aspects of magnocellular neurons
Molecular genetic study for the regulatory mechanism of social behavior regulated by GABAergic neurons, expressing oxytocin receptor
Nishimori, K.1, Miyazaki, S.1 , Hiraoka, Y.1 , Hidema, S.1 , Radulovic, J.2
1Department of Molecular and Cell Biology, Graduate School of Agricultural Sciences, Tohoku University, Miyagi, Japan, 2Department of Psychiatry and Behaviorail Sciences, Northwestern University, Feinberg School of Medicine, Chicago, USA
We previously observed the impairment of social behaviors in oxtr(-/-) mice (1). On the other hand, various clinical groups have reported curing effects of oxt, when it was administrated to ASD patients. We carried out experiments using mice with modification of oxtr gene, to elucidate the neurons, to which externally administrated oxytocin targeted in the therapy of ASD, and also to clarify the neural circuit, whose impairment might cause ASD-like symptom. We considered oxtr gene KO mouse as a model ASD-like animal, and compared various nuclei with c-fos positive neurons of oxtr(-/-) mice with those of wt mice, when exposed to social stimuli, by immunostaining of c-fos(+) cells. We then analyzed the co-localization of Venus in c-fos(+) neurons, in Oxtr-Venus mice by double immunohistochemistry for c-fos(+) and venus(+) cells. We observed highly efficient co-localization of Venus (= OXTR) with c-fos(+) signal in the neurons in MeA (Medial amygdaloidal nucleus).
We injected AAV-Cre vector into MeA of Oxtr(fx/fx) mice, to induce region-specific deletion of oxtr gene, and observed impaied social memory with those mice. On the other hand, we carried out rescue experiment by injection of AAV-Oxtr-IRES-Venus vector into MeA of OXTR(-/-) mice, and confirmed the recovery of social memory with them. We also characterized the subtype of neuron expressing OXTR in MeA. Oxtr (+) neurons in MeA were suspected to be GABAergic or glutamatergic, and we first carried out double immunostaining (for calbindin and venus), and combination of in situ hybridization and immunostaining (for calretinin or somatostain and venus). Result showed that 30% of oxtr-expressing neurons in MeA were calretinin(+), inidicating that major populstion of oxtr-expressing neurons in MeA were GABAergic interneurons.
We hypothesized that the neurons expressing oxtr with regulatory function onto social memory might be GABAegic, and introduced Vgat-IRES-Cre mice line to our Lab. to induce GABAergic neurons-specific deletion of oxtr gene in mice. Resultant double mutant mice (Vgat-IRES-Cre; Oxtr(fx/fx)) showed abnormal behavior in social novelty test by three chamber arena test. On the other hand, collaborative study with Dr. Radulovic, showed also impaired social novelty with Oxtr(fx/fx) mice, whose lateral septal nucleus (LS) was infected with AAV-Cre vector, to conditionally delete oxtr gene (3). Our original data also indicated that most neurons expressing oxtr (~100%) were GABAergic (calretinin (+)) in LS nuclei, and we are continuously studying whether the abnormal social behavior observed in Vgat-IRES-Cre: Oxtr(fx/fx) mice is caused by deletion of oxtr gene in LS nuclei, or in MeA nuclei.
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1. Takayanagi, Y., Yoshida, M., Bielsky, I.F., Ross H.E., Kawamata, M., Onaka, T., Yanagisawa, T., Kimura T, Matzuk MM, Young LJ, Nishimori K. (2005) Pervasive social deficits, but normal parturition, in oxytocin receptor-deficient mice. Proc Natl Acad Sci USA, 102:16096-101
2. Yoshida, M., Takayanagi, Y., Inoue, K., Kimura, T, Young, L.J., Onaka, T., Nishimori, K. (2009) Evidence that oxytocin exerts anxiolytic effects via oxytocin receptor expressed in serotonergic neurons in mice. J Neurosci, 29:2259-71
3. Mesic,I. Guzman, Y.F., Guedea, A.L., Jovasevic,V., Corcoran,K.A., Leaderbrand,K., Nishimori,K., Contractor,A., Radulovic, J. (2015) Double Dissociation of the Roles of Metabotropic Glutamate Receptor 5 and Oxytocin Receptor in Discrete Social Behaviorsm., Neuropsychopharmacol.
Central axonal oxytocin pathways: pain, fear and sociality
Grinevich, V.
Schaller Research Group on Neuropeptides, German Cancer Research Center/DKFZ and University of Heidelberg, Heidelberg, Germany
Using virus-based vectors, we explored the anatomical features of magnocellular oxytocin (magnOT) neurons in rodents, showing that these neurons are projecting to the forebrain, especially to regions controlling fear, such as the central nucleus of amygdala (CeA). We developed genetic tools (named Genetic Activity-Induced Tagging, vGAIT) to express genes of interest in only those OT neurons, which have been exclusively activated by pain and fear in the contextual fear conditioning setup. Taking advantage of this tool, in rats we were able to tag OT neurons activated by fear acquisition (pairing of a series of electric shocks with the context) separately from OT neurons activated by fear extinction (e.g. by the expectation of pain in the context). Despite the small number of tagged magnOT neurons (less than 15%), optogenetic stimulation of their axons within the CeA drastically reduced freezing time in the conditioning chamber. Importantly, the magnitude of the inhibition of fear response was comparable with our previous experiments1 based on tagging of the entire population (~ 9000) of OT neurons. In the course of the present study, we observed that, in addition to magnoOT neurons, a phenotypically-distinct population of OT neurons has been tagged only during fear acquisition associated with a painful component (e.g. electric shocks) was applied. Our ongoing experiments demonstrate that these neurons are pre-autonomic parvocellular OT (parvOT) neurons, which exert analgesic effects via simultaneous activation of hypothalamic magnoOT neurons and nociceptive neurons in the spinal cord. Finally, using vGAIT technique, we are on the way to tag magnOT and parvOT neurons activated by social stimuli and to trace their projections through the forebrain, brainstem and spinal cord. In conclusion, our preliminary results allow us to speculate about anatomical and functional specialization within the central OT system, which are orchestrating distinct forms of behaviour, such as pain, fear and social behaviours.
1. Knobloch, H.S., Charlet, A., Hoffmann, L.C., Eliava, M., Khrulev, S., Cetin, A.H., Osten, P., Schwarz, M. K., Seeburg, P.H., Stoop, R., Grinevich, V (2012). Evoked axonal oxytocin release in the central amygdala attenuates fear response. Neuron. 2008, 73: 553-566.
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Electrophysiological study combined with fluorescent imaging and optogenetic approaches in rat vasopressin neuron
Ueta, Y.
Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, 807-8555 Japan
We generated the transgenic rats that express the arginine vasopressin (AVP)-enhanced green fluorescent protein (eGFP) fusion gene in the supraoptic (SON) and paraventricular nuclei (PVN) of the hypothalamus and the posterior pituitary (PP)1. We also generated another transgenic rat that express the oxytocin (OXT)-monomeric red fluorescent protein 1 (mRFP1) fusion gene in the SON, PVN and the PP2. These transgenic rats are very useful to identify AVP- and OXT-producing magnocellular neurosecretory cells (MNCs) in in vitro preparations under fluorescent microscopy.
Double transgenic rats that express the AVP-eGFP and OXT-mRFP1 fusion genes were obtained by mating an AVP-eGFP transgenic rat with an OXT-mRFP1 transgenic rat. The SON and PVN in double transgenic rats contain AVP-eGFP positive and OXT-mRFP1 positive MNCs. Thus, it is possible to obtain reliable data by examining the physiological differences between AVP-eGFP and OXT-mRFP1 MNCs simultaneously under the same preparation. We showed one example that discovered the different effects of acid on AVP-eGFP and OXT-mRFP1 MNCs isolated from the SON and PVN in double transgenic rats3. The whole-cell patch-clamp recordings in voltage clamp mode revealed that the acid-induced inward currents in AVP-eGFP MNCs were significantly larger than those in OXT-mRFP1 MNCs.
Recently, we have challenged to regulate the neuronal activities of AVP MNCs by a light-activated ion channel in transgenic rats that express the AVP-eGFP and channelrhodopsin 2 (ChR2) fusion gene. The ChR2-eGFP was mainly localized in the membrane of the AVP MNCs in the SON and PVN of the transgenic rats. Using whole-cell patch-clamp recordings in in vitro preparations, blue light evoked action potentials in current clamp mode, and caused inward currents in voltage clamp mode.
These transgenic rats give us advantage to identify AVP and OXT MNCs in living, and optogenetic approaches to regulate neuronal activities of AVP MNCs.
1. Ueta, Y. Fujihara, H. Serino, R. Dayanithi, G. Ozawa, H. Matsuda, K. Kawata, M. Yamada, J. Ueno, S. Fukuda, A. & Murphy, D. (2005) Transgenic expression of enhanced green fluorescent protein enables direct visualization for physiological studies of vasopressin neurons and isolated nerve terminals of the rat. Endocrinology 146(1): 406-413
2. Katoh, A. Fujihara, H. Ohbuchi, T. Onaka, T. Hashimoto, T. Kawata, M. Suzuki, H. & Ueta, Y. (2011) Highly visible expression of an oxytocin-monomeric red fluorescent protein 1 fusion gene in the hypothalamus and posterior pituitary of transgenic rats. Endocrinology 152(7): 2768-2774
3. Ohkubo, JI. Ohbuchi, T. Yoshimura, M. Maruyama, T. Hashimoto, H. Matsuura, T. Suzuki, H. & Ueta, Y. (2014) Differences in acid-induced currents between oxytocin-mRFP1 and vasopressin-eGFP neurons isolated from the supraoptic and paraventricular nuclei of transgenic rats. Neuroscience Letters 583(7): 1-5
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Transcription factor CREB3L1 mediates the cAMP regulation of the vasopressin
Greenwood, M.1, Greenwood, M.P.1, Paton, J.F.R.2, Murphy, D.1
1School of Clinical Sciences, University of Bristol, Bristol, UK, 2School of Physiology and Pharmacology, University of Bristol, Bristol
Arginine vasopressin (AVP) is synthesised in the magnocellular (MCN) and parvocellular neurons of the hypothalamus in response to hyperosmotic stress. cAMP and glucocorticoids have repeatedly been shown to positively and negatively regulate Avp expression, respectively; however the exact mechanism remains unclear (1). Recently, we identified CREB3L1 (cAMP-responsive element binding protein 3 like 1), a transcription factor in CREB/ATF families, as a putative regulator of Avp transcription in the rat hypothalamus (2). To further investigate the mechanisms of CREB3L1 action on AVP expression, we used the acute hyperosmotic stimulation mediated by intraperitoneal injection of hypertonic saline solution to study the dynamics of Creb3l1 expression during hyperosmotic stress. An increase in Creb3l1 mRNA abundance was observed as early as 1 hour after hypertonic injection. The change in localization of CREB3L1 protein, from Golgi to cytosol and nucleus, which indicates activation of CREB3L1, was observed at 4 hours after the injection. The effect of cAMP activation on Creb3l1 was investigated in both hypothalamic organotypic culture and AtT20 cells. The expression of Creb3l1 was increased by treatment with forskolin (FSK; cAMP activator). This FSK-induced increase in Creb3l1 expression was diminished by combined treatment with dexamethasone, suggesting that cAMP and glucocorticoid effects on Avp are mediated by CREB3L1. Activation of cAMP by FSK increased Avp promoter activity in AtT20 cells. This effect was blunted by deletion of a putative CREB3L1 binding site (G box) in rat AVP promoter, and shRNA mediated silencing of Creb3l1,, suggesting that activation of Creb3l1 via the cAMP pathways is regulating AVP transcription.
1. Yoshida M. Gene regulation system of vasopressin and corticotropin-releasing hormone. Gene Regul Syst Bio. 2008; 271-88.
2. Greenwood M, Bordieri L, Greenwood MP, Rosso Melo M, Colombari DS, Colombari E, Paton JF, Murphy D. Transcription factor CREB3L1 regulates vasopressin gene expression in the rat hypothalamus. J Neurosci. 2014; 34(11): 3810-20.
Sunday morning
Plenary Lecture 2
Vasopressin neurons in the suprachiasmatic nuclei (SCN): Critical signaling inside and outside the biological clock
Kalsbeek, A.
Department of Endocrinology and Metabolism, Academic Medical Center (AMC), University of Amsterdam & Hypothalamic Integration Mechanisms, Netherlands Institute for Neuroscience (NIN), Amsterdam, The Netherlands
The discovery of the hypothalamic suprachiasmatic nuclei (SCN) as the seat of the central biological clock in 1972 was made almost at the same time as the discovery of the neurotransmitter function of neuropeptides in general and of vasopressin in particular. Only a few years after its discovery it was demonstrated, in 1975, that next to the well-known vasopressin production sites in the paraventricular and supraoptic nuclei (PVN and SON), also the SCN contained a prominent
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population of vasopressin-producing neurons. In the first 10-15 years after its discovery in the SCN vasopressin mainly served as an SCN marker, as the vasopressin-containing subpopulation turned out to be a characteristic feature of the SCN in many species, including humans. However, in this presentation I will show that in the years after the vasopressin neurons in the SCN have been at the heart of the deciphering and understanding of the mechanism of the circadian timing system.
As the activity of the vasopressin neurons in the SCN had been found to show a pronounced daily variation in its activity that even could be demonstrated in human post-mortem brains, the next 10-15 years concentrated on the significance of the SCN vasopressin neurons for the functional output of the biological clock. Animal experiments showed an important role for SCN-derived vasopressin in the control of neuroendocrine day/night rhythms such as that of the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal axes. The remarkable correlation between a diminished presence of vasopressin in the SCN and a deterioration of sleep/wake rhythms during ageing and depression make it likely that, also in humans, the vasopressin neurons contribute considerably to the rhythmic output of the SCN.
More recently attention has shifted to the role of vasopressin signaling within the SCN. Indeed a high proportion of the SCN neurons (>40%) can be excited through the vasopressin V1a receptor. Moreover, microdialysis studies have clearly proven that vasopressin is also released within the SCN. By now a number of studies have demonstrated that the vasopressin neurons in the SCN are critical for interneuronal coupling within the SCN. As a consequence mice that lack vasopressin V1a and V1b receptors are resistant to jet lag, i.e., these mutant mice shift almost instantaneously to a new environmental light/dark cycle. More physiological models indicate that an absence of rhythmic intra SCN vasopressin signaling may allow animals to be active during their usual sleep period, when necessary.
Symposium 2: Oxytocin and behaviour
Oxytocin as a secretagogue of behaviours: Where does feeding regulation fit in the puzzle?
Olszewski, P.K. 1, Klockars, A.1, Levine, A.S.
1Department of Biological Sciences, University of Waikato, Hamilton, New Zealand, 2Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN, USA
Already during the early stages of research on the involvement of oxytocin (OT) in food intake regulation, OT was clearly defined as an anorexigen whose chief function is to protect the organism from dangerously elevated plasma osmolality, excessive stomach distention or toxicity. However, it soon became apparent that its anorexigenic effects should be seen in a much broader context, mainly due to OT’s role in key physiological and behavioural processes other than energy intake control: OT has been found to promote sexual behaviour, lactation, parturition and social bonding in family and non-family groups. Intuitively, interruption of these processes by the drive to consume food, under certain conditions, might not be advantageous from the evolutionary point of view. Hence, we propose that the context of termination of food intake facilitated by OT goes beyond maintaining internal milieu and it includes balancing physiological and behavioural responses to both internal and external challenges.
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Oxytocin therapeutics for psychiatric disorders: Applications and mechanisms
Guastella, A.J.
Brain & Mind Centre, Central Clinical School, University of Sydney, Sydney, Australia
Oxytocin and its associated analogues have great potential for the treatment of many psychiatric disorders, including autism and associated developmental disorders, schizophrenia, and addiction. The aim of this presentation is to provide a general overview of the potential and challenges facing human therapeutic applications of oxytocin treatment for psychiatric disorders moving from the benchside into the clinic. Human clinical trials will be presented suggesting potential benefit of oxytocin treatments for psychiatric disorders and in some cases, to provide first medical treatments for these conditions. Significant theoretical and practical challenges remain however and the limitations of the literature and discussion of future required advances will be discussed.
When she’s gone: Emotional consequence of partner loss due to suppression of oxytocin signaling in the NAc shell in male prairie voles
Bosch, O.J.1, Dabrowska, J.2, Modi, M.E.3,4, Keebaugh, A.C.4, Barrett, C.E.4,5, Johnson, Z.V.4,5, Ahern, T.H.4,6, Guo, J.D.4,5, Grinevich, V.7, Rainnie, D.G.4,5, Neumann, I.D.1, Young, L.J.4,5
1Department of Behavioural and Molecular Neurobiology, University of Regensburg, Regensburg, DE, 2Department of Neuroscience, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA, 3Pfizer Neuroscience Research Unit, Pfizer Inc. Cambridge, MA, USA, 4Center for Translational Social Neuroscience, Department of Psychiatry, Emory University, Atlanta, GA, USA, 5Silvio O. Conte Center for Oxytocin and Social Cognition, Emory University, Atlanta, GA, USA, 6Center for Behavioral Neuroscience, Department of Psychology, Quinnipiac University, Hamden, CT, USA, 7Schaller Research Group on Neuropeptides, German Cancer Research Center DKFZ, Cell Network Cluster of Excellence, University of Heidelberg, Heidelberg, DE
Social bonds positively impact physical and mental health, such that their absence or sudden loss increases the susceptibility to emotional dysfunctions. In monogamous male prairie voles (Microtus ochrogaster), which form enduring and selective pair bonds, separation from the pair-bonded female partner increases passive stress-coping1, indicative of depressive-like behavior. This emotional consequence is mediated by the corticotropin releasing factor (CRF) system1. Interestingly, CRF-expressing neurons from the bed nucleus of the stria terminalis (BNST) project to the nucleus accumbens (NAc) shell, where CRF-R2 are co-expressed with oxytocin receptors (OT-R). Therefore, we hypothesized that the OT and CRF system in the NAc shell counterbalance passive stress-coping after partner-loss.
We induced pair bonds by pair-housing naïve male and female prairie voles for 5 days, after which they were separated or remained paired for another 3 days until testing1. Separation caused decreased OT mRNA and OT-R binding in the PVN and in the NAc shell, respectively. Furthermore, OT and CRF-R2 showed nearly complete somatodendritic immunoreactive overlap in PVN neurons and within the sparse large-caliber neuronal fibres that course through the NAc shell. Chronic pharmacological administration of OT or CRF-R2 antagonist into the NAc shell decreased passive stress-coping following separation. Likewise, in males that remained paired, chronic infusion of OT-R
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antagonist or CRF-R2 agonist increased passive stress-coping. Furthermore, local OT-R knock down by shRNA caused the same effect. Finally, a combined icv injection/local microdialysis approach in naïve male prairie voles revealed that activating and blocking CRF-R2 decreased and increased, respectively, local OT release in the NAc shell. Electrophysiological data from the PVN suggests that CRF-R2 activation decreases glutamate drive and excitability of the OT neurons
The negative emotional consequences of partner-loss are mediated by CRF-R2 inhibiting the OT system in the NAc shell; local application of OT can buffer against the partner-loss induced passive stress-coping. We propose that the suppression of OT signaling is likely adaptive during short separations to encourage reunion with the partner and may have evolved to maintain long-term partnerships. Additionally, therapeutic strategies targeting these systems should be explored for the treatment of social loss-mediated depression.
Supported by NIH grants MH-077776 and MH096983 to LJY, OD P51OD011132 to YNPRC, DFG NE 465/16-1 to IDN.
1. Bosch, O.J., Nair, H.P., Ahern, T.H., Neumann, I.D. and Young, L.J. (2009). The CRF system mediates increased passive stress-coping behavior following the loss of a bonded partner in a monogamous rodent. Neuropsychopharmacology. 34: 1406-15.
Oxytocin inhibits ethanol consumption and intoxication in rats: interactions with dopamine and extrasynaptic GABAA receptors
Bowen, M.T.1, Peters, S.T.2, Absalom, N.3, Chebib, M.3, Neumann, I.D.2, McGregor, I.S.1
1School of Psychology, University of Sydney, Australia, 2Department of Behavioral and Molecular Neurobiology, University of Regensburg, Germany, 3Faculty of Pharmacy, University of Sydney, Australia
Alcohol is one of the most widely abused recreational drugs and is arguably the most harmful. Current treatment options lack efficacy and a breakthrough in our approach is sorely needed. A growing number of preclinical studies indicate that the neuropeptide oxytocin may have substantial utility in the treatment of alcohol use disorders. Work in the 1980s demonstrated that oxytocin inhibits ethanol tolerance and withdrawal but little further work was conducted on oxytocin-ethanol interactions over the proceeding decade and a half. Our initial studies demonstrated that administration of oxytocin causes a lasting decrease in ethanol consumption and preference in rats. More recently, we have demonstrated that oxytocin effects on alcohol consumption are centrally mediated and we have linked these effects of oxytocin to a hitherto unknown ability of the neuropeptide to reduce ethanol stimulation of the dopamine reward system. While conducting this work we discovered that oxytocin causes a striking inhibition of the acute motor impairing effects of ethanol. Ethanol’s effects at extrasynaptic δ subunit-containing GABA A receptors are heavily involved in the motor impairing effects of ethanol, and in ethanol reward and tolerance. Using electrophysiological techniques, we have subsequently demonstrated that oxytocin exerts a highly specific and complete blockade of ethanol actions at these δ subunit-containing GABAA receptors. Furthermore, these effects of oxytocin are due to a direct, non-oxytocin receptor mediated action at δ subunit-containing GABAA receptors. These recent findings therefore indicate that oxytocin substantially interferes with ethanol’s primary actions in the CNS, providing novel insights into possible mechanisms driving oxytocin’s interference with ethanol ataxia, tolerance and reward.
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Sunday afternoon
Young Investigator Prize Presentations
Alison Douglas Young Investigator Award Winner
Diverse morphology and structural plasticity of glial cells in the supraoptic nucleus
Choe, K.Y.1,2, Farmer, W.T.2, Murai, K.K.2, Bourque, C.W.2
1Department of Neurology, Geffen School of Medicine, UCLA, Los Angeles, CA, USA, 2Centre for Research in Neuroscience, McGill University Health Centre, Montreal, QC, Canada
It is well established that astrocytes in the supraoptic nucleus (SON) possess a unique ability to retract their fine processes in response to a wide range of physiological stimulations including chronic hypernatremia1. Various studies have demonstrated that this dramatic form of structural plasticity has a functional impact on excitability of magnocellular neurosecretory cells (MNCs), of which there are two types – vasopressin (VP) and oxytocin (OT) neurons. Surprisingly, several important questions regarding the properties of this astrocytic structural plasticity still remain unanswered. For instance, it is still a matter of debate whether the withdrawal occurs universally in the SON2 or is restricted to processes located in the vicinity of OT neurons3. Moreover, astrocytes have been shown to exist in different shapes and sizes in the brain, but currently there is very little information on the types of astrocytes that reside in the SON and which of these undergo process withdrawal. In order to address such questions, we combined a variety of cell labeling techniques with fluorescence microscopy to examine whether there exists a geographical and/or cell-type dependency in the degree of astrocytic process withdrawal in the SON, as well as classify these astrocytes based on their morphologies and investigate their abilities to retract the processes in response to chronic salt-loading in vivo.
In the SON of 7-day salt-loaded rats (2% saline, SL), anti-GFAP immunolabeling revealed a significant reduction in staining intensity as expected (EU: 1.0 ± 0.055, n=24; SL: 0.71 ± 0.068, n=25; P=0.001). Double immunolabeling with antibodies against GFAP and neurophysin-VP or neurophysin-OT (markers of VP or OT neurons, respectively) was then performed to examine whether the retraction is restricted to astrocytic processes near a particular MNC type. We observed that GFAP staining intensity was significantly decreased around the OT neurons of the SON (EU: 1.0 ± 0.010, n=18; SL: 0.42 ± 0.057, n=28; P<0.001) as well as around the VP neurons (EU: 1.0 ± 0.11, n=14; SL: 0.41 ± 0.016, n=14; P<0.001). Next, in order to investigate the detailed morphology of SON astrocytes at a single-cell resolution, we used the diolistic labeling approach with a gene-gun mediated delivery of DiI-coated microbeads. The microbead delivery was aimed at the ventral glial limitans area of the SON which is rich with astrocytic cell bodies. Morphological analysis of these putative astrocytes indicated that there exist at least three different types in the SON – “protoplasmic (n=24)”, “lamellar (n=4)” and “fibrous (n=11)” astrocyte type. Salt loading reduced the average area of “protoplasmic” astrocytes in the X-Y plane (EU: 18044.5 ± 1603.9 μm2, n=31; SL: 10853.8 ± 1997.1 μm2, n=12; P=0.02) but not the other types (P=0.53 for “lamellar”; 0.54 for “fibrous”). In summary, these results suggest that while the SL-induced retraction of SON astrocytes occur without the discrimination of the neuronal identity around them, only the “protoplasmic” subtype of astrocytes account for the morphological plasticity. This study was supported by CIHR grant MOP9939 to CWB.
1. Tweedle, C.D. and Hatton, G.I. (1976). Ultrastructural comparisons of neurons of supraoptic and circularis nuclei in normal and dehydrated rats. Brain Res Bull. 1(1) 103-21.
2. Marzban, F., Tweedle, C.D., and Hatton, G.I. (1992). Reevaluation of the plasticity in the rat supraoptic nucleus after chronic dehydration using immunogold for oxytocin and vasopressin at the ultrastructural level. Brain Res Bull. 28(5) 757-66.
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3. Chapman, D.B., Theodosis, D.T., Montagnese, C., Poulain, D.A., and Morris, J.F. (1986). Osmotic stimulation causes structural plasticity of neurone-glia relationships of the oxytocin but not vasopressin secreting neurones in the hypothalamic supraoptic nucleus. Neuroscience. 17(3) 679-86.
Glenn Hatton Postdoctoral Award Winner
Vasopressin enhances social memory: Illuminating a PVN to hippocampal CA2 circuit
Smith, A.S., Williams Avram, S.K., Cymerblit-Sabba, A., Song, J., Young, W.S.
Section on Neural Gene Expression, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA
Peptide action in the brain propagates social living by influencing complex social behaviors. Vasopressin (Avp) is a neuropeptide that regulates a wide range of social behaviors from pair bonding to aggression. Two subtypes of Avp receptors are expressed in the brain (Avpr1a and Avpr1b); however, we are only beginning to learn about the role of Avpr1b in regulating social behavior. Previously, we noted Avpr1b is prominently expressed in the CA2 region of the hippocampus1, afferent fibers from AVP-expressing neurons in the paraventricular nucleus of the hypothalamus (PVN) innervate this region2, and unconditional deletion of the Avpr1b gene impairs
social memory3. Thus, it is reasonable to postulate that the AvpPVN➜CA2 pathway may be involved in processing social memory.
To test this, we virally targeted expression of the light-sensitive receptor channelrhodopsin-2 to Cre recombinase-expressing AVP neurons in the PVN of transgenic mice and assessed social memory in these mice. Social memory in mice is defined as a decrease in spontaneous investigation behaviors observed in a mouse reexposed to a conspecific, and memory from a brief encounter (5 minutes) does not extend beyond 1 h in mice. Thus, we exposed our transgenic mice to a conspecific over two 5-minute trials, optically stimulating only the AVP fibers within the CA2, and measured the amount
of investigation behavior. Excitation of the AvpPVN➜CA2 pathway dramatically prolonged social memory in mice from 30 minutes to at least 7 days. In addition, we observed a marked increase in neuronal activity in the PVN and CA2, as measured by enhanced immediate-early gene expression. No changes were observed in sociability or object memory. Interestingly, social memory was only
improved when the AvpPVN➜CA2 pathway was optically stimulated during memory acquisition, not retrieval. Memory enhancement was lost by pharmacological blockage of Avpr1b in the CA2 during the optical stimulation, confirming receptor-specific function of this pathway.
Together, our data demonstrate that the strength or salience of social memories is enhanced by
selectively targeting the excitation of the AvpPVN➜CA2 pathway. The hippocampus is essential for encoding declarative memory; however, the CA2 region, which is selectively enriched with Avpr1b expression, has largely been ignored since it was first described over 80 years ago. Our work provides new knowledge about how the CA2 is integrated into a circuit regulating such memories.
This research was supported by the intramural research program of the NIMH (ZIA-MH-002498-24).
1. Young, W.S., J. Li, S.R. Wersinger and M. Palkovits (2006). The vasopressin 1b receptor is prominent in the hippocampal area CA2 where it is unaffected by restraint stress or adrenalectomy. Neuroscience 143: 1031-9.
2. Cui, Z., C.R. Gerfen and W.S. Young (2013). Hypothalamic and other connections with dorsal CA2 area of the mouse hippocampus. J Comp Neurol 521: 1844-66.
3. Wersinger, S.R., E.I. Ginns, A.M. O'Carroll, S.J. Lolait and W.S. Young (2002). Vasopressin V1b receptor knockout reduces aggressive behavior in male mice. Mol Psychiatry 7: 975-84.
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Glenn Hatton Student Award Winner
Blockade of the anti-opioid effects of the Neuropeptide FF system using a novel antagonist reverses morphine tolerance in vasopressin neurons
Kim, J.S.1, Brown, C.H.2, Anderson, G.M.1
Centre for Neuroendocrinology, Departments of 1Anatomy and 2Physiology, University of Otago, New Zealand
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 µg/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 responses to morphine (i.v. 30 µg/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.
Monday morning
Plenary Lecture 3
There is more to vasopressin than meets the eye
Ludwig, M
Centre for Integrative Physiology, University of Edinburgh, Edinburgh, UK
Novel transgenic rat models and viral transfection protocols have helped to identify previously unknown populations of vasopressin and oxytocin neurons and their projections. We recently identified vasopressin-expressing neurons in the main and accessory olfactory bulb and in the anterior olfactory nucleus. Blocking the actions of vasopressin in the main olfactory bulb impairs the social recognition abilities of rats suggesting that that the vasopressin expressing neurons process olfactory signals relevant to social discrimination.
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The vasopressin neurons in the AON are neurochemically different to the vasopressin neurons in the main olfactory bulb. Exposure of adult rats to a conspecific juvenile or a heterospecific predator odour leads to increases in Egr-1 expression in the AON in a subregion specific manner. However, only exposure to a juvenile increases Egr-1 expression in AON vasopressin neurons, suggesting that vasopressin neurons in the AON, may be selectively involved in the coding of social odour information.
We are currently working on a population of vasopressin expressing retinal ganglion cells (RGCs). These cells respond to light, but only ~5% of the vasopressin-RGCs express melanopsin. Intravitreal injections of an rAAV-expressing Venus under the vasopressin promoter showed that the majority of vasopressin-RGCs project to the core part of the suprachiasmatic nucleus (SCN) terminating on VIP and GRP cells. Microdialysis data showed an increase in intra-SCN vasopressin concentration in response to electrical stimulation of the optic nerve or light stimulation of the eye. Furthermore, in response to optic nerve or light stimulation a subpopulation of SCN neurons showed an excitatory response which was blocked by icv injection of a vasopressin V1 receptor antagonist. Our data suggest that activation of vasopressin-RGCs may mediate light cues by releasing vasopressin into the SCN to excite SCN neurons, altering SCN activity and thus regulating neuroendocrine hormonal rhythms.
Symposium 3: Vasopressin secretion in health and disease
Inhibitory influence of median preoptic/medial septal/diagonal band region on suppression of vasopressin secretion and rapid excretion of a water load
McKinley, M.J., Pennington, G.L.
Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria, Australia
In addition to an adequate water intake, the secretion of arginine vasopressin (AVP) is essential for maintaining normal bodily hydration. Conversely, reduced AVP secretion in response to overhydration is necessary for the prevention or amelioration of dangerous hyponatremic/hyposmolar states. Evidence that an active suppression of AVP occurs even before ingested water has been absorbed into the systemic circulation has been obtained in experimental animals and humans. These data suggest that the suppression of AVP release is more than just the removal of hypertonic and hypovolemic stimuli, but caused in part by direct inhibitory neural influences on the magnocellular vasopressin-containing neurons of the supraoptic and paraventricular nuclei of the hypothalamus. Little is known of the neural pathways that mediate the rapid inhibition of vasopressin release associated with drinking. As well, it is not known whether the reduction in vasopressin secretion that occurs with hypo-osmolar overhydration is due just to withdrawal of osmotic or volemic stimuli, or whether there is active inhibition of vasopressin-secreting magnocellular neurons of the supraoptic and paraventricular nuclei. It is known that there are strong GABAergic inhibitory neural inputs from the median preoptic nucleus (MnPO)1 and diagonal band2 to the supraoptic nucleus. The MnPO also sends excitatory neural projections to the supraoptic and paraventricular nuclei that mediate in part osmoregulatory vasopressin release.
Our data, obtained from experiments in conscious, water-loaded sheep, suggest that such reduction of vasopressin secretion involves an active inhibition of the neuroendocrine magnocellular neurons of the supraoptic and paraventricular nuclei that secrete vasopressin. We have observed that the excretion of a water load in sheep is greatly impaired if tissue is ablated in the region of the lamina terminalis, that includes the MnPO, medial septum and diagonal band (DB). Vasopressin release is not suppressed adequately in these animals when they are overhydrated. As a result, hyponatremia
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and hypoosmolality ensued. We also observed that the water diuresis and inhibition of plasma AVP levels that normally results from experimental reduction of cerebrospinal Na concentration caused by intracerebroventricular infusion of 0.3M mannitol is blocked in sheep with such MnPO/medial septal/DB lesions. Our experiments show that not only is this region necessary for relaying excitatory signals to vasopressin secreting magnocellular neurons, but that an inhibitory pathway from MnPO/medial septum/DB region to supraoptic and probably paraventricular nuclei is essential for inhibiting vasopressin secretion in overhydrated animals.
1. Nissen, R and L.P. Renaud (1994). GABA receptor mediation of median preoptic nucleus-evoked inhibition of supraoptic neurosecretory neurons in rat. Journal of Physiology 479: 207-16.
2. Jhamandas, J.H. and L.P. Renaud (1986). A γ-aminobutyric-acid-mediated baroreceptor input to supraoptic vasopressin neurons in the rat. Journal of Physiology 381: 595-606.
Blood-brain barrier disruption activates vasopressinergic PVN neurons
Yao, S.T.
Florey Institute of Neuroscience and Mental Health, University of Melbourne
The blood-brain barrier (BBB) acts primarily as a selective diffusion barrier consisting of cerebral microvascular endothelium, pericytes, astrocytes and the extracellular matrix. In addition, tight cell-to-cell junctions, a lack of fenestrations and low pinocytotic activity restrict the passage of substances accessing the brain1. The BBB also regulates the delivery of nutrients and removal of metabolites from the central nervous system; hence, a functional BBB is critically important for normal brain function2.
The BBB is thought to be affected by a number of diseases and conditions including hypertension. Disruption of the BBB in hypertensive animals has been attributed partly to the effects of mechanical shear stress on the vascular walls with increased pressure3. Whether this disruption allows the entry of substances such as Ang II into the brain is unclear. In order to address this, we experimentally disrupted the BBB using hypertonic mannitol (1.4M) injected into the carotid artery of anaesthetised (sodium pentobarbitone, 60mg/kg i.p.) male Sprague-Dawley rats. Following the administration of mannitol we observed extensive extravasation of Evan’s blue dye in the paraventricular nucleus of the hypothalamus (PVN). Administration of a subpressor dose of AngII (5ng/kg) increased the number of vasopressin and oxytocin positive PVN neurons in the mannitol compared with saline-treated control animals. Pre-treatment with the angiotensin type 1 receptor antagonist, losartan, attenuated this increase in PVN cell activation. These results suggest that disruption of the BBB allows the entry of circulating Ang II into the PVN to activate both vasopressin and oxytocin expressing neurons.
1. Persidsky Y, Ramirez SH, Haorah J, Kanmogne GD (2006) Blood–brain barrier: structural components and function under physiologic and pathologic conditions. J Neuroimmune Pharmacol 1: 223–236.
2. Bernacki J, Dobrowolska A, Nierwinska K, Malecki A (2008) Physiological and pharmacological role of the blood–brain barrier. Pharmacol Rep 60: 600–622.
3. Kaya M, Kalayci R, Kucuk M, Arican N, Elmas I, Kudat H, Korkut F (2003) Effect of losartan on the blood–brain barrier permeability in diabetic hypertensive rats. Life Sci 73: 3235–3244.
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Regulation of vasopressin secretion during septic shock
Carnio, E.C.
Laboratory of Physiology at Nursing Faculty of Ribeirao Preto, University of Sao Paulo, SP-Brazil
Septic shock is a multifaceted syndrome, which imposes an elevated morbidity and mortality in intensive care units, reaching a mortality rate of nearly 50%, despite adequate vasoconstrictor and antibiotic therapy. It is characterized by peripheral arteriolar vasodilatation, hypotension, hyporeactivity to vasoconstrictor agents, and inadequate tissue perfusion, leading to multiple organ dysfunction and death1. The prevalent hypothesis regarding its mechanism is that the syndrome is caused by an excessive defensive and inflammatory response.
During the acute phase some signalling mechanisms are activated, particularly hormone release, which function to restore the host homeostasis that has been disturbed by the infection. Since the neuroendocrine and immune systems are functionally associated, so the contact to antigens induces a coordinated response, which lets the organism to successfully endure immunology alterations. An important characteristic of this communication comprises the advent of proteins released into the circulation by stimulated immune cells. These proteins, called cytokines can enter the circulation and reach neuroendocrine organs, where they act either themselves or through the release of intermediates such as prostaglandin, catecholamines and nitric oxide. Besides that, the synthesis of these substances may be induced into the brain as a consequence of infection and may alter the function of the hypothalamic-pituitary axis2.
In this presentation it will be discussed the physiologic roles of the nitric oxide in central nervous system controlling the regulation of vasopressin during the pathophysiology of sepsis.
1. Annane, D., Bellissant, E., & Cavaillon, M. (2005) Septic Shock. Lancet, 365: 63-78.
2. Landry, D.W., Levin, H.R., Gallant, E.M., Ashton, R.C., Seo, S., D'Alessandro, D., & Oliver, J.A. (1997). Vasopressin deficiency contributes to the vasodilation of septic shock. Circulation, 95: 1122-1125
Oxidative stress in vasopressinergic neurons during sepsis
Rocha, M.J.A.
School of Dentistry of Ribeirão Preto, São Paulo University, Ribeirão Preto, SP, Brazil
Sepsis and its consequences, septic shock and, organ dysfunction, represent a serious public health problem. Besides the high costs, causes a high rate of mortality and the survivors, after hospital discharge, may have cognitive impairments resulting in future hospitalizations further burdening the national health care system. During sepsis there is release of cytokines, prostaglandins, leukotrienes, nitric oxide, etc.., which may contribute to the cognitive changes and hormonal dysfunctions observed during or after illness. In our laboratory, by using cecal ligation and puncture we have seen, as already reported in clinical studies, an impaired secretion of vasopressin despite persistent hypotension in the late stage of sepsis. In addition, we recently observed that there is an increase in IL-1β, iNOS, HIF-1 and caspase-3 in the supraoptic nucleus of septic rat which may affect vasopressin synthesis. Therefore, oxidative stress may be responsible for the hormonal dysfunction observed. We are currently analyzing the action of drugs in preventing this oxidative stress that may improve vasopressin secretion and survival in sepsis.
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Monday afternoon
Young Investigator Presentations
The anxiolytic effect of neuropeptide S is mediated via the oxytocin system
Grund, T., Neumann, I.D.
Department of Behavioural and Molecular Neurobiology, Institute for Zoology, University of Regensburg, Regensburg, Germany
Anxiety disorders are highly prevalent and despite substantial research novel therapeutics are still required. Neuropeptides such as neuropeptide S (NPS) and oxytocin (OXT) both represent potential targets due to their robust anxiolytic activity in rodents1,3. In order to extend our understanding of the molecular and cellular mechanisms of action of NPS we studied its possible effects on the nonapeptide OXT within the hypothalamic paraventricular nucleus (PVN) of male Wistar rats.
We assessed the effect of local infusion of NPS into the PVN on anxiety-related behaviour, a region known to express high levels of the NPS receptor (NPSR). Using the elevated plus-maze, we observed that rats treated with NPS displayed an increased percentage of time spent on the open arms, indicative of an anxiolytic-like effect. Further, qRT-PCR analysis revealed that central NPS induced the local expression of OXT mRNA in relation to the reference gene GAPDH after 3h.
Due to the lack of a specific NPSR antibody2, a potential direct interaction between these systems using an immunohistochemical approach cannot be examined. Therefore, we studied, whether icv NPS (1 and 5 nmol) induces OXT release within the PVN using microdialysis and in combination with a highly sensitive radioimmunoassay. We found that NPS dose dependently stimulates local OXT release. As both, NPS and OXT3 exert strong anxiolytic activity after intra-PVN administration, we further analysed, whether the anxiolytic activity of NPS is dependent on the oxytocinergic system. Thus, we applied a specific OXT receptor antagonist (icv, 0.75 µg) prior to icv infusion of NPS (1 nmol). Both in the light/dark-box and the open field we revealed that inhibition of the OXT receptor prevented the NPS-induced anxiolysis .
In conclusion, our results indicate a role for the oxytocinergic system in mediating the anxiolytic effect of NPS. Based on these findings we currently investigate the site of possible interaction between the NPS and the OXT systems.
Taken together, these data fill an important gap in our knowledge of the underlying mechanisms of the anxiolytic-like effects of NPS.
1. Xu, Y.L. et al. (2004). Neuropeptide S: a neuropeptide promoting arousal and anxiolytic-like effects. Neuron. 43: 487-97.
2. Slattery, D.A. and Naik, R.R. et al. (2015). Selective breeding for high anxiety introduced a synonymous SNP that increases neuropeptide s receptor activity. Journal of Neuroscience. 35: 4599-613.
3. Blume, A. et al. (2008). Oxytocin reduces anxiety via ERK1/2 activation: local effect within the rat hypothalamic paraventricular nucleus. European Journal of Neuroscience. 27:1947-1956
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Molecular effects of oxytocin on stress: Oxytocin delays CRF gene expression through CRTC3
Jurek, B., Slattery, D.A., Hiraoka, Y., Liu, Y., Nishimori, K., Aguilera, G., van den Burg, E.H., Neumann, I.D.
Department of Behavioural and Molecular Neurobiology, Institute for Zoology, University of Regensburg, Regensburg, Germany
Acute threatening stimuli evoke fear and physiological stress responses, which become pathological when exaggerated or prolonged, and can lead to severe psychological and somatic disorders including depression and anxiety. The hypothalamic corticotropin-releasing factor (CRF) is a key regulator of fear, anxiety and stress responses and controls the hypothalamo-pituitary-adrenal (HPA) axis. Neuronal CRF expression is regulated by the transcription factor CREB and its cofactors CRTC2/3 (CREB regulated transcription coactivator, also known as TORC) that, upon an appropriate stimulus, translocates from the cytoplasm to the nucleus to activate gene transcription by binding to the promoter region. We have shown that the neuropeptide oxytocin exerts anxiolytic [1] and anti-stress [2] effects, for example within the hypothalamic paraventricular nucleus (PVN). However, the underlying molecular mechanisms of these effects are unknown, yet are important for the quest for pharmacological intervention that may dampen (exaggerated) stress responses. We hypothesized that oxytocin inhibits HPA axis activity via altering CRF gene expression and prevention of CRTC translocation, and assessed this using a translational approach of several in vivo and in vitro assays.
In male rats, oxytocin (1 nmol, icv) delayed the restraint stress-induced increase in CRF hnRNA levels in the PVN as assessed by qPCR, but was without effect under non-stress conditions. CRF hnRNA levels were parallel to hypothalamic nuclear CRTC3 levels, and could be reduced at time points 10-30 min by the specific OT agonist TGOT in rats as well as in C57Bl/6 mice. In vitro studies revealed that in primary hypothalamic Wistar rat neurons, a Sprague Dawley rat hypothalamic and amygdala, as well as a human neuroblastoma cell line, TGOT (10 nM) delayed forskolin- (1 or 50µM) induced CRF transcription, comparable to oxytocin effects on stress-induced CRF expression. Selective knock-down of CRTC3 by siRNA prevented these effects of TGOT on CRF. ChIP analyses revealed that forskolin induced binding of CRTC2 and CRTC3 to the CRF promoter, and that TGOT specifically prevented binding of CRTC3 to the CRF promoter.
These results indicate that oxytocin controls CRF gene expression by modulating nuclear CRTC3 levels, thus attenuating/delaying CRF synthesis during the initial phase of an acute stress response. Taken together, our findings indicate that oxytocin acts on the CRF gene to modulate the acute response of the system to a stressor. Therefore, drugs, such as oxytocin, that target CRTC3 activity may represent novel candidates for the treatment of stress-related disorders.
1. Jurek, B., Slattery, D.A., Maloumby, R., Hillerer, K., Koszinowski, S., Neumann, I.D., van den Burg, E.H., 2012. Differential contribution of hypothalamic MAPK activity to anxiety-like behaviour in virgin and lactating rats. PloS one 7, e37060.
2. Neumann, I.D., Wigger, A., Torner, L., Holsboer, F., Landgraf, R., 2000. Brain oxytocin inhibits basal and stress-induced activity of the hypothalamo-pituitary-adrenal axis in male and female rats: partial action within the paraventricular nucleus. Journal of neuroendocrinology 12, 235-243.
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Neural mechanisms of oxytocin and serotonin interaction in non-human primates
Lefevre, A., Jazayeri, M., Richard, N., Ballanger, B., Beuriat, P.A., Duhamel, J.R., Sirigu, A.
Institut des Sciences Cognitives Marc Jeannerod, UMR 5229, CNRS and Université Claude Bernard Lyon 1, Bron, France
Oxytocin is increasingly studied for its therapeutic potential in psychiatric disorders which are supposedly caused by a deregulation of several neurotransmission systems. Hence investigating neurotransmitters’ interaction is a relevant step towards treatment. Studies in rodents have shown that serotonin (5-HT) is released following oxytocin (OXT) administration (1). Using PET-scan in humans we have recently found that 5-HT 1a receptor (5ht1a-r) function is modified after intra-nasal oxytocin intake (2). However, whether OXT modulates 5ht1a-r receptors through 5-HT release, receptor externalization or increased G protein coupling is still unclear.
To understand these mechanisms we tested 2 macaque monkeys using [18F]MPPF and [11C]DASB, PET radiotracers, two markers of the 5ht1a-r and the serotonin transporter, respectively. Oxytocin (1IU in 20µL of artificial cerebro-spinal fluid) or placebo was injected into the lateral ventricle 45 minutes before scans.
Preliminary results on 12 scans in one monkey, show that oxytocin significantly reduced [11C]DASB binding potential in the right hippocampus and posterior putamen whereas [18F]MPPF binding potential tended to increase in insula, amygdala and hippocampus.
These results indicate that oxytocin administration in primates influences serotoninergic neurotransmission via at least two ways: first by provoking a release of serotonin in key limbic regions and second, by increasing the number of available 5ht1a-r receptors in both limbic and cortical areas. Getting a full picture of the interaction effects between these two major neuromodulators would help to elaborate new pharmacological therapies for mental disorders such as autism or depression.
1. Dölen G, Darvishzadeh A, Huang KW, Malenka RC (2013): Social reward requires coordinated activity of nucleus accumbens oxytocin and serotonin. Nature. 501: 179–184.
2. Mottolese R, Redouté J, Costes N, Le Bars D, Sirigu A (2014): Switching brain serotonin with oxytocin. Proc Natl Acad Sci U S A. 111: 8637–8642.
Aldosterone activated mineralocorticoid receptor regulation of epithelial sodium channels (ENaC) in rat hypothalamic vasopressin-synthesizing neurons
Mills, N.J., Haque, M., Teruyama, R.
Department of Biological Sciences, Louisiana State University, USA
Vasopressin (VP) is synthesized in magnocellular neurosecretory cells (MNCs) and is released from the neurohypophysis to maintain a precise homeostatic state. Our previous study showed that functional epithelial sodium channels (ENaCs) and the known regulator of ENaC, the mineralocorticoid receptor (MR), are present in VP-synthesizing MNCs. However, it remains unknown if ENaC expression in MNCs is regulated by aldosterone, the primary mineralocorticoid, due to MR's equal binding affinity for both glucocorticoids and mineralocorticoids. To determine if MR in MNCs are regulated by aldosterone, in situ hybridization and immunocytochemistry was performed to examine whether 11β-hydroxysteriod dehydrogenase 2 (11β-HSD2), an enzyme that degrades glucocorticoids, is present in MR-expressing MNCs. Results showed that MR-
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immunoreactivity and 11β-HSD2 mRNA are co-localized in MNCs. Single-cell RT-PCR also revealed that individual MNCs co-express MR and 11β-HSD2 mRNA. These results suggest the MR in MNCs is protected from glucocorticoids by 11β-HSD2. ENaC mediation through aldosterone activation of MR was also examined in acute, coronal hypothalamic slices incubated with aldosterone or corticosterone. Real-time PCR analysis showed that both treatment with aldosterone and corticosterone resulted in a significant upregulation of γENaC mRNA with no change to the α and βENaC subunit mRNA. In addition, the MR antagonist, RU-28318, and the glucocorticoid receptor (GR) antagonist, RU-486, attenuated γENaC upregulation via aldosterone and corticosterone, respectively. These results indicate that MNC γENaC expression is mediated by aldosterone and corticosterone through the MR and GR, respectively. The present study suggests aldosterone and corticosterone independently modulate the firing patterns of MNCs via ENaC activity, which ultimately control the secretion of VP.
1. Teruyama, R., Sakuraba, M., Wilson, L.L., Wandrey, N.E., and Armstrong, W.E. (2012) Epithelial Na⁺ sodium channels in magnocellular cells of the rat supraoptic and paraventricular nuclei. Am J Physiol Endocrinol Metab. 302(3):E273-85.
Central activation of oxytocin neurons by Melanotan-II
Paiva, L., Sylvester, R., Tamborska, A., Ludwig, M.
Centre for Integrative Physiology, University of Edinburgh, UK
Central oxytocin release modulates several social behaviours in animals and humans. Moreover, variations in oxytocin levels have been associated with neuropsychiatric disorders such as anxiety and autism disorders. The administration of exogenous oxytocin has been proposed as a potential therapeutic. However, it is not clear whether oxytocin penetrates the blood brain barrier in neuroactive amounts1. An alternative approach to avoid this limitation is to stimulate endogenous oxytocin release using melanocortins2,3. Alpha-melanocortin stimulating hormone has been shown to be a potent stimulus to induce dendritic oxytocin release while inhibiting its systemic release2, but it also does not cross the blood brain barrier in physiological significant amounts. Thus, experiments were performed to test whether exogenous administration (intravenous or intranasal) of the synthetic melanocortin agonist, Melanotan-II (MT-II), is able to induce neural activation of oxytocin neurons. We used the immediate early gene product c-Fos as a marker of this, and showed that intravenous injections of MT-II induce Fos expression in magnocellular oxytocin neurons of the supraoptic nucleus (SON) and paraventricular nucleus (PVN) as well as in putative vasopressin cells of the SON. MT-II induced Fos expression was significantly reduced by central administration of the melanocortin antagonist SHU-9119. Finally, no significant change in Fos expression was detected in the PVN, SON and other brain regions analysed following intranasal administration of MT-II. These results suggest that intravenous administration of MT-II is potentially a pharmacological tool to induce activation of oxytocin neurons in the brain. Whether MT-II is able to induce central oxytocin release and oxytocin-dependant behaviours is currently under investigation.
1. Ludwig M, Tobin VA, Papadaki E, Callahan MF, Becker A, Engelmann M, Leng G. 2013. Intranasal application of vasopressin fails to elicit changes in brain immediate early gene expression, neural activity and behavioural performance of rats. J Neuroendocrinol 25: 655-667.
2. Sabatier N, Caquineau C, Dayanithi G, Bull P, Douglas AJ, Guan XM, Jiang M, Van der Ploeg L, Leng G. 2003. Alpha-melanocyte-stimulating hormone stimulates oxytocin release from the dendrites of hypothalamic neurons while inhibiting oxytocin release from their terminals in the neurohypophysis. J Neurosci 23:10351-58.
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3. Modi ME, Inoue K, Barrett CE, Kittelberger KA, Smith DG, Landgraf R, Young LJ. 2015. Melanocortin receptor agonists facilitate oxytocin-dependent partner preference formation in the prairie vole. Neuropsychopharmacology doi: 10.1038/npp.2015.35.
Emergence of a kisspeptin projection from the periventricular nucleus to the perinuclear zone of the supraoptic nucleus during pregnancy
Seymour, A.J., Piet, R., Campbell, R.E., Brown, C.H.
Centre for Neuroendocrinology and Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand
Kisspeptin is the endogenous ligand for the GPR54 (Kiss1R) receptor and is well established as a positive regulator of the hypothalamic-pituitary-gonadal axis. Using in vivo electrophysiology, we have shown that kisspeptin might also play a role in regulation of oxytocin neurons in late pregnancy. Oxytocin neurons from late pregnant (day 18 – 21) rats increase their firing rates following intracerebroventricular kisspeptin administration, whereas oxytocin neurons from virgin or early pregnant rats are unaffected.
Here we have further explored this relationship using immunohistochemistry and in vitro electrophysiology. Sections containing the supraoptic nucleus (SON) from virgin and day 21 pregnant (P21) rats were double-labelled for kisspeptin and oxytocin. The density of kisspeptin-positive fibres in the perinuclear zone (PNZ) of the SON was significantly higher in P21 rats than in virgin rats. We investigated expression of kisspeptin in hypothalamic cellular populations and showed a higher expression of kisspeptin-positive cell bodies in the anteroventricular periventricular nucleus (AVPV) and periventricular nucleus (PeN) but a lower expression in the arcuate nucleus (ARN) of P21 rats compared to virgin rats.
Kisspeptin-positive fibres in the PNZ did not co-label with neurokinin B, a peptide that is co-expressed with kisspeptin in cells from the ARN only. To identify the origin of PNZ kisspeptin-positive fibres, we stereotaxically injected a green fluorescent retrograde tracer into the PNZ and labelled AVPV, PeN and ARN containing sections for kisspeptin. Cells expressing both kisspeptin and the tracer were only found in the PeN population. Furthermore, there was a significant increase in cells expressing both kisspeptin and tracer in P21 rats compared to virgin rats, while the number of cells expressing tracer only was similar in both groups, suggesting that kisspeptin synthesis is increased in pregnancy.
To identify whether kisspeptin causes activation of oxytocin neurons in slices, we used cell-attached recordings to measure firing rates of magnocellular neurosecretory cells (MNCs). We found that kisspeptin did not affect firing rates in either late-pregnant or virgin rats. Because most MNCs were silent in these experiments, kisspeptin-induced subthreshold effects might have been missed. Therefore, we next used whole-cell recordings to examine whether kisspeptin affects excitatory synaptic transmission in MNCs from virgin and late pregnant rats. We found that kisspeptin did not affect spontaneous or miniature excitatory post-synaptic current frequency or amplitude in MNCs from virgin rats or late pregnant rats.
The results from the immunohistochemistry experiments suggest that kisspeptin peptide synthesis is upregulated during pregnancy and transported to the PNZ fibres where it might be involved in oxytocin neuronal excitation. However, the results from the electrophysiology suggest that the kisspeptin-induced increase in oxytocin neuronal activity in late pregnancy does not occur in slices. The effect might be mediated via upstream receptors that have had their connections to oxytocin neurons excised during slicing.
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Symposium 4: Neurohypophysial hormones acting at the hippocampal CA2 regulate social behaviours
Hypothalamic vasopressin system innervation of the hippocampus: Characteristics and functional implications
Zhang, L.
Department of Physiology, Faculty of Medicine, Universidad Nacional Autónoma de México, Mexico City, Mexico
The remarkable presence of arginine-vasopressin immunoreactive (AVPir) fibres in hippocampus has long been known. However little is known about where these projections come from, which neurons they act on and what the functional implications are. We aim to answer these questions using anatomical and electrophysiological approaches.
In one project, we used light and electron microscopy to trace the origin, distribution and synaptic relationship of AVPir fibres in the rat hippocampus. AVPir fibres were present in all areas (CA1-3, dentate gyrus) of the whole septo-temporal extent of the hippocampus; they exhibited highest density in the CA2 region, strongly increasing in density towards the ventral hippocampus. Two types of fibres were identified. Type A had large varicosities packed with AVPir large-granulated peptidergic vesicles (AVP-dcv) and few small clear vesicles forming type I synaptic junctions with pyramidal neuron dendrites, spines and axonal spines. Type B had smaller varicosities containing mostly small clear vesicles and only a few AVP-dcv and established type II synapses, mainly with identified interneuron dendrites. Using fluorogold retrograde tracing and anatomical analysis, we defined that the hypothalamo-hippocampal AVPir axons entered the hippocampus mostly through a ventral route, also innervating amygdala and to a lesser extent through the dorsal fimbria fornix, in continuation of the septal AVP innervation. Thus, it appears the AVPir neurons of the magnocellular hypothalamic nuclei serve as an important source for hippocampal AVP synaptic innervation, challenging the long-standing hypothesis that the AVPir parvocellular cells from BNST and amygdala were the main sources for hippocampal AVP innervation.
Using in vivo dual electrode extracellular recording and juxtacellular labelling in CA2 of the dorsal and ventral hippocampus, and PVN combined with signal analysis, we observed that AVP magnocellular neurons exhibit spontaneous REM like transitions and REM episodes. An osmotic stressor activates the magnocellular neurons of the PVN and increases the low-theta power and numbers of REM like episodes. The theta oscillation coherence between ventral versus dorsal hippocampus and ventral hippocampus versus PVN also increased.
These findings will be discussed within a broader context reflecting subcortical AVP containing circuits’ influence on hippocampal function.
Supported by grants PAPIIT-UNAM 216214 and CONACYT 238744 and 176916.
1. Zhang L and V.S. Hernández (2013). Synaptic innervation to rat hippocampus by vasopressin-immuno-positive fibres from the hypothalamic supraoptic and paraventricular nuclei. Neuroscience, 228:139-62.
2. Hernandez VS, S. Ruíz-Velazco and L. Zhang, (2012). Differential effects of osmotic and SSR149415 challenges in maternally separated and control rats: the role of vasopressin on spatial learning. Neurosci Lett. 528(2):143-7.
3. Hernandez V.S., E. Vazquez-Juarez, M.M. Marquez, F. Jauregui-Huerta, R.A. Barrio and L. Zhang. Extra-neurohypophyseal axonal projections of individual vasopressin-containing magnocellular neurons of rat hypothalamus. Under review.
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A role for the Avpr1b in social behaviours: What we have learned from Avpr1b knockout mice and lesion studies
Caldwell, H.K.
Laboratory of Neuroendocrinology and Behaviour, Department of Biological Sciences and School of Biomedical Sciences, Kent State University, Kent, OH, USA
It is well established that central vasopressin (Avp) acts via Avp 1a receptors (Avpr1a) to modulate social behaviours across species. However, work over the last 15 years has demonstrated that Avp can also affect social behaviours by signalling through the Avp 1b receptor (Avpr1b). While the Avpr1b has proven to be more difficult to visualize than the Avpr1a, it is more discretely localized than the Avpr1a with prominent expression in the CA2 region of the hippocampus. While this region of the hippocampus is largely unexplored, it does have distinct neurochemistry, connectivity, and structure. Thus, the presence of the Avpr1b here is intriguing. This talk will focus on what genetic knockout and site-specific lesion studies have taught us about the contributions of the Avpr1b and the CA2 region to the neural regulation of social behaviours, including social forms of aggressive behaviour and social memory.
Oxytocin and vasopressin induce synaptic potentiation in CA2 pyramidal neurons
Dudek, S.M.
Neurobiology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA
The hippocampal formation is critical for certain forms of memory, but until recently, little has been known about area CA2. Previous work had suggested a role for CA2 in aggressive behaviour and social recognition memory1- 3, but how synapses in this part of the brain change was unknown. From experiments based on the finding that CA2 pyramidal neurons appeared to be resistant to cell death4, we discovered that that Schaffer collateral synapses in CA2 are similarly resistant to induction of long-term potentiation (LTP) using stimulation protocols typical for induction of synaptic plasticity in the other sub-regions of the hippocampus. Using whole-cell patch clamp recordings from pyramidal neurons in slices of rat and mouse hippocampus, we have identified several redundant mechanisms that serve as brakes on the canonical form of activity-induced LTP in CA2, including robust calcium extrusion. The question that followed was whether and how synaptic plasticity occurs at Schaffer collateral synapses in CA2, if not through the usual routes. We have found that layered on this suppression of typical LTP at CA2 synapses is an increased capacity for potentiation induced by other means; the social neuropeptides oxytocin and vasopressin and adenosine A1 receptor antagonists such as caffeine all induce potentiation in CA2 at concentrations that are ineffective in CA1. Interestingly, the mechanisms by which oxytocin and vasopressin caused potentiation in CA2 neurons were very similar to that underlying typical LTP in CA1: NMDA receptors, calcium, and CaMKII activity. This pathway was distinct from that underlying adenosine A1 receptor antagonist-induced potentiation, which relies on adenylyl cyclase activity, cAMP, and PKA activity. These results indicate that CA2 Schaffer collateral synapses are indeed plastic and further suggest that social neuropeptides like oxytocin and vasopressin have the potential to influence CA2 function in vivo. These studies may inspire renewed interest to an area of the brain with possibly important
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roles in social recognition memory and in pathologies associated with social memory dysfunction.
1. Wersinger, S.R., Ginns, E.I., O'Carroll, A.M., Lolait, S.J. & Young, W.S., 3rd. (2002). Vasopressin V1b receptor knockout reduces aggressive behavior in male mice. Mol. Psychiatry 7: 975–984.
2. Stevenson, E.L. & Caldwell, H.K. (2014). Lesions to the CA2 region of the hippocampus impair social memory in mice. Eur. J. Neurosci. 40: 3294–3301.
3. Hitti, F. L. & Siegelbaum, S. A. (2014). The hippocampal CA2 region is essential for social memory. Nature 508: 88–92.
4. Caruana, D.A., Alexander, G.M. & Dudek, S.M. (2012). New insights into the regulation of synaptic plasticity from an unexpected place: hippocampal area CA2. Learn. Mem. 19: 391–400.
5. Pagani, J. H. et al. (2014). Role of the vasopressin 1b receptor in rodent aggressive behavior and synaptic plasticity in hippocampal area CA2. Mol. Psychiatry 20: 490-499.
Vasopressin innervation of the hippocampal CA2 area enhances social memory
Young, W.S.
Section on Neural Gene Expression, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA
My laboratory has been interested in the role of the vasopressin 1b receptor (Avpr1b) in behavior since we discovered that its inactivation in mice reduces social aggression, memory and motivation1,2. Further investigations revealed that in females, maternal aggression is also reduced and that normal pregnancy block is eliminated3,4. Furthermore, memory for temporal order is impaired5.
We found that the Avpr1b is expressed exclusively in area CA2 and immediately adjacent portion of area CA3 of the hippocampus as well as the likely developmentally related area fasciola cinereum6,
unpublished. Replacement of Avpr1b in the KO restores aggression7. With the discovery that the paraventricular nucleus of the hypothalamus of rats and mice projects to the CA2 region8,9, we embarked on a series of studies that examine how the vasopressinergic innervation of CA2 influences social behaviors. I will report on the progress we have made using optogenetics and DREADDs to show how AVP and the Avpr1b in the CA2 regulate social behavior.
This research was supported by the intramural research program of the NIMH (ZIA-MH-002498-24).
1. Wersinger, S. R., Ginns, E. I., O'Carroll, A. M., Lolait, S. J., & Young, W. S. (2002). Vasopressin V1b receptor knockout reduces aggressive behavior in male mice. Mol Psychiatr, 7: 975–984.
2. Wersinger, S. R., Kelliher, K. R., Zufall, F., Lolait, S. J., O'Carroll, A.-M., & Young, W. S. (2004). Social motivation is reduced in vasopressin 1b receptor null mice despite normal performance in an olfactory discrimination task. Hormones Behav, 46: 638–645.
3. Wersinger, S. R., Caldwell, H. K., Christiansen, M., & Young, W. S. (2007). Disruption of the vasopressin 1b receptor gene impairs the attack component of aggressive behavior in mice. Genes Brain Behav, 6: 653–660.
4. Wersinger, S. R., Temple, J. L., Caldwell, H. K., & Young, W. S. (2008). Inactivation of the oxytocin and the vasopressin (Avp) 1b receptor genes, but not the Avp 1a receptor gene, differentially impairs the Bruce effect in laboratory mice (Mus musculus). Endocrinology, 149: 116–121.
5. DeVito, L. M., Konigsberg, R., Lykken, C., Sauvage, M., Young, W. S., & Eichenbaum, H. (2009). Vasopressin 1b receptor knock-out impairs memory for temporal order. J Neurosci, 29: 2676–2683.
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6. Young, W. S., Li, J., Wersinger, S. R., & Palkovits, M. (2006). The vasopressin 1b receptor is prominent in the hippocampal area CA2 where it is unaffected by restraint stress or adrenalectomy. Neuroscience, 143: 1031–1039.
7. Pagani, J. H., Zhao, M., Cui, Z., Williams Avram, S. K., Caruana, D. A., Dudek, S. M., & Young, W. S. (2015). Role of the vasopressin 1b receptor in rodent aggressive behavior and synaptic plasticity in hippocampal area CA2. Mol Psychiatr, 20: 490–499.
8. Zhang, L., & Hernandez, V. S. (2013). Synaptic innervation to rat hippocampus by vasopressin-immuno-positive fibres from the hypothalamic supraoptic and paraventricular nuclei. Neuroscience, 228: 139–162.
9. Cui, Z., Gerfen, C. R., & Young, W. S. (2013). Hypothalamic and other connections with dorsal CA2 area of the mouse hippocampus. J Comp Neurol, 521: 1844–1866.
Tuesday morning
Plenary Lecture 4
Selenoether oxytocin analogues are analgesic in a mouse model of chronic abdominal pain
de Araujo, A.D.1, Mobli, M.1,2, Castro, J.3,5, Harrington, A.M.3,5, Vetter, I.1, Dekan, Z.1,
Muttenthaler, M.1, Wan, J.J.1, Lewis, R.J.1, King, G.F.1, Brierley, S.M.3,4,5, Alewood, P.F.1
1Institute for Molecular Bioscience and 2Centre for Advanced Imaging, The University of Queensland, St Lucia QLD, Australia, 3Nerve-Gut Research Laboratory, Discipline of Medicine and 4Discipline of Physiology, Faculty of Health Sciences, The University of Adelaide, Adelaide SA, Australia, 5Department of Gastroenterology & Hepatology, Hanson Institute, Royal Adelaide Hospital, Adelaide SA, Australia
Poor oral availability and susceptibility to reduction and protease degradation is a major hurdle in
peptide drug development. However, druggable receptors in the gut present an attractive niche for peptide therapeutics. Here we demonstrate, in a mouse model of chronic abdominal pain, that oxytocin receptors are significantly up-regulated in nociceptors innervating the colon. Correspondingly, we develop novel chemical strategies to engineer non-reducible and therefore more stable oxytocin analogues. Chemoselective selenide macrocyclization yields stabilized analogues equipotent to native oxytocin. Ultra-high-field nuclear magnetic resonance structural analysis of native oxytocin and the seleno-oxytocin derivatives reveals that oxytocin has a pre-organized structure in solution, in marked contrast to earlier X-ray crystallography studies. Finally, we show that these seleno-oxytocin analogues potently inhibit colonic nociceptors both in vitro and in vivo in mice with chronic visceral hypersensitivity. Our findings have important implications for clinical use of oxytocin analogues and disulfide-rich peptides in general.
Symposium 5: Integration of central and peripheral systems
Salt loading increases blood pressure via BDNF-mediated downregulation of KCC2 and impaired baroreceptor inhibition of vasopressin neurons
Choe, K.Y.1, Han, S.Y.2, Gaub, P.3, Shell, B.4, Voisin, D.L.5, Knapp, B.A.4, Barker, P.A.3, Brown, C.H.2, Cunningham, J.T.4, Bourque, C.W.1
1Centre for Research in Neuroscience, McGill University, Montreal QC, Canada, 2Centre for Neuroendocrinology and Department of Physiology, University of Otago, Dunedin, New
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Zealand, 3Montreal Neurological Institute, Montreal QC, Canada, 4Department of Integrative Physiology, University of North Texas Health Sciences Centre, Fort Worth TX, USA, 5Neurocentre Magendie, INSERM U862, 33077 Bordeaux, France
The mechanisms by which dietary salt promotes hypertension are unclear. Previous work has shown that plasma [Na+] and extracellular fluid osmolality rise in proportion with salt intake and thus promote release of vasopressin (VP) from the posterior pituitary. Although an increase in the circulating concentration of VP can significantly increase arterial pressure1, this action is normally prevented by a potent and GABAA receptor dependent inhibition of VP neurons by aortic baroreceptors (BR)2. Previous work has shown that inhibitory signalling by BRs can be attenuated in the deoxycorticosterone acetate (DOCA) salt model of hypertension3. Therefore in this study we examined the effect of simply increasing salt intake via salt-loading (SL) on rats (drinking fluid = 2%NaCl for 7 days). Whole cell perforated patch clamp recordings in vitro showed that SL impairs the inhibition of VP neurons mediated via the pathway that relays the BR afferents due to a depolarization of the equilibrium voltage for chloride, which attenuated inhibitory postsynaptic potentials. Extracellular recordings in vivo also showed that BR mediated inhibition of VP neurons is eliminated by SL. Experiments described in this talk will show that the loss of baroreceptor inhibition of rat VP neurons is mediated by a brain-derived neurotrophic factor (BDNF)-dependent activation of TrkB receptors and downregulation of KCC2 expression which causes increased intracellular chloride and prevents inhibitory GABAergic signalling. We also find that SL increases the spontaneous firing rate of VP neurons in vivo and that circulating VP contributes significantly to the elevation of arterial pressure under these conditions. Thus high levels of dietary salt intake can increase blood pressure by inducing neurotrophin dependent plasticity in central homeostatic circuits.
1. Yosten, G. L. and Samson, W. K. (2012) Pressor doses of vasopressin result in only transient elevations in plasma peptide levels. Peptides. 33: 342-345.
2. Grindstaff, R. R. and Cunningham, J. T. (2001) Cardiovascular regulation of vasopressin neurons in the supraoptic nucleus. Experimental Neurology. 171: 219-226.
3. Kim, Y. B., Kim, Y.S., Kim, W.B., Shen, F.Y., Lee, S.W., Chung, H.J., Kim, J.S., Han, H.C., Colwell, C.S. and Kim, Y.I. (2013) GABAergic excitation of vasopressin neurons: possible mechanism underlying sodium-dependent hypertension. Circulation Research. 113: 1296-1307.
Stress induced oxytocin release – modulation by corticotropin releasing factor peptide family
Dabrowska, J., Martinon, D.
Department of Cellular and Molecular Pharmacology and Department of Neuroscience, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA
Oxytocin (OT) is a hormone and neuromodulator produced primarily in the paraventricular nucleus of the hypothalamus (PVN). OT is implicated in the regulation of social and anxiety-like behavior as well as stress-coping mechanisms. We have recently shown that OT neurons from the PVN send oxytocinergic projections to the oval nucleus of the bed nucleus of the stria terminalis (BNSTov), a forebrain region critically involved in a long-duration fear response, that resemble anxiety-like behavior. Importantly, these OT terminals in the BNSTov express corticotropin releasing factor (CRF) receptor type 2 (CRFR2), which suggests that CRFR2 might modulate OT release in the BNST. Therefore the aim of this study was to determine 1) whether OT is released in the BNST in response to an acute stress and 2) whether OT release could be modulated by CRFR2. Therefore we have
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employed microdialysis in freely-moving rats to determine if an acute stress and CRFR2 manipulation by a selective (Urocortin 3), non-selective (CRF) agonist; or antagonist (Astressin 2B), could modulate OT release. Our results demonstrate that exposure to a single session of acute stress produced an increase in the OT release specifically in the BNSTov. We have also shown that blocking CRFR2 by Astressin 2B delivered locally via reverse dialysis (retrodialysis) caused an instant and significant increase in the OT release in the BNST, while CRFR2 activation by Urocortin 3 caused a delayed and only moderate decrease in the OT release. This suggests that Urocortin 3 in the BNST might be involved in a tonic inhibition of OT release. Interestingly, a non-specific CRFR2 agonist, CRF, did not have an immediate effect on the OT release, but instead caused a delayed and significant increase in the OT content in the BNST, suggesting a potential feedback mechanism via a postsynaptic CRFR1 activation. The latter observation is in agreement with our previously published findings that a reciprocal circuit exists between the neurons of the BNST and oxytocin neurons in the PVN. It also suggests that CRF (via CRFR1) and Urocortin 3 (via CRFR2) might exert opposite effects on OT release in the BNST, which further highlights differential roles of respective CRF peptide family members in modulation of stress-coping mechanisms. Together, our results suggest a fine-tuned control of OT release by the members of the CRF peptide family and by the feedback loop between the BNST and PVN. Understanding how the PVN-BNST circuit modulates oxytocin release will be critical for our understanding of the oxytocin effects on stress-induced anxiety-like behavior.
Mouse preoptic kisspeptin neurons integrate estrogen feedback and vasopressin signaling in female mice: implications for the generation of the preovulatory gonadotropin releasing hormone surge
Piet, R.1, Fraissenon, A.1, Boehm, U.2, Herbison, A.E.1
1Centre for Neuroendocrinology and Department of Physiology, University of Otago, Dunedin, NZ, 2Department of Pharmacology and Toxicology, University of Saarland School of Medicine, Homburg, Germany
Fertility in all mammals is controlled by a complex neuronal network, of which the gonadotropin releasing hormone (GnRH) neurons are the final output. In females, the rising concentrations of circulating estrogen during the proestrous stage of the estrous cycle activate the GnRH neuronal network, causing the GnRH surge that triggers ovulation. In nocturnal rodents, the preovulatory surge occurs specifically at the end of the proestrous afternoon. Precise timing of the surge relies on integration within the GnRH neuronal network of daily signals arising from the central circadian clock in the suprachiasmatic nucleus (SCN) with the cyclical fluctuations in circulating estrogen concentrations. Kisspeptin neurons of the preoptic area, a population of cells upstream of the GnRH neurons, play a central role in the generation of the preovulatory surge and have recently emerged as a critical node where circadian and gonadal hormone cues might be integrated. These neurons express the estrogen receptors required for the surge and are innervated by SCN neurons that produce arginine vasopressin (AVP), a neuropeptide thought to play a key role in generating the preovulatory surge.
We combined electrophysiology and calcium imaging in acute brain slices obtained from female mice in which kisspeptin neurons express the green fluorescent protein or the genetically encoded calcium indicator GCaMP3 to investigate the cellular mechanisms governing the integration of humoral and circadian cues in these neurons. We first examined the effect of AVP on the activity of preoptic kisspeptin neurons. Bath applications of AVP significantly and reversibly increased spontaneous action potential firing and intracellular calcium concentration in the majority of preoptic kisspeptin neurons. This effect was mediated directly at the kisspeptin neuron and required the activation of type 1a AVP receptors (V1aRs). These
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data reveal that SCN neurons drive the activity of preoptic kisspeptin neurons via activation of V1aRs in these neurons. To generate the preovulatory surge only on the proestrous afternoon, SCN-derived daily signals to the GnRH neuronal network are thought to be gated in a time of day- and estrous cycle stage-dependent manner. We therefore tested whether this is achieved through regulation of AVP-V1aR signaling in preoptic kisspeptin neurons. The effect of AVP on kisspeptin neuron action potential firing was similar hours prior to (zeitgeber time ZT4-6) or just before (ZT10) the expected time of the proestrous preovulatory surge, suggesting that AVP-V1aR signaling in these neurons is not time of day-dependent. Further, we found that AVP-induced increases in firing were not significantly different in slices from diestrous and proestrous females, suggesting that fluctuating estrogen levels do not alter AVP signaling in kisspeptin neurons. Remarkably, however, the effect of AVP was found to be critically dependent on circulating estrogen as it was markedly reduced in slices from ovariectomized (OVX) mice and restored to ‘ovary-intact’ levels by in vivo treatment of OVX mice with estrogen implants.
Together these data identify AVP-V1aR signaling as a pathway through which SCN neurons may control the activity of preoptic kisspeptin neurons. However, our results show that V1aR functioning on kisspeptin neurons cannot alone account for the circadian and estrogen gating of the daily signal to the afternoon of proestrus. Instead, we find that circulating estrogen exerts a permissive effect on AVP-V1aR signaling in kisspeptin neurons enabling but not causing their suspected enhanced activity prior to the surge.
Regulation of vasopressin neurons and fluid balance in a model of dilutional hyponatremia
Cunningham, J.T.1, Nedungadi, T.P.1, Saxena, A.1, Shell, B.1, Walch, J.D.1,2
1Department of Integrative Physiology and Anatomy and the Cardiovascular Research Institute, UNT Health Science Center, Fort Worth, TX, 2Department of Pharmacology and Neuroscience, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA
Hyponatremia is associated with negative outcomes in many chronic disease states such as congestive heart failure and cirrhosis. In both liver and heart failure, changes in drinking behavior and arginine vasopressin (AVP) release contribute to water retention that leads to dilutional hyponatremia which increases morbidity and mortality. The CNS mechanisms responsible for elevated drinking and AVP release in the face of escalating hyponatremia are not known. We have used chronic bile duct ligation (BDL) as a model of liver failure to study the mechanisms leading to increase water intake and AVP release. As in the human condition, these rats have elevated circulating vasopressin and increased drinking behavior accompanied by significantly decreased plasma osmolality, increased plasma renin activity, and hypervolemic ascites formation. In this condition we have also observed increased FosB and TRPV4 expression in AVP neurons in the supraoptic nucleus (SON) of the hypothalamus.
FosB is a member of the AP-1 family of transcription factors and has been linked to neuroplasticity. An adeno-associated viral vector containing a dominant negative antagonist against FosB was injected into the SON of BDL rats to test the role of this transcription factor in hyponatremia. Dominant negative inhibition of FosB in the SON significantly increased urine output and reduced urine sodium in BDL rats without influencing water intake. In addition, plasma osmolality and hematocrit were normalized in BDL rats treated with the dominant negative construct. These effects on urine output suggest that FosB may mediate changes in gene expression necessary for sustained AVP release in BDL rats.
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Changes in TRPV4 expression in BDL rats were significantly correlated with plasma renin activity and circulating AVP suggesting that it could contribute to AVP release during cirrhosis. We tested the effect of angiotensin on TRPV4 function in vitro using a vasopressin expressing hypothalamic IVB cell line. After a 1-h incubation in ANG II (100 nM), 4B cells showed increased TRPV4 abundance in the plasma membrane fraction, and this effect was prevented by the angiotensin type 1 receptor antagonist losartan (1 uM) and by a Src kinase inhibitor, PP2 (10 uM). Ratiometric calcium imaging experiments demonstrated that angiotensin incubation potentiated TRPV4 agonist (GSK 1016790A, 20 nM)-induced calcium influx. These effects were also blocked by losartan and PP2. Based on these results, we tested the role of central angiotensin receptors in hyponatremia in BDL rats. We observed that BDL was associated with increase expression of the AT1aR in the subfornical organ (SFO). Chronic infusions of icv losartan blocked the increase in AT1aR and reduced water intake in BDL rats but did not significantly affect plasma osmolality or hematocrit. Virally mediated knockdown of AT1aR in the SFO produced similar effects on water intake and plasma osmolality. AT1aR knockdown in the SFO did not significantly affect FosB staining in vasopressin neurons of the SON.
Together these data indicate that angiotensin receptors in the SFO are necessary for increased water intake in BDL rats but not for changes in vasopressin release or FosB expression in the SON that contribute to hyponatremia.
Plenary Lecture 5
Clinical and translational applications of vasopressin and oxytocin: Groundbreaking past, exciting future
Verbalis, J.G.
Georgetown University, Washington DC, USA
This talk will summarize the past history, current applications, and future potential of clinical use of agonists and antagonists of the arginine vasopressin (AVP) and oxytocin (OT) receptors. The history of the development of new therapeutic uses for AVP and OT can truly be described as groundbreaking, as early studies of the biological actions and structure of AVP and OT were quickly translated into new therapies. AVP from pituitary extracts was used in 1913 to treat diabetes insipidus, which represented the first use of a hormone to treat a hormonal deficiency disorder. Similarly, OT from pituitary extracts was used to stimulate labor as early as 1947. The subsequent elucidation of the structure of AVP and OT by du Vigneaud in 1953 led to safer use of more targeted synthetic peptides, and the eventual successful synthesis of the first selective AVP V2R agonist, desmopressin, in 1978, which was also later found to be the first peptide that could be administered orally. More recently, antagonists of the AVP V2R, known as vaptans, have been developed and approved for clinical use as alternatives or supplements to current therapies for the treatment of both acute and chronic hyponatremia due to water retention, and antagonists of OTR to prevent or delay preterm labor. At the current time, V2R agonists (desmopressin) and antagonists (conivaptan and tolvaptan) are important tools of therapy for patients with disorders of water metabolism, and OT agonists (pitocin, syntocinon) and antagonists (atosiban) are widely used for induction of labor, reduction of post-partum hemorrhage prevention of pre-term labor, and somewhat less successfully for enhancement of lactation. Building upon this rich history of effectively translating physiological actions of AVP and OT into therapeutic treatments, emerging data implicating these peptides in a variety of unexpected actions will provide abundant opportunities for future novel applications of both agonists and antagonists of AVP and OT receptors. Among those that will be discussed include the use of V2R antagonists to treat polycystic kidney disease, differential actions of AVP and OT on bone, effects of OT on social behaviors and affiliative diseases such as autism, use if V1bR
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antagonists as anxiolytics, and effects of OTR agonists on appetitive behaviors and diseases of body weight homeostasis such as the Prader-Willi syndrome. All of these areas, and others, are at different degrees of translation into human therapies, but it seems certain that many, if not all, will result in further contributions to human health in the near future.
Poster Abstracts
Poster 1
Induction of angiotensin II-dependent hypertension does not activate microglia in the hypothalamic supraoptic and paraventricular nuclei
Korpal, A.K.1, Schwenke, D.O.2, Brown, C.H.1
1Centre for Neuroendocrinology and 2Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand
Arginine vasopressin (AVP) magnocellular neurosecretory cells of the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON) release AVP into the bloodstream to cause vasoconstriction and reabsorption of water from the collecting ducts in the kidneys. Both of these actions increase arterial blood pressure. Under basal conditions, AVP secretion is inhibited by a tonic GABAergic input to AVP neurons that is activated by a relay of afferent inputs that are, in turn, activated by arterial baroreceptors, which detect acute moment-to-moment changes in blood pressure. Low blood pressure decreases this GABAergic inhibition of AVP neurons, allowing AVP release, which helps to return blood pressure back towards normal.
Paradoxically, some patients suffering from essential hypertension have increased plasma AVP concentrations. We have found that AVP neuron firing rate is increased early in the induction of angiotensin (ANG) II-dependent hypertension in rats. Microglial activation has been observed in the PVN of rats with severe chronic ANG II-dependent hypertension. We hypothesised that microglial activation might drive the increased AVP neuron activity at the onset of hypertension.
To test this this hypothesis, we determined whether microglia are activated in the PVN and SON during the onset of moderate hypertension using 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 (I3C) over 7 days. The increase in arterial blood pressure over 7 days was confirmed using tail-cuff plethysmography to take serial measurements of systolic blood pressure (SBP) in rats fed ordinary diet vs rats fed I3C diet over the 7 day treatment period. Rats fed I3C diet exhibited a rise in SBP from an average baseline of 138 mmHg on day 0 to 172 mmHg on day 7. These rats were designated as ‘hypertensive’. Rats fed ordinary diet maintained a relatively constant SBP, with a baseline of 138 mmHg on day 0 and 133 mmHg on day 7. These rats were designated as ‘non-hypertensive’.
Immunohistochemistry for ionized calcium-binding adapter molecule 1 (Iba1), a microglial marker, was carried out to quantify microglial activation. The area fraction for Iba1 positive immunolabelling was calculated for the PVN and SON. There was no increase in Iba1 immunolabelled area in the SON (non-hypertensive = 9.2% vs. hypertensive = 11.3%; P = 0.19 unpaired t-test; n = 5 - 7). Similarly, no change was observed in the PVN (non-hypertensive = 12.2% vs. hypertensive = 12.3%; P = 0.95 unpaired t-test; n = 6). In conclusion, we found that microglial activation does not occur in the SON or PVN during the induction of moderate angiotensin II-dependent hypertension. In conclusion, microglial activation is unlikely to cause the increased AVP neuron activity evident early in the onset of hypertension, refining the scope for identifying key targets in AVP neuron activation.
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Poster 2
Love drug to treat abdominal pain
De Araujo, A.D.1, Mobli, M.1,2, Castro, J.3,5, Harrington, A.M.3,5, Vetter, I.1, Dekan, Z1,
Muttenthaler, M.1,6, Wan, J.1, Lewis, R.J.1, King, G.F.1, Brierley, S.M.3,4,5, Alewood, P.F.1
1Institute for Molecular Bioscience and 2Centre for Advanced Imaging, The University of Queensland, St Lucia QLD, Australia, 3Nerve-Gut Research Laboratory, Discipline of Medicine and 4Discipline of Physiology, Faculty of Health Sciences, The University of Adelaide, Adelaide SA, Australia, 5Department of Gastroenterology & Hepatology, Hanson Institute, Royal Adelaide Hospital, Adelaide SA, Australia, 6Current address: The Institute for Research in Biomedicine, Departments of Chemistry and Molecular Pharmacology, C/ Baldiri Reixac 10, Barcelona, Spain.
Poor oral bioavailability and susceptibility to reduction and protease degradation is a major hurdle in peptide drug development. However, druggable receptors in the gut present an attractive niche for peptide therapeutics. Here we demonstrate, in a mouse model of chronic abdominal pain, that oxytocin receptors are significantly up-regulated in nociceptors innervating the colon. We developed novel chemical strategies to engineer non-reducible and therefore more stable oxytocin analogues, featuring a robust selenoether crosslink. Chemoselective selenide macrocyclization yielded analogues equipotent to native oxytocin. Ultra-high-field nuclear magnetic resonance structural analysis of native oxytocin and the seleno-oxytocin derivatives reveals that oxytocin has a pre-organized structure in solution, in marked contrast to earlier X-ray crystallography studies. Finally, we demonstrated that these seleno-oxytocin analogues potently inhibit colonic nociceptors both in vitro and in vivo in mice with chronic visceral hypersensitivity.1
1. de Araujo et al (2014) Selenoether oxytocin analogues have analgesic properties in a mouse model of chronic abdominal pain, Nature Communications 5, 3165.
Poster 3
Effects of arginine vasopressin (AVP) in the long term regulation of arterial blood pressure in the rat
Quintanar-Stephano, A.1, Villanueva-Rodríguez, M.1, Organista-Esparza, A.1, Viñuela-Berni, V.1, Huerta-Carreón, P.1, Chavira-Ramírez, R.2, Tinajero-Ruelas, M.1, Kovacs, K.3
1Departamento de Fisiología y Farmacología, Centro de Ciencias Básicas, Laboratorio de Neuroinmunoendocrinología, Universidad Autónoma de Aguascalientes, México, 2Laboratorio de Hormonas Esteroides, Instituto Nacional de Ciencias Médicas y Nutrición ¨Salvador Zubirán¨, México, DF, 3Department of Laboratory Medicine, Division of Pathology, St. Michael’s Hospital, University of Toronto, Toronto, Ontario. Canada
Previously, we described that neurointermediate pituitary lobectomy (NIL) in rats, induced an early but transient diabetes insipidus and permanent low BP levels without changes in both Na and K serum levels and aldosterone (1). Thus, the vascular and kidney mechanisms involved in the hydroelectrolytic homeostatasis in these low BP animals are unknown. In order to obtain a deeper insight into the effects of NIL on AVP and oxytocin (OT) serum levels and some kidney mechanisms
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involved in the survival of these animals, we evaluated the AVP and Oxytocin (OT) serum levels through the time after NIL, water intake and urine output (diabetes insipidus), serum and urine sodium and potassium and mean BP immediately (0 to 6 hours post NIL) and 3, 15, 45 and 90 days post NIL. The renin, angiotensin II and aldosterone system (RAAS) was assessed at 3 and 45 days post NIL. Values were compared with the respective sham operated (SHAM) groups. Results: NIL caused significant and permanent low AVP and OT serum level (10.6 ± 0.8 versus (vs) 2.4 ± 0.16 (pg/ml) (SE), SHAM vs NIL), transient diabetes insipidus (15-30 days of duration) and a significant and permanent decrease of the MBP in 70% respect to NIL animals (106.8 ± 3.15 vs 73.8 ± 3.2 mmHg). Hypotension was apparent 30 minutes after NIL. No significant differences in the RAAS and serum electrolytes were evident whereas urine electrolytes were lower in NIL vs SHAM rats (Na; 33.08 ± 6.177 vs 58.99 ± 15.061 and K; 67.148 ± 12.515 vs 126.9 ± 26.97 respectively). We concluded that: 1) the permanent deficiency of AVP is responsible of the long term low BP levels. 2) At the present we suppose that the normality of the Na and K serum levels in the NIL animals is due to the remnant basal concentrations of AVP (2.4 ± 0.16) in the NIL animals which are enough to maintain the electrolytes concentration in the blood. 3) The decreased Na and K levels in the urine of the NIL animals may be explained by the AVP deficiency which is not enough to maintain the antidiuretic effect of AVP. 4) Further studies are needed to explain why some animals did not develop hypotension, and to determine how cardiovascular, renal and hormone adaptive mechanisms in the NIL group support their survival.
Supported by UAA-PIFF 14-1 grant (AQS) and CONACYT 221262 (AQS) and PhD scholarship (GMV), México and the Jarislowsky and Lloyd Carr-Harris Foundations(KK), Canada.
1. Andrés Quintanar-Stephano, Citlalli Contreras-Romo, Adriana Rodriguez-Peralta Claudia R. González-Ruiz Irma G. Reynoso Andeola, Raúl Ponce-Gallegos and Kalman Kovacs. The role of neurohypophysis in the long-term blood pressure regulation in rats. FASEB Meeting. 2012.
Poster 4
Effects of arginine vasopressin on innate immunity in the rat
Quintanar-Stephano, A.1, Viñuela-Berni, V.1, Organista-Esparza, A.1, Tinajero-Ruelas, M.1, Huerta-Carreón, E.P. 1, Kovacs, K.2, Berczi, I.1,3
1Departamento de Fisiología y Farmacología, Centro de Ciencias Básicas, Laboratorio de Neuroinmunoendocrinología, Universidad Autónoma de Aguascalientes, México, 2Department of Laboratory Medicine, Division of Pathology, St. Michael’s Hospital, University of Toronto, Toronto, Canada, 3Department of Immunology, Faculty of Medicine, University of Manitoba, Canada
Immune and vascular endothelial cells possess arginine vasopressin (AVP) receptors (V1a and V2 and V2 respectively). AVP is an important stimulating/regulatory hormone in acquired immunity1, whereas its role on innate immunity (INIM) is not well known. Neurointermediate pituitary lobectomy (NIL) in the rat causes permanent low AVP serum levels. Here, we investigate the AVP and INIM relationship in NIL rats (3 weeks after surgeries), subject to two INIM tests. In experiment 1, the ability of peritoneal macrophages (Mθ) for erythrophagocytosis was assessed, whereas in the experiment 2, the skin Evans blue extravasation-histamine doses-response test was evaluated. In experiment 1, NIL group was compared against Intact control (IC), sham operated (SHAM) and anterior pituitary lobectomysed (AL) groups. In the experiment 2, NIL group was compared against IC, NIL+desmopressin (DP) (a synthetic analog of AVP) and IC+conivaptan (CON) (an antagonist of V1a-V2 AVP receptors) groups. Results: Experiment 1 showed that NIL induced a significant decrease in macrophages erithrophagocytosis as compared with the IC and AL groups. In experiment 2 a
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significant increase of skin edema (histamine doses-dependent) occurred in NIL and IC+CON groups, whereas DP administration to NIL animals block the increased edema observed in the NIL and IC+CON groups (non-statistical differences between IC vs NIL+DP groups).
This work shows that uggests that Conclusions: AVP plays an important role in phagocytic activity of the peritoneal Mθ and for the stabilization of the vascular endothelial cells permeability during histamine inflammation.
Supported by UAA-PIFF 14-1 and CONACYT-221262, México and the Jarislowsky and Lloyd Carr-Harris Foundations. Canada.
1. Quintanar-Stephano A., Organista-Esparza A., Chavira-Ramírez R., Kovacs K and Berczi I. (2012). Effects of Neurointermediate Pituitary Lobectomy and Desmopressin on Acute Experimental Autoimmune Encephalomyelitis in Lewis Rats. Neuroimmunomodulation. 19:148–157
Poster 5
Oxytocin and familiarity: preferences for conspecifics and objects in male mice
Klockars, A.1, Levine, A.S.2, Olszewski, P.K.1
1 Department of Biological Sciences, University of Waikato, Hamilton, New Zealand,2 Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN, USA
Oxytocin (OT) has been previously associated with the development of social and object preferences, however, the majority of studies performed to date have focused on the prairie vole as a model of social behaviour. In the current set of studies, we wished to investigate how OT receptor blockade affects preference for novel vs familiar conspecifics in mice and whether this parallels the effects on preference for a novel vs familiar object. Male group-housed mice were habituated to short-term sessions in the 3-chamber sociability apparatus. Before experimental sessions, a blood-brain barrier penetrant OT receptor antagonist, L-368,899, was administered intraperitoneally (saline was used as a control). Each animal tested in the apparatus during the experiment could choose between chambers containing a familiar vs novel conspecific or familiar vs novel object or a “neutral” (middle) chamber. OT receptor blockade was associated with a decrease in time spent with a familiar conspecific and a familiar object and it had no effect on the amount of time spent in the chamber containing a novel animal or novel object. In a subsequent study, mice exposed to familiar or novel conspecifics vs objects, were sacrificed and their brains processed immunohistochemically for an immediate-early gene product, c-Fos. We found a partial overlap in brain activity in object vs conspecific-exposed mice in both novelty and familiarity scenarios, which suggests that responses to novel and familiar stimuli of a social and non-social nature are governed by a shared circuitry.
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Poster 6
Study of combined action of vasopressin receptor agonists and GLP-1 mimetic on renal sodium transport
Kutina, A.V., Marina, A.S.
Laboratory of Renal Physiology, Sechenov Institute of Evolutionary Physiology and Biochemistry the Russian Academy of Sciences, Saint-Petersburg, Russia
Vasopressin plays a primary role in the concentration of urine to maintain body fluid homeostasis. The data on the effect of vasopressin on renal sodium handling are contradictory. It was shown that lower vasopressin levels stimulate V2-receptor-mediated sodium reabsorption, whereas higher levels stimulate V1a-receptor-mediated natriuresis1. It was hypothesized that abovementioned vasopressin effects are mediated by its action in distal nephron. To test this hypothesis and to evaluate a value of vasopressin-regulated sodium reabsorption it was of interest to investigate vasopressin action on renal sodium excretion under inhibition of proximal reabsorption. Recently we have shown that incretin, glucagon-like peptide-1, is secreted after water and NaCl consumption; this hormone and its mimetic, exenatide, exert natriuretic effects in rats2 mediated by Na/H-exchanger inhibition in renal proximal tubules3. The aim of the present study was an investigation of combined action of vasopressin receptor agonists and glucagon-like peptide-1 mimetic on renal sodium transport.
Experiments were carried out using Wistar rats. We applied exenatide (1.5 nmol/kg, intramuscularly), V1a agonist (1 nmol/kg Phe2-Ile3-Orn8-vasopressin, Bachem, Switzerland) and V2 agonist (0.01 nmol/kg desmopressin, Sigma-Aldrich, USA). Urine samples were collected using metabolic cages. Blood samples were obtained from the carotid artery under zoletil (50 mg/kg) anesthesia. Sodium concentration in urine and serum was measured using a Sherwood-420 flame photometer (UK), creatinine concentration was determined by kinetic method using Erba XL-200 (Czech Republic).
Desmopressin, agonist of V2 receptors, did not affect urinary sodium excretion (0.05±0.01 vs. 0.07±0.02 mmol/kg/2h, p>0.05). Stimulation of V1a receptors led to natriuresis (1.5±0.2 mmol/kg/2h, p<0.05), sodium fractional excretion (FENa) rose from 0.15±0.04% to 8±2%. Incretin mimetic exenatide sharply increased urinary sodium excretion (3.3±0.2 mmol/kg/2h, p<0.05) and FENa (9±1%, p<0.05). Desmopressin significantly reduced natriuresis stimulated by exenatide. Urinary sodium excretion and FENa after coadministration of exenatide and desmopressin were 2.5±0.2 mmol/kg/2h and 7±1%, respectively (p<0.05). The opposite effect was revealed after coinjection of exenatide with V1a-agonist – natriuresis enhanced up to 5.2±0.2 mmol/kg/2h (p<0.05), FENa – up to 18±2% (p<0.05). The data obtained suggested that vasopressin-regulated sodium reabsorption could amount significant part of filtered sodium (up to 8%) and takes place in distal segment of nephron. In conditions of decreased proximal tubular fluid reabsorption by exenatide vasopressin could essentially modulate renal sodium excretion by activating different types of V-receptors. Thus, interaction of effects of neurohypophyseal hormones and incretins could be significant in regulation of sodium balance.
This work was supported by the Russian Scientific Foundation (project 14-15-00730).
1. Perucca, J., Bichet, D.G., Bardoux, P., Bouby, N. and L. Bankir (2008). Sodium excretion in response to vasopressin and selective vasopressin receptor antagonists. J. Am. Soc. Nephrol. 19: 1721–31.
2. Kutina, A.V., Marina, A.S. and Y.V. Natochin (2011). A novel natriuretic factor in hypervolemia. Dokl. Biol. Sci. 441: 360–2.
3. Thomson, S.C., Kashkouli, A. and P. Singh (2013). Glucagon-like peptide-1 receptor stimulation increases GFR and suppresses proximal reabsorption in the rat. Am. J. Physiol. Renal Physiol. 304: F137–44.
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Poster 7
Visualization of vasopressin neurons in hypothalamic organotypic cultures using vasopressin-Venus transgenic mice
Hagiwara, D.1, Tochiya, M.1, Lu, W.J.1, Azuma, Y.1, Shiota, A.2, Arima, H.1
1Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, Nagoya, Japan, 2Institute of Immunology Co., Ltd., Utsunomiya, Japan
In the hypothalamo-neurohypophysial system, arginine vasopressin (AVP) is synthesized in the magnocellular neurons (MCNs) in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus and transported through the axon to the posterior pituitary. Because there were no useful homologous cell lines to study the AVP-MCNs, hypothalamic organotypic cultures were developed1. Furthermore, various electrophysiological examinations of AVP-MCNs have been performed using the AVP-eGFP transgenic rats2. On the other hand, AVP-MCNs are more vulnerable in rats than in mice, and it is shown that AVP-MCNs cannot survive well in the rat hypothalamic organotypic cultures3.
In order to establish an ex vivo system to study the AVP-MCNs, we generated a bacterial artificial clone (BAC) transgenic mouse line expressing yellow fluorescent protein variant, Venus, in the AVP neurons. In the AVP-Venus BAC transgenic mice, the robust expression of Venus was observed in the SON, PVN, and suprachiasmatic nucleus as well as posterior pituitary. Double immunofluorescence staining for Venus and AVP or oxytocin (OXT) in the SON and PVN revealed that almost all of the Venus-positive neurons expressed AVP but not OXT. The Venus expression in the SON and PVN was increased at both mRNA and protein levels after water deprivation for 48 h, suggesting that the transcriptional activity of AVP-Venus BAC transgenes was properly regulated by plasma osmolality.
Using 7-day-old AVP-Venus BAC transgenic mice, we performed hypothalamic organotypic cultures. The Venus-expressing AVP-MCNs in the SON of the explants were visible at least for 7 d in serum containing medium (containing 25% heat-inactivated horse serum) and for additional 4 d in serum free medium without any neurotrophic factors. Furthermore, the incubation of hypothalamic slices with K+ (50 mM) increased the Venus fluorescence in the AVP-MCNs.
In conclusion, we generated the AVP-Venus BAC transgenic mice in which AVP neurons were labelled with Venus. Furthermore, our data suggest that the hypothalamic organotypic cultures using the AVP-Venus BAC transgenic mice would be useful to study the AVP-MCNs.
1. House, S.B., Thomas, A., Kusano, K. and Gainer, H. (1998). Stationary organotypic cultures of oxytocin and vasopressin magnocellular neurones from rat and mouse hypothalamus. Journal of Neuroendocrinology. 10(11): 849-61
2. Ueta, Y., Fujihara, H., Serino, R., Dayanithi, G., Ozawa, H., Matsuda, K., Kawata, M., Yamada, J., Ueno, S., Fukuda, A. and Murphy, D. (2005). Transgenic expression of enhanced green fluorescent protein enables direct visualization for physiological studies of vasopressin neurons and isolated nerve terminals of the rat. Endocrinology. 146(1): 406-13.
3. Arima, H., House, S.B., Gainer, H., Aguilera, G. (2001). Direct stimulation of arginine vasopressin gene transcription by cAMP in parvocellular neurons of the paraventricular nucleus in organotypic cultures. Endocrinology. 142(11): 5027-30
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Poster 8
Vasopressin receptors and renal effects of arginine-vasopressin and arginine-vasotocin in rat kidney
Golosova, D.V., Natochin, Yu. V.
Department of Renal Physiology, Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences, Saint-Petersburg, Russia
Osmotic and ionic homeostasis depends on renal functions modification under the influence of the hormones of neurohypophysis. In evolution of vertebrates the key hormone of neurohypophysis regulating water-salt balance arginine-vasotocin (AVT) was substituted by arginine-vasopressin (AVP) in mammals. V1a-, V1b- and V2-receptors were discovered in rat kidney. The aim of the study was to compare AVT and AVP effects on cation excretion by rat kidney and to elucidate functional role of subtypes of V-receptors in the in vivo effects of the nonapeptides.
The study was performed on female Wistar rats weighting between 160-230 g. Diuresis was recorded at spontaneous urination for 4 h. Blood samples were obtained from carotid artery under zoletil anaesthesia (5 mg/100g body weight (bw), Zoletil, Virbac, France). Osmolality (Micro-Osmometer 3300, Advanced Instruments, USA), concentrations of Na+ and K+ (automated clinical chemistry analyzer Erba XL-200, Erba Lachema, Czech Republic; and Sherwood-420 flame photometer, Sherwood Scientific, Cambridge), Ca++ and Mg++ (AA-6200 atomic absorption spectrophotometer, Shimadzu, Japan) and creatinine (automated clinical chemistry analyzer Erba XL-200) were measured in each sample. Results were normalized per 100 g bw and calculated for 90 min period. Agonists of V-receptors were injected at maximal effective doses.
The injection of 0.05 nmol/100g bw of AVP (Sigma Chemichal, USA) contributed to double increase of К+ excretion, diuresis and solute free water reabsorption; 5-fold decrease of Mg++ excretion was observed with no change in renal Na+ and Ca++ excretion. The injection of 0.05 nmol/100g bw of AVT (Sigma Chemical, USA) led to triple rise of diuresis, 12-fold increase of Na+ excretion; K+ excretion and solute free water reabsorption more than doubled with no change in Mg++ and Ca++ excretion. 0.05 nmol/100g bw of deamino-vasotocin (dAVT) (Sintez Peptidov, Russia) led to 34-fold rise of Na+ excretion, 9-fold increase of K+ excretion+, 3-fold rise of Mg++ excretion and 32-fold increase of Ca++ excretion. 0.1 nmol/100g bw of agonist of V1a-receptors [Phe2,Ile3,Orn8]-vasopressin (Bachem, Switzerland) promoted 12-fold diuresis rise due to almost 30-fold increase of Na+ excretion, 4-fold – K+ excretion, 3-fold – Mg++ excretion, 16-fold – Ca++ excretion. The 70-fold increase of fractional excretion of Na+ was observed and it achieved 14% at maximum diuresis. 0.05 nmol/100g bw of agonist of V1b-receptors [deamino-Cys1,Leu4,Lys8]-vasopressin (Bachem, Switzerland) only led to double increase of K+ excretion; diuresis and Na+, Mg++and Ca++ excretion remained at the control level. 0.05 nmol/100g bw of agonist of V2-receptors desmopressin (Sigma Chemical, USA) led to 3-fold decrease of diuresis, but contributed to triple K+ excretion rise; Na+, Ca++ and solute free water reabsorption remained on a high control level.
The particular quantities of studied cations excretion and solute free water reabsorption and the comparison of AVP and AVT effects and agonists of V-receptors action proves that the key role in AVP action lies in V2- and V1b-receptors stimulation while AVT predominantly activates V1a- and V2-receptors. The data obtained could explain the necessity of substitution of AVT by AVP during evolution in vertebrates. In mammals AVP was more preferable due to its significant water conservation action and low effect on Na+ excretion.
This work was supported by the Russian Foundation for Basic Research (project 14-04-00990) and the Program of the Department of Physiological Sciences of the Russian Academy of Sciences.
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Poster 9
Whole body pharmacokinetic distribution of intranasally delivered oxytocin in rats
Carson, D.S.,1,2 Hanson, L.R.,3 Frey II, W.H.,3 Yeomans, D. C.2,4
1Department of Psychiatry, Stanford University, CA, USA, 2Trigemina, Inc., Division of Pain Therapeutics, Moraga, CA, USA, 3 Health Partners Institute for Education and Research, Center for Memory and Aging, Regions Hospital, Saint Paul, MN, USA, 4Department of Anesthesiology, Stanford University, CA, USA
A vast body of research now supports the therapeutic potential of intranasal oxytocin (OT) in the treatment of various central nervous system (CNS) disorders. However, there remains little evidence confirming the ability of intranasally delivered OT to reach the CNS. Here we quantified the concentration and distribution pattern of OT in the CNS and peripheral tissues after intranasal delivery to anesthetized rats. We examined the distribution of 125I-radiolabelled OT (0.4 mg/48 uL/animal) 30 min after intranasal delivery. Results showed that intranasal OT successfully reaches the CNS with very high concentrations in the trigeminal nerve branches and trigeminal ganglion. The trigeminal ganglion contained an average concentration of 311 nM while the three major branches, maxillary, mandibular, and ophthalmic, contained average concentrations of 264 nM, 291 nM, and 320 nM, respectively. The average concentration of olfactory bulb samples was 33 nM. The high concentrations in these tissues confirm delivery to the brain along the olfactory and trigeminal nerve pathways. The highest delivery of OT in the brain was observed in the caudate/putamen (41 nM), parietal cortex (28 nM), septal nucleus (28 nM), pons (26 nM), and medulla (25nM, Table 01). Trigeminal nerve entry into the brainstem may account for the high concentrations in the pons and medulla. The lowest concentration of OT in the brain was found in the cerebellum at 10 nM. The dorsal and ventral dura/meninges also contained high concentrations of OT at 152 and 271 nM, respectively. As the Kd for high affinity OT receptors is ~1 nM and the Kd for low affinity OT receptors is ~ 50 nM, this data suggests that at the dose administered, high affinity OT receptors would be activated in all CNS tissues and both high and low affinity receptors would be activated in the trigeminal system. The concentration of OT in the respiratory epithelium averaged 680,567 nM leading to effective delivery along the trigeminal nerve pathway. The average concentration of OT in the upper cervical spinal cord was higher than anticipated at 33 nM. The remaining spinal cord segments, lower cervical, thoracic, and lumbar, contained average concentrations of 4 nM, 5 nM, and 5 nM respectively. Again, these concentrations are high enough to activate high affinity OT receptors. Although our measurements were made 30 minutes after initiating intranasal treatment, intranasal OT likely reaches the trigeminal nerves within a minute or two following administration to the nasal cavity, and likely reaches the brain within 10 minutes. Thirty minutes after initiation of intranasal delivery, the concentration of radiolabel in the blood was 61 nM. As the half-life of OT in the blood is about 10 minutes, a significant portion of this radiolabel will represent degraded OT. Irrespective of the amount of degradation in the blood, the OT concentration is at least five-times higher in the trigeminal ganglion than in the blood at this time. Qualitative autoradiography studies confirmed that intranasal OT successfully reached the CNS. The distribution of cold OT in autoradiography experiments was similar to that identified by gamma counting, with the greatest intensity in the trigeminal ganglion, olfactory bulbs, septal nucleus, and frontal cortex. This study provides strong evidence for intranasal OT reaching the CNS.
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Poster 10
A network model of vasopressin neurons – using dendritic communication to regulate heterogeneous population response
MacGregor, D.J., Leng G.
Centre for Integrative Physiology, University of Edinburgh, Edinburgh, UK
The magnocellular vasopressin neurons of the hypothalamus form part of the homeostatic systems that maintain osmotic pressure. Experiments show a robust linear relationship between osmotic pressure and vasopressin hormone secretion despite the 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 model1 that this heterogeneity actually helps to produce a linear population response, despite the non-linear response in individual neurons.
The problem that we are now considering 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 this does not appear to be sufficient to match the high rates of secretion. It has been suggested2 that the neurons may use dendritic communication to coordinate their response, taking turns at being the most active, and allowing stores to recover.
To test this idea we have added synthesis to our model, based on a previous model using spike activity dependent upregulation of mRNA stores3, and dendritic communication, turning the unconnected population model into a coordinated network. Dendritic vasopressin secretion acts to suppress the inputs of neighbouring neurons by reducing the magnitude of post-synaptic potentials, 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 is successful in cycling 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, similar to the ‘small world’ network structure previously used in the oxytocin network model4.
1. MacGregor D.J. and G. Leng (2013) Spike triggered hormone secretion in vasopressin cells; a model investigation of mechanism and heterogeneous population function. PLoS Computational Biology 9(8): e1003187.
2. Leng, G., C. Brown, N. Sabatier and V. Scott (2008). Population dynamics in vasopressin cells. Neuroendocrinology 88 (3), 160–172.
3. Fitzsimmons M.D., M.M. Roberts, T.G. Sherman and A.G. Robinson (1992) Models of neurohypophyseal homeostasis. Am J Physiol Regul Integr Comp Physiol 262:R1121-R1130.
4. Rossoni, E., J. Feng, B. Tirozzi, D. Brown, G. Leng and F. Moos (2008). Emergentsynchronous bursting of oxytocin neuronal network. PLoS Computational. Biology 4(7): e1000123.
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Poster 11
Oxytocin affects feeding for energy and reward via the nucleus accumbens core
Herisson, F.M.1, Levine, A.S.2, Olszewski, P.K.1
1Department of Biological Sciences, University of Waikato, Hamilton, New Zealand, 2Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN, USA
A nonapeptide oxytocin (OT) promotes termination of food intake. Though it is widely recognized that anorexigenic effects of OT are mediated via the central nervous system, relatively few brain sites have been directly linked with OT-driven hypophagia. One of the feeding-related areas expressing a high density of OT receptors is the nucleus accumbens (Acb). We therefore sought to investigate whether infusions of OT into the core (AcbC) or shell (AcbSh) of the Acb in male rats affect feeding for energy and for reward. We found that only AcbC injections of the peptide supported early termination of consummatory behaviour. AcbC OT dose-dependently reduced intake of standard chow in overnight-deprived animals and it decreased consumption of sweet, palatable solutions (sucrose, saccharin) in non-deprived rats. These effects were reversed by the pre-treatment with an OT receptor antagonist. OT in the AcbSh did not alter ingestive behaviour in any of the paradigms. Finally, by using c-Fos immunohistochemistry, we found that AcbC infusions of OT lead to changes in activity within a widespread network of feeding-related brain sites.
Poster 12
Insect neuropeptides as novel ligands for human oxytocin and vasopressin receptors
Di Giglio, G.1, Muttenthaler, M.2, Gruber, C.W.1
1Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria, 2Institute for Molecular Bioscience, The University of Queensland, Australia
Oxytocin (OT) and arginine vasopressin (AVP) are neuropeptide hormones implicated in the regulation of crucial physiological functions such as uterine contraction, fluid homeostasis, and social behaviour. In humans, these peptides signal via four known G protein-coupled receptors – the OT, V1a, V1b and V2 receptors [1]. Sequence homology of the ligands and high structural similarity among the different receptor subtypes pose several challenges for the design and development of selective agonists and antagonists. Certain invertebrate animals contain a closely related neuropeptide signalling system, which appears to be important for the regulation of reproduction and learning processes (summarized in Ref. [2]). For this reason, OT/AVP-like peptides from natural sources are thought to have great potential for the development of potent and selective ligands to human receptors [1, 3]. Our aim is to study the OT/AVP signalling system of insects and to harness its molecular and evolutionary conservation as a proof-of-concept for the characterization of novel peptide analogues with improved functional selectivity on human OT and AVP receptors. In particular, we aim to design novel peptide ligands based on their natural templates for studying structure-activity relationships and to develop lead compounds for therapeutic use.
We utilized transcriptome sequencing to obtain peptide and receptor sequences of our model ant species Lasius. The cognate receptor sequence was validated by PCR and the specific interaction with its native ligand inotocin, a structural homolog of human OT, was confirmed by in vitro
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pharmacology. To improve the stability of the inotocin peptide in vivo, several peptides with D-amino acid substitutions were synthesized. The affinity and efficacy of these analogues for the human OT and AVP receptors were determined via displacement binding experiments and quantitative second messenger analysis. These studies demonstrate that the invertebrate ligand is able to selectively activate human AVP receptors in a concentration-dependent manner at a nanomolar range. Moreover, D-enantiomer replacement of specific residues within the endogenous insect peptide sequence yields a functional selectivity switch [4] at the human vasopressin V1b and V1a receptors. Our results indicate that affinity vs. activity can be selectively modulated revealing a rational strategy for the design of selective antagonists with enhanced affinity to V1bR and V1aR compared to OT and V2 receptors. In summary we demonstrate that naturally-occurring neuropeptides can be used as pharmacological tools to modulate activity and affinity on human receptors, offering an evolutionary advantage over traditional combinatorial chemistry approaches with potential opportunity for drug design applications.
1. Gruber, C.W., Muttenthaler, M. & Freissmuth, M. (2010) Ligand-based peptide design and combinatorial peptide libraries to target G protein-coupled receptors. Curr. Pharm. Des. 16: 3071–88.
2. Gruber, C.W. (2014) Physiology of invertebrate oxytocin and vasopressin neuropeptides. Exp. Physiol. 99: 55-61.
3. Gruber, C.W., Koehbach, J. & Muttenthaler, M. (2012) Exploring bioactive peptides from natural sources for oxytocin and vasopressin drug discovery. Future Med. Chem. 4: 1791–8.
4. Dutertre, S., Croker, D., Daly, N.L., Andersson, A., Muttenthaler, M., Lumsden, N.G., Craik, D.J., Alewood, P.F., Guillon, G., Lewis, R.J. (2008) Conopressin-T from Conus tulipa reveals an antagonist switch in vasopressin-like peptides. J. Biol. Chem. 11: 7100-18.
Poster 13
Contrasting the organizational vs. activational roles of the oxytocin system in the regulation of zebrafish social behavior
Nunes, A.R.1,2, Gliksberg, M.1, Anbalagan, S.1, Wircer, E.1, Oliveira, R.2, Levkowitz G.1
1Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel, 2Integrative Behavioural Biology Lab, Instituto Gulbenkian de Ciência, Oeiras, Portugal
The “organizational hypothesis” claiming that hormones could shape the structure of the developing nervous system and as a result alter the adult animal behavior was suggested in 1959 by Phoenix, Young et al. [Organizing action of prenatally administered testosterone propionate on the tissues mediating mating behavior in the female guinea pig. 1959, Endocrinology 65, 369–382]. Thus, organizational effects refer to long-term, irreversible impact of hormones on tissue differentiation and/or neuronal circuits formation that can either directly or indirectly influence behavior. On the other hand, activational effects refer to short-term, reversible hormonal impact, which occur in the mature/adult organism, e.g. responses to specific homeostatic challenges. Oxytocin-like neurohormones (isotocin in fish, mesotocin in birds, reptiles and amphibian, and oxytocin in mammals) have been implicated in the regulation of social behavior across vertebrates1-3. Using the zebrafish model we developed new genetic tools that allow us to contrast the organizational vs. activational roles of oxytocin in social behavior. We first characterized the ontogeny of sociality, measured by zebrafish shoal preference, and showed that shoal preference emerges around the third week post fertilization. To explore how oxytocin affects the acquisition and modulation of sociality, we have specifically perturbed oxytocinergic neuronal circuits and examined how loss of function of these neurons during embryonic development, or throughout the organism’s lifetime modifies social behavior. Thus, we have generated an oxytocin neurons-specific transgenic system
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for conditional (i.e. temporal) and cell-specific ablation of oxytocin neurons. Using this system, we demonstrated that ablation of these neurons at a critical developmental time window but not during adulthood significantly alter shoal preference behavior. Our data suggest a unique developmental organizational rather than activational effect of the oxytocin neuronal system on a specific social behavior trait.
1. Wircer E., S. Ben-Dor and G. Levkowitz. (in press). Non Mammalian Models for Neurohypophyseal Peptides. In: International Neuroendocrine Federation (INF) Masterclass Series: Molecular Neuroendocrinology: “From genome to physiology”, David Murphy and Harold Gainer, Eds. (Chichester:Wiley&Sons, Ltd).
2. Oliveira, R.F. (2013). Mind the fish: zebrafish as a model in cognitive social neuroscience. Frontiers in neural circuits 7, 131.
3. Goodson JL, Thompson RR. (2010). Nonapeptide mechanisms of social cognition, behavior and species-specific social systems. Curr Opin Neurobiol, 20:784-94.
Poster 14
Osmotic responsiveness of supraoptic nucleus neurons in virgin and lactating rats
Bouwer, G.T., Jaquiery, Z., Augustine, R.A., Brown, C.H.
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 and oxytocin in the kidney to cause anti-diuresis and natriuresis, respectively. Vasopressin and oxytocin secretion is stimulated by increased osmolality. During pregnancy and lactation, plasma osmolality is reduced and plasma volume is increased. Despite the decreased plasma osmolality, circulating vasopressin is paradoxically increased during lactation. Vasopressin and oxytocin 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. The activity of isolated supraoptic nucleus 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 and oxytocin 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, our preliminary data suggest that ruthenium red does not affect the firing rate of supraoptic nucleus oxytocin magnocellular neurosecretory neurons in virgin or lactating rats (four neurons from virgin rats and one from lactating rats).
Hence, it appears that the spontaneous activity of vasopressin neurons is driven, at least in part, by TRPV1 activity in vivo and that this intrinsic osmotic drive remains during lactation despite the decreased plasma osmolality evident during lactation. While vasopressin neurons were consistently inhibited by ruthenium red, oxytocin neurons were not, suggesting that oxytocin neurons might be less osmosensitive that vasopressin neurons in vivo.
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Poster 15
The effect of P2X7 receptor blockers on ATP release from rat hypothalamic slices
Svobodová, I., Vávra, V., Bhattacharya, A., Zemková, H.
Department of Cellular and Molecular Neuroendocrinology, Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic
In mammals, circadian rhythms are driven by a pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The rhythm continues in constant darkness and is dependent on cell-cell communication, involving both neurons and glia. Previous studies have shown that rhythm of total ATP content and extracellular level negatively correlate with electrical activity and arginine vasopressin (AVP) secretion rhythm, indicating that ATP and AVP are released from different cells. Here we tested a hypothesis that ATP acts as extracellular messenger in the hypothalamus and is released by plasma membrane purinergic P2X7 receptor. We measured circadian release of ATP from the hypothalamic SCN organotypic slices and examined the effect of N-[2-[[2-[(2–hydroxyethyl)amino]ethyl]amino]-5-quinolinyl]-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide dihydrochloride (AZ 10606120) and 3-[[5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl]methyl] pyridine hydrochloride (A 438079), which inhibit P2X7 receptor, and tetrodotoxin (TTX), which inhibits voltage-gated sodium channels. Coronal sections (~ 250 µm thick) containing the SCN and supraoptic nuclei (SON) were removed from rats of 14-15 postnatal days of age between CT 2 and CT 8. In our experimental protocol spanning for 52 hours, samples of medium above slices were collected every 4 hours and ATP was measured by bioluminescence assay. Control group of organotypic slices exhibited circadian rhythm in ATP release with peak between CT 18 and CT 22 which was disrupted in the presence of AZ 10606120 and A 438079, indicating involvement of P2X7 receptor in maintaining the rhythm, Application of TTX also disrupted ATP release rhythm indicating involvement of neuronal activity. The fact that ATP rhythm is inhibited by P2X7 blockers might indicate that ATP is primarily stored and released from glia through activated P2X7 channel. In agreement with this, electrophysiological recordings of revealed that hypothalamic glia cels, not neurons, respond to P2X7 receptor-specific agonist (3'-O-(4-benzoyl)benzoyl denosine 5'-triphosphate, BzATP) with inward current and RT-PCR analysis showed that P2X7 receptor is the second most expressed P2X subunit in hypothalamic tissue (P2X2>P2X7>P2X4). Inhibition of ATP release by TTX suggests that glia function is under control of activity of neurons.
Supported by the Grant Agency of the Czech Republic (grant No. P304/12/G069) and the project ‘BIOCEV – Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University’ (CZ.1.05/1.1.00/02.0109), from the European Regional Development Fund.
Poster 16
Kiss1r expression in the rat hypothalamic oxytocin neurons
Higo, S., Honda, S., Iijima, N., Ozawa, H.
Department of Anatomy and Neurobiology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
Kisspeptin and its receptor GPR54 play an essential role in reproduction as one of potent modulators of the GnRH neuron. In addition to its significance to the reproductive function, kisspeptin-GPR54 system is also involved in extra-HPG axis systems, including oxytocin secretion. Previous studies
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demonstrated that kisspeptin administration increased the plasma oxytocin level1, and also increased the firing rate of oxytocin neurons in the supraoptic nucleus (SON)2. Although these studies strongly suggest the prominent role of kisspeptin in oxytocin secretion, these studies did not show the neuroanatomical evidence of direct neuronal communication between kisspeptin neurons and oxytocin neurons. The aim of this study is to show the neuroanatomical evidence of direct neuronal interaction between these two neuronal populations in rats.
First, we performed double immunohistochemistry using antibodies against kisspeptin and oxytocin. Kisspeptin immunoreactive (kiss-ir) fibers were observed in the upper part of the SON, and were also observed broadly in periventricular area including the medial part of the paraventricular nucleus (PVN). Close apposition of these kiss-ir fibers and oxytocin neurons were observed in both the SON and PVN. Majorities of the close apposition of kiss-ir fibers were restricted in upper part of the SON and medial part of the PVN. Kiss-ir fibers in these areas did not show the immunoreactivity against neurokinin B (NKB), suggesting kiss-ir fibers which project to oxytocin neurons were came from the kisspeptin neurons in the anteroventralperiventricular (AVPV) subpopulation.
Next, we examined the expression of the kisspeptin receptor in oxytocin neurons. Little has been reported about the histocmemical distribution of kisspeptin receptor or Kiss1r mRNA expression in the rat brain, probably because of the difficulty in detecting its low level expression of the mRNA or in development of the antibody with sufficient sensitivity and specificity. In this study, we visualized Kiss1r mRNA by using in situ hybridization with careful optimization of probe design and experimental procedure. The neurons with strong Kiss1r expression were observed in the olfactory bulb, diagonal band of Broca, and throughout the preoptic area. To validate the correctness of the in situ hybridization signals, we performed immunohistochemistry using antibody against GnRH, and confirmed Kiss1r positive neurons in these areas were GnRH neurons. Moderate expression of the Kiss1r mRNA was also observed in the SON and PVN. Additional immunohistochemistry revealed that Kiss1r mRNA expressing cells in the SON and PVN were oxytocin neurons.
These results strongly suggest that kisspeptin affect the oxytocin secretion via direct neuronal connection. Present study provides a fundamental anatomical basis for further examination of the kisspeptin-kisspeptin receptor system in extra-HPG axis as well as reproductive function.
1 Kotani, M. et al. The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. J Biol Chem 276, 34631-34636, doi:10.1074/jbc.M104847200 (2001).
2 Scott, V. & Brown, C. H. Kisspeptin activation of supraoptic nucleus neurons in vivo. Endocrinology 152, 3862-3870, doi:10.1210/en.2011-1181 (2011).
Poster 17
Social deficits in dysbindin-1 knockout mice are ameliorated by intranasal oxytocin
Huang, H.1, Michetti, C.2, Gigliucci, V. 3,4, Buselli, M. 3,4, Gentili, G.5, Giancardo, L.7, Hilario, C.
6, Sona, D.6, Murino, V.6, Chini, B.4, Scattoni, M.L.2, Papaleo, F.1
1Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, Genova, Italy, 2Behavioural Neuroscience Section, Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy, 3Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Milan, Italy, 4CNR, Institute of Neuroscience, Milan, Italy, 5Dipartimento di Scienze del Farmaco, Università degli Studi di Padova, Padova, Italy, 6Pattern Analysis and Computer Vision, Istituto Italiano di
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Tecnologia, Genova, Italy, 7Madrid-MIT M+Vision Consortium, RLE, Massachusetts Institute of Technology, Cambridge, MA, USA
The aim of our studies is to investigate, through the use of clinically-relevant animal models of schizophrenia, the therapeutical potential and related molecular mechanisms of intranasal administration of oxytocin (OXT). OXT is a neurohypophysial peptide strongly implicated in the mediation of social behaviours1,2, which has led to the proposal of its use as an adjunctive therapeutic treatment in neuropsychiatric disorders characterised by social behavioural alterations such as autism and schizophrenia3. However, despite promising initial studies in humans, it is not yet clear the specificity of the behavioural effects induced by intranasal OXT nor its mechanisms of action.
Our recent study provided the first evidence of implementing chronic and acute intranasal administration of OXT in C57BL/6J mice4. Notably, these experiments demonstrated that chronic intranasal OXT in a healthy system has detrimental effects specifically on social behaviour, concomitant with a reduction in the expression of OXT receptors throughout the brain.
Conversely, our new findings show that chronic, as well as acute intranasal OXT treatment, consistently ameliorated social behavioural deficits in the schizophrenia-relevant dysbindin-1 mouse model, where both heterozygous and homozygous dysbindin-1 knockout mice have clear deficits in a number of social settings (i.e. male-female, male-male, female-female, unfamiliar/familiar interactions). Furthermore, dysbindin-1 mutant mice show an alteration in the expression of OXT receptors in specific brain areas which is, thereafter, rescued by intranasal OXT treatments.
Overall, these results indicate that while intranasal OXT might be detrimental in subjects with normal levels of social behaviours, it might ameliorate social deficits in psychiatric diseases characterised by social impairments such as schizophrenia. Importantly, our studies of the genetic interaction between OXT and dysbindin-1 may help to pave the way for increased specificity in clinical testing and consequently, the development of more effective and personalised healthcare in the treatment of social deficits in schizophrenia.
1. Winslow, J.T. and Insel, T.R. (2002). The social deficits of the oxytocin knockout mouse. Neuropeptides. 36(2-3):221-9.
2. M. Sala, D. Braida, D. Lentini, M. Busnelli, E. Bulgheroni, V. Capurro, A. Finardi, A. Donzelli, L. Pattini, T. Rubino, D. Parolaro, K. Nishimori, M. Parenti, and B. Chini. (2011). Pharmacologic rescue of impaired cognitive flexibility, social deficits, increased aggression, and seizure susceptibility in oxytocin receptor null mice: a neurobehavioral model of autism. Biol Psychiatry, 69(9):875-82.
3. Striepens, N., Kendrick, K.M., Maier, W., and Hurlemann, R. (2011). Prosocial effects of oxytocin and clinical evidence for its therapeutic potential. Front Neuroendocrinol. 32(4):426-50.
4. Huang, H., Michetti, C., Busnelli, M., Giancardo, L., Managò, F., Sannino, S., Scheggia, D., Sona, D., Murino, V., Chini, B., Scattoni, M.L., Papaleo, F. (2014). Chronic and acute intranasal oxytocin produce divergent social effects in mice. Neuropsychopharm. 39(5):1102-14.
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Poster 18
Can acute intranasal oxytocin modulate neural responses to socio-emotional cues in a neurodegenerative population?
Labuschagne, I.1, Poudel, G.2,3, Kordaschia, C.2, Wu, Q.3, Georgiou-Karistianis, N.2, Churchyard, A.2,4, Stout, J.C.2
1School of Psychology, Faculty of Health Sciences, Australian Catholic University, Fitzroy, Melbourne, Australia, 2School of Psychological Sciences, Faculty of Medicine, Nursing and Health Sciences, Monash University, Melbourne, Australia, 3Monash Biomedical Imaging, Monash University, Melbourne, Australia, 4Huntington’s Disease Service, Calvary Health Care Bethlehem, Melbourne, Australia
Deficits in core social cognitive functions, such as emotion recognition, are evident in the very early stages of Huntington’s disease (HD), with declines worsening with an increase in disease severity. Little is known about the underlying mechanisms involved in declines of social cognitive functions in HD. The neuropeptide oxytocin has shown to gate behavioural and physiological responses to social cognitive cues. No study to date has examined the role of oxytocin in the neurobiology of a neurodegenerative disease such as HD. The current study therefore aimed to examine if an acute dose of intranasal oxytocin can modulate social brain regions in response to an emotional face matching task in non-medicated HD carriers compared to controls.
In a double-blind placebo-controlled study, non-medicated gene-positive for HD and age-matched control participants completed two fMRI scans approximately one week apart. Prior to each scan, participants self-administered an acute intranasal treatment of 24 IU of either oxytocin or placebo sprays. An emotional face matching task consisting of six basic emotions and neutral was completed in the scanner. Whole-brain voxel-wise analysis of variance were conducted for each individual emotion. Results showed significant group x drug interactions mostly strongly for disgust, and also for angry and happy emotions in fronto-parietal regions. Under placebo, HD carries (vs. controls) showed significantly reduced fronto-parietal activations. In contrast, oxytocin (vs. placebo) significantly increased fronto-parietal activations within the HD carriers but reduced such activations within the control group.
The findings provide the first evidence of oxytocin’s role in a neurodegenerative disease. In particular, the direction of the effects suggests that oxytocin may have ‘normalisation’ effects in HD and provide a brain-based mechanism of the impact of oxytocin in modulating reduced processing of social cues in patients with HD. This intriguing initial evidence is novel and provides evidence of a broader physiological role of oxytocin in humans. Additional research using larger samples is needed.
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Poster 19
Discovery of an oxytocin-like plant peptide as template for oxytocin and vasopressin receptor ligand design
Koehbach, J.1,2, Muttenthaler, M.3,4, Gruber, C.W.1
1Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria, 2School of Biomedical Sciences, The University of Queensland, St. Lucia, Australia, 3 Institute for Research in Biomedicine, Barcelona Science Park, Barcelona, Spain, 4Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Australia
Natural products are still one of the biggest resources for the discovery of novel pharmaceutical lead compounds. Over the recent years a family of cyclic plant peptides has attracted much interest in drug development approaches. These so-called cyclotides comprise a circular backbone and three interlocked disulfide bonds that confer them with exceptional stability. Due to their high sequence variability they are often regarded as natural combinatorial peptide library. Originally, they have been discovered in Oldenlandia affinis based on their use in traditional African medicine to accelerate labor. Following a bioactivity-guided fractionation approach we analysed plant peptide fractions and purified peptides and identified the cyclotide kalata B7 as potent uterotonic agent. Radioligand displacement and second messenger-based reporter assays confirmed the oxytocin and vasopressin V1a-receptors, members of the G protein-coupled receptor (GPCR) family, as molecular targets for this cyclotide. Furthermore we demonstrated that cyclotides can serve as templates for the design of selective GPCR ligands by generating an oxytocin-like peptide with nanomolar affinity and enhanced receptor selectivity. This nonapeptide elicited concentration-dependent contractions of human myometrium. These observations provide a proof-of-concept for cyclotide-based ligand design1. Our results are encouraging and confirm that mining of natural peptide libraries enables the discovery of novel GPCR ligands. GPCRs are promising drug targets and nearly 50% of all currently marketed drugs act on these receptors. Taken together the variety of naturally-occurring cyclotides and the number of human GPCRs these findings open new avenues for the development of other peptide-based therapeutics.
1. Koehbach, J. et al. (2013) Oxytocic plant cyclotides as templates for peptide G protein-coupled receptor ligand design. Proceedings of the National Academy of Sciences of the United States of America. 110: 21183-88.
Poster 20
Modulatory role of Zinc on neuropeptide release from neurohypophysis
Lemos, J.R.1, Chau, Q.C.3, Custer, E.E.1, Ortiz-Miranda, S.1,2
1Department Microbiology and Physiological Systems, Massachusetts Medical School, Worcester, MA, 2Tufts Cumming’s School of Veterinary Medicine, N. Grafton, MA, 3La Salle University, Philadelphia, PA, USA
Zinc (Zn2+) is an essential trace metal and Zn2+ deficiency has long been recognized as playing a role in a number of physiological brain disorders. Since many synaptic vesicles/neurosecretory granules (“zincosomes”) appear to contain Zn2+, and previous studies have shown the presence of Zn2+ in the hypothalamus as well as uptake within the Neurohypophysis, the Hypothalamic Neurohypophysial System is an excellent model structure to study its mechanism of action. Furthermore, we have
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preliminary evidence that vasopressin (AVP) and oxytocin (OT) Neurohypophysial terminals possess Zn2+and its receptor (GPR39).
AVP and OT release from isolated neurohypophysial terminals of transgenic Wistar rats was examined in the presence and absence of Zn2+ using ELISAs. An exogenous concentration of 6-50 μM Zn2+ was necessary to initiate neuropeptide release and a maximal response was obtained at a concentration of ≥200 μM. Since Zn2+ is a known activator of P2X2, P2X3, and/or P2X4 receptors, present exclusively in AVP terminals, the release effects could possibly be the result of Zn2+ activation of these purinergic receptors. Interestingly, when PPADS, an antagonist for P2X2 and P2X3 receptors, was used in the presence of Zn2+ not only was the release AVP inhibited but OT release was inhibited as well. Only AVP release is blocked in the presence of PPADS alone. Alternatively, it has been reported that PPADS is also able to inhibit G-protein coupled receptor-induced IP3 release, and thus reduce [Ca2+]i; an effect that could impact the release of both types of neuropeptides. In agreement with this mechanism, exogenous Zn2+ stimulation caused neuropeptide release in zero extracellular Ca2+ conditions. Furthermore, 10 mM Ca-EDTA, a Zinc chelator, reduced High K+-induced release of both AVP and OT release.
These results lead us to hypothesize that Zn2+, most likely from endogenous sources, can modulate AVP and OT release by influencing intracellular calcium levels in neurohypophysial terminals.
Supported by UMMS Grant P60037094900000 (SOM) and NIH Grant NS29470 (JRL).
Poster 21
Re-examining the importance of vasopressin receptors in myometrial contraction
Arrowsmith, S.1, Wray, S.1, Busnelli, M.2,3, Chini, B.2, Muttenthaler, M.4
1Department of Cellular and Molecular Physiology, University of Liverpool, UK, 2CNR, Institute of Neuroscience, Milan, Italy, 3 Department of Biotechnology and Translational Medicine, Università degli Studi di Milano, Milan, Italy, 4 Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia
Introduction: Oxytocin receptor (OTR) antagonists have been proposed as tocolytic agents for preterm labor (PTL). However, OT can also activate the three vasopressin receptor (AVPR) subtypes including V1aR, which is expressed in myometrium and remains highly expressed throughout gestation. Atosiban (AT) possesses high affinity for both V1aR and OTRs and it is not clear if its tocolytic activity is also mediated via vasopressin antagonism, whilst the OTR-selective antagonist barusiban was not effective in delaying delivery in women with PTL. Using newly developed human OTR- and AVPR subtype-selective agonists and antagonists, we re-visited this long-standing question to investigate the role of the individual AVPR subtypes in human myometrial contraction.
Methods: Peptides were synthesized by manual Boc-SPPS, folded and purified by RP-HPLC. Ligand selectivity was established by measuring the Ca (OTR, V1aR, V1bR) and cAMP (V2R) response in CHO cell lines heterologously expressing the different human receptor subtypes. Isometric tension recordings were performed on strips of myometrium obtained from women delivering by pre-labor, elective Caesarean section with informed consent (n=≥5/ligand). Contractile activity [integral, contraction frequency, duration and amplitude] was measured and the effect of rising molar concentrations (1nM-1µM) of OT, human OTR and V1aR/V1bR-specific peptides were compared and examined in the presence of the hOTR-selective antagonist Antag III (provided by Ferring) or AT (100nM).
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Results: All ligands dose-dependently increased all contraction parameters except frequency. OT and the OTR-specific ligand, [Se-Se]-OT-OH induced the greatest increase in activity and [Se-Se]-OT-OH was as efficacious as OT. In the presence of Antag III the response to OT and [Se-Se]-OT-OH was completely inhibited, the response to AVP or to FE 201874 (hV1a/V1bR agonist, Ferring) however was not, indicating some involvement of the AVPRs. AT only inhibited the response to ligands at ≤100nM confirming its competitive mechanism.
Conclusion: Data suggests that contraction in the human myometrium is mainly driven by the hOTR. The observed involvement of hV1a/V1bR-selective ligands however warrants further investigation since this might be crucial for the much needed development of better and safer tocolytic agents for PTL.
Poster 22
Unveiling the mechanism of action and superagonism of oxytocin receptors bivalent ligands
Busnelli, M.1,2, Passoni, I.2, Kleinau, G.3, Muttenthaler, M4, Stoev, S.5, Manning, M.5, Di Lascio, S.2, Braida, D.2, Sala, M.E.1,2, Rovati, G.E.6, Bellini, T.2, Chini, B.1
1Institute of Neuroscience, CNR, Milan, Italy, 2Department of Medical Biotechnology and Translational Medicine, University of Milan, Italy, 3Institute of Experimental Pediatric Endocrinology, Charité-Universitätsmedizin Berlin, Berlin,Germany, 4The Scripps Research Institute, La Jolla, CA, USA, 5Department of Biochemistry and Cancer Biology, University of Toledo, USA, 6 Department of Pharmacological and Biomolecular Sciences, University of Milan, Italy
The neuropeptide oxytocin (OXT) regulates a number of socio-emotional behaviors in mice, including parental care, pair-bonding, social memory, and social aggression by activating OXT receptors (OXTR). These receptors are expressed at the plasma membrane and, in response to OXT, can activate different G-proteins1. Like many other G-protein coupled receptors (GPCRs), OXTR can exist as monomer and dimers in cell systems and in native tissues; i.e. the mammary gland and the brain2,3. We synthesized a series of bivalent ligands, consisting of two identical OXT-like ligands joined by spacers with different lengths, and performed homology modeling, ligand-docking and receptor mutagenesis analysis.
We identified a specific OXTR dimeric arrangement that allows for the simultaneous binding of the two pharmacophores of bivalent ligands with specific spacer lengths into the two orthosteric sites of the dimer. For these ligands, we obtained EC50 values three orders of magnitude lower than those for monovalent analogs in Gq-protein activation assays. In parallell, we perfomed in vivo studies and we found that a single i.c.v injection of the OXTR bivalent ligand, in the Oxtr+/- genetically modified mice, that displayed social impairments4, restored, in a three-chamber test, sociability at a 100-folds lower dose in comparison to OXT. To further confirm the effect of bivalent ligands on social behaviors, we used zebrafish, and observed their shoaling behavior; a social behavior that we previously showed to be modulated by several OXT-related peptides5. We demonstrated that the OXTR bivalent ligand significantly increased the sociality, measured as shoaling preference to non-conspecific fish, already at a 40-folds lower dose as compared to the endogenous OXT-homologous peptide isotocin.
Our results suggest that bivalent ligands target a specific OXTR dimeric conformation coupled to Gq with a remarkable gain in binding and activation efficiency, both in vitro and in vivo. Notably, the increased potency of bivalent ligands that we have observed in vivo is consistent with the presence
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of OXTR homo-dimers in native tissue and support their presence in the CNS. Moreover, the findings that OXTR bivalent ligands strongly promote social behaviors both in mice and zebrafish, can pave the way toward new therapeutic strategies for disorders associated with social impairments, such as autism spectrum disorders (ASD) and schizophrenia.
1. Busnelli, M., Saulière, A., Manning, M., Bouvier, M., Galés, C. and B. Chini (2012) Functional selective oxytocin-derived agonists discriminate between individual G protein family subtypes. J Biol Chem. 2012 3;287(6):3617-29
2. Albizu, L., Cottet, M., Kralikova, M., Stoev, S., Seyer, R., Brabet, I., Roux, T., Bazin, H., Bourrier, E., Lamarque, L., Breton, C., Rives, M.L., Newman, A., Javitch, J., Trinquet, E., Manning, M., Pin, J.P., Mouillac, B. and T. Durroux (2010) Time-resolved FRET between GPCR ligands reveals oligomers in native tissues. Nat Chem Biol. 2010 epub
3. Romero-Fernandez, W., Borroto-Escuela, D.O., Agnati, L.F. and K. Fuxe (2013) Evidence for the existence of dopamine D2-oxytocin receptor heteromers in the ventral and dorsal striatum with facilitatory receptor-receptor interactions. Mol Psychiatry, epub
4. Sala, M., Braida, D., Donzelli, A., Martucci, R., Busnelli, M., Bulgheroni, E., Rubino, T., Parolaro, D., Nishimori, K. and B. Chini (2013). Mice heterozygous for the oxytocin receptor gene (Oxtr(+/-) ) show impaired social behaviour but not increased aggression or cognitive inflexibility: evidence of a selective haploinsufficiency gene effect. J Neuroendocrinol. 2013; 25: 107-18
5. Braida, D., Donzelli, A., Martucci, R., Capurro, V., Busnelli, M., Chini, B. and M. Sala (2012) Neurohypophyseal hormones manipulation modulate social and anxiety-related behavior in zebrafish. Psychopharmacology; 220(2):319-30.
Poster 23
Upregulation of Gonadotrophin Inducible Transcription Factor 1 mRNA in the PVN and SON by cAMP Pathways
Greenwood, M.P.1, Greenwood, M.1, Paton, J.F.R.2, Murphy, D.1
1School of Clinical Sciences, University of Bristol, Bristol, UK, 2School of Physiology and Pharmacology, University of Bristol, Bristol
Using microarray technology, we identified increased gonadotrophin inducible transcription factor 1 (Giot1) mRNA expression in both the paraventricular (PVN) and supraoptic nuclei (SON) of dehydrated vs. control rats (1). Giot1 is Kruppel-type zinc finger protein originally identified as being rapidly induced by gonadotrophins in immature rat ovary granulosa cells. It is thought to function as a transcriptional repressor, with the N-terminal located Kruppel-associated box-A domain being responsible for this activity. However, neither the functions nor the mechanisms of regulation of this gene in the SON and PVN are understood. We thus investigated the activation of this gene both in vitro and in vivo. The activity of the Giot1 promoter has been shown to be induced by cAMP intracellular signalling pathways in vitro (2). It is well documented that cAMP pathways are upregulated in the SON and PVN after dehydration (3, 4), suggesting that increased intracellular cAMP may be responsible for the observed increase in Giot1 expression. Indeed, we previously reported that adenoviral mediated overexpression of protein kinase inhibitor alpha, a potent and specific peptide inhibitor of protein kinase A, delivered specifically into the PVN, attenuates the upregulation of Giot1 mRNA by dehydration. To extend our earlier findings we have used the sensitive technique of qPCR to measure the time course of Giot1 activation in rat hypothalamic 4B cells and in rat hypothalamic organotypic cultures. To increase intracellular cAMP levels, cultures were treated with adenylyl cyclase activator forskolin (10 µM) or vehicle (DMSO). In both culture models Giot1 expression was significantly induced by forskolin treatment consistent with the
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proposed activation of this gene by cAMP pathways. We next assessed the expression profile of Giot1 in the rat itself at different time points (10 min, 30 min, 1, 2, 4) after intraperitoneal administration of 1.5 ml/100g body weight of 1.5 M NaCl. In the PVN and SON Giot1 expression increased as early as 1 h after 1.5 M NaCl administration and continued to increase for the duration of the experiment. Together, these findings support a role for Giot1 in the acute response to hyperosmotic stress and support the activation of this gene by cAMP pathways.
1. Qiu J, Yao S, Hindmarch C, Antunes V, Paton J, Murphy D 2007 Transcription factor expression in the hypothalamo-neurohypophyseal system of the dehydrated rat: upregulation of gonadotrophin inducible transcription factor 1 mRNA is mediated by cAMP-dependent protein kinase A. J Neurosci 27:2196-2203
2. Yazawa T, Mizutani T, Yamada K, Kawata H, Sekiguchi T, Yoshino M, Kajitani T, Shou Z, Miyamoto K 2003 Involvement of cyclic adenosine 5'-monophosphate response element-binding protein, steroidogenic factor 1, and Dax-1 in the regulation of gonadotropin-inducible ovarian transcription factor 1 gene expression by follicle-stimulating hormone in ovarian granulosa cells. Endocrinology 144:1920-1930
3. Carter DA, Murphy D 1989 Cyclic nucleotide dynamics in the rat hypothalamus during osmotic stimulation: in vivo and in vitro studies. Brain research 487:350-356
4. Young WS, 3rd, Shepard EA, Burch RM 1987 Plasma hyperosmolality increases G protein and 3',5'-cyclic adenosine monophosphate synthesis in the paraventricular and supraoptic nuclei. Molecular endocrinology (Baltimore, Md 1:884-888
Poster 24
Systemic vasopressin V1a receptor antagonism blocks oxytocin's attenuation of reinstatement of methamphetamine-seeking behaviour in rats
Everett, N.A.1, McGregor, I.S. 2, Baracz, S.J.1, Cornish, J.L.1
1Department of Psychology, Macquarie University, NSW, Australia, 2School of Psychology, University of Sydney, NSW, Australia
Methamphetamine (METH) is a potent and highly addictive psychostimulant. Systemic administration of the neuropeptide oxytocin attenuates METH-related reward and METH-seeking behaviour. However, selective antagonism of the oxytocin receptor only partially reversed the inhibitory effects of oxytocin on METH-primed reinstatement, suggesting mediation by receptors other than the oxytocin receptor. Oxytocin has a high affinity for the vasopressin V1a receptor, antagonism of which has been shown to reduce some of oxytocin’s prosocial effects. Using a self-administration paradigm of drug reinstatement, we aimed to determine whether oxytocin’s attenuation of METH-primed reinstatement of METH-seeking behaviour could be reduced by co-administering SR49059, a selective vasopressin V1a receptor antagonist. 14 male Sprague-Dawley rats were surgically implanted with an intravenous jugular vein catheter and trained to self-administer METH (0.1mg/kg/infusion) by lever pressing during daily 2-hr fixed ratio 1 scheduled sessions for 20 days. Following extinction of a preference for the active lever, rats were then tested for the effects of systemically administered oxytocin (1.0mg/kg), SR49059 (0.3/mg/kg, 1.0mg/kg), or co-administration of oxytocin (1.0mg/kg) and SR49059 (1.0mg/kg) on METH-primed (1mg/kg, i.p.) reinstatement of METH-seeking behaviour. As expected, systemic oxytocin substantially reduced METH-primed reinstatement of lever pressing, as well as METH-induced hyperactivity. Importantly, co-administration of SR49059 blocked the inhibitory effects of oxytocin on METH primed reinstatement and METH-induced hyperactivity. These findings highlight the ability of systemic oxytocin to activate vasopressin V1a receptors to have its important modulatory effects on relapse to METH-seeking behaviour.
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Poster 25
Synthesis of [lys8]-oxytocin dendrimers that maintain potency and structure and inhibit visceral nociceptive responses
Wan, J.1, Mobli, M.2, Brust, A.1, Muttenthaler, M.1, Andersson, Å.1, Ragnarsson, L.1, Castro, J.3, Vetter, I.1, Huang, J.1, Nilsson, M.4, Brierley, S.M.3, Cooper, M.A.1, Lewis, R.J.1, Alewood, P.F.1
1Institute of Molecular Bioscience, The University of Queensland, St Lucia QLD, Australia, 2Centre for Advanced Imaging, The University of Queensland, St Lucia QLD, Australia, 3The University of Adelaide, South Australian Health and Medical Research Institute (SAHMRI), Adelaide, SA, Australia; 4School of Chemistry, University of Manchester, Manchester, UK
Bioactive peptide dendrimers comprise a new class of precisely defined macromolecules that in principle may demonstrate enhanced pharmaceutical properties via enabling polyvalent interaction between multiple receptors, enhanced resistance to proteolysis and delayed renal clearance due to their higher molecular weights. Here, we describe the synthesis, structure, binding affinity, selectivity, functional activity and anti-nociceptive properties of [lys8]-oxytocin (LVT) dendrimers containing up to 16 copies of LVT. These were generated using a CuAAc reaction with azido-pegylated LVT peptides on an alkyne-polylysine scaffold. 2D NMR analysis demonstrated that each attached LVT ligand was freely rotating and maintained identical 3D structures in each dendrimeric macromolecule. The binding affinity of the LVT dendrimers increased with low generation dendrimers and decreased in higher generations without affecting selectivity with increases ~17-, ~12-, ~3- and ~1.5- fold respectively for the 2-, 4-, 8- and 16-mer dendrimeric LVT conjugates suggesting a multivalent interaction. A similar trend in affinity was also observed at the related human V1a, V1b and V2 receptors, with no significant selectivity change observed across this family of receptors. All LVT dendrimers were functionally active in vitro on human OT receptors and potently inhibited colonic nociceptors in a mouse model of chronic abdominal pain.
Poster 26
Kisspeptin regulation of oxytocin neurons in pregnant mice
Augustine, R.A., Bouwer, G.T., Brown, C.H.
Centre for Neuroendocrinology and Department of Physiology, University of Otago, New Zealand
Oxytocin secretion induces uterine contractions, which 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. 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 rats 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
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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 the response of oxytocin neurons in the PVN to endogenous administration of kisspeptin via an ICV cannula inserted into anaesthetized mice. Preliminary results suggest ICV kisspeptin activates Fos protein in oxytocin neurons in late pregnant mice, but not in non-pregnant mice. Hence, it appears that kisspeptin excitation of oxytocin neurons emerges over pregnancy and might contribute to oxytocin neuron excitation at parturition.
Poster 27
Myocardial infarction increases Fos protein expression in the paraventricular and supraoptic nuclei
Roy R.K., Brown, C.H., Schwenke, D.O.
Department of Physiology, University of Otago, Dunedin, New Zealand
Acute myocardial infarction (MI), including ischemic heart disease, is a global health problem costing around 7.4 million lives every year. One of the main pathophysiologies contributing to the high morbidity and mortality associated with acute MI is increased cardiac sympathetic nerve activity (SNA) (Malpas, 2010). Once increased cardiac SNA is established, this sympathetic hyper-excitation is irreversible and is difficult to reduce even using sympathetic inhibitors such as beta blockers. Hence, the increased drive for SNA might be of central origin. However, the brain areas (nuclei) involved in the generation of increased SNA after MI are unknown. This study set out to investigate the activation of specific brain areas immediately following MI, which might contribute to the increased cardiac sympathetic nerve activity.
To investigate this issue, we first established a robust time course of changes in cardiac SNA following acute MI by using a standard multi fiber direct nerve recording technique. Cardiac SNA began to increase immediately following acute MI and became significantly elevated (by 37.44 ± 8.27%) at 16 min post-MI (p=0.022, one way ANOVA) and remained elevated throughout the four hours of recording. We next determined the activation of specific brain areas in response to acute MI. Rats were transcardially perfused under deep anesthesia 90 min following the induction of MI by ligation of the left anterior descending coronary artery. Because the immediate early gene c-fos has been used as a robust marker of neuronal activation, immunohistochemistry for Fos protein was performed on brain sections to determine neuronal activation. Ninety min after acute MI there were significantly higher (p=0.0002, unpaired t-test) numbers (397 ± 37) of Fos-positive cells in the paraventricular nucleus (PVN) compared to sham operated rats (163±19), with significantly higher (p=0.0012) Fos protein expression (161 ± 15) in the parvocellular division of PVN of MI rats compared to sham operated rat (81 ± 26). There was also a significantly higher (p=0.0002) number of Fos-positive neurons in the magnocellular division of the PVN of MI rats (236±26) compared to sham operated rats (82 ± 9). In the supraoptic nucleus (SON), there was also a significantly higher (p<0.0001) number of Fos-positive neurons (248 ± 7) in response to acute MI compared to sham operated rat (158 ± 11). Hence, acute MI activates magnocellular and parvocellular neurons in the PVN, as well as magnocellular neurons in the SON. Because the parvocellular PVN neuron population contains pre-autonomic neurons (Stern, 2001), activation of these neurons might drive the increase the cardiac SNA immediately following MI.
1. Malpas SC (2010) Sympathetic nervous system overactivity and its role in the development of cardiovascular disease. Physiological reviews 90: 513-557
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2. Stern JE (2001) Electrophysiological and morphological properties of pre-autonomic neurones in the rat hypothalamic paraventricular nucleus. The Journal of physiology 537: 161-177
Poster 28
Genetic evidence for people’s attitudes towards time
Yang, G.1, Chew, S.H.1, Ebstein, R.P.2
1Department of Economics, National University of Singapore, Singapore, 2Department of Psychology, National University of Singapore, Singapore
People’s attitudes towards time play an important role when making decisions regarding the future. Beyond psychology, where addictive behaviors, temptation, self-control, and personality traits such as impulsivity have been widely studied from different perspectives, the degree of impatience is an essential dimension to be included when studying dynamic decision making in economics. For instance, a person offered a check that can be paid in one week would be tempted perhaps ‘on the spot’ to accept a somewhat discounted amount of money. If the check could be cashed in one month a person might be willing to accept a greater discounting of the initial sum. This phenomenon is known as temporal or delay discounting (DD). Furthermore, the discounting rate for the same length of time, like one week or one month, tends to be smaller in the remote future or the far horizon than in the near future. This phenomenon is known as near-term bias. Importantly, considerable individual variations in DD and near-term bias have been documented in the literature, which suggests that hard wiring or genes may play a partial role in time related attitudes. Although there have been some genetic and neural imaging studies of DD, there are to our knowledge no studies comparing DD and gene expression in peripheral blood (‘blood genomics’ strategy1). Gene expression studies capture both environmental as well as hard wired gene variations and hence are complementary and perhaps even more informative than simple gene association studies. Moreover, there are few if any studies linking DD to an individual’s overall conceptualization or perception of time. We selected RARA (retinoic acid receptor-α) as a novel candidate gene for explaining individual differences in DD based on the emerging new role for retinoic acid (RA) as a regulator of biological rhythms within the CNS2. Vitamin A (all trans RA) is an essential component of the mammalian diet that circulates in the blood in the form of retinol. Interestingly, there is a reduction in maximum pineal melatonin synthesis under vitamin A-deficient conditions. Several components of the RA signaling pathway oscillate according to photoperiodic changes in light conditions. For example, increased day length leads to enhanced retinoid signaling within the hypothalamus. Hence, it makes ‘biological sense’ that there is a relationship between RARA, retinoids and DD. Such inter-temporal preferences have been attributed to impulsivity, to differences in cognitive representations between near and future events as well as to dissimilarities in time orientation. Our main findings are that the expression level of the gene RARA in peripheral blood is positively correlated with the degree of impatience in the remote future (β= 0.156, p=0.018, N=229) but negatively correlated with the degree of near-term bias (β=-0.137, p=0.039, N=229). The significance of these correlations is robust when we control for demographic characteristics including gender, age and IQ. Of notable interest is the biologically plausible finding that for the first time a gene known to be involved as a mediator of biological rhythms in the brain has been implicated in temporal decision making elicited in terms of impatience in the remote future and near-term bias. Finally, this is likely the first study to include measures of gene expression to investigate choice behavior elicited from incentivized decision making tasks.
1. Tang, Y. et al. Human blood genomics: distinct profiles for gender, age and neurofibromatosis type 1. Brain Res Mol Brain Res 132, 155-67 (2004).
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2. Ransom, J., Morgan, P.J., McCaffery, P.J. & Stoney, P.N. The rhythm of retinoids in the brain. Journal of Neurochemistry 129, 366-376 (2014).
Poster 29
Selective localization of oxytocin receptors and vasopressin 1a receptors in human brain tissue
Freeman, S.M.1, Smith, A.L.2,3, Goodman, M.M.3, Bales, K.L.1
1Department of Psychology, University of California-Davis, Davis, CA, USA, 2Center for Translational Social Neuroscience, Emory University, Atlanta, GA, USA, 3Center for Systems Imaging, Emory University, Atlanta, GA, USA
Human studies have shown that intranasal oxytocin affects a suite of social behaviors, such as trust1, eye contact2, emotion recognition3, and pair-bonding-related behaviors4,5. However, it is still unclear where oxytocin receptors (OXTR) and the structurally related vasopressin 1a receptors (AVPR1a) are expressed in the human brain. We have previously described a reliable, pharmacologically informed receptor autoradiography protocol for visualizing these receptors in post-mortem nonhuman primate brain tissue6,7. We used this optimized technique in human brainstem tissue (n=7) to specifically identify the neural targets of oxytocin and vasopressin in humans. To determine binding selectivity of the OXTR radioligand and AVPR1a radioligand in human brainstem, sections were co-incubated in four conditions: radioligand alone, with 10 nM of the selective AVPR1a competitor SR49059, and with a low (100 nM) and high (1 uM) concentration of the selective OXTR competitor ALS-II-69. Binding density across conditions was analyzed with a repeated measures one-way ANOVA for each region and radioligand. We found selective OXTR binding in the spinal trigeminal nucleus, a conserved region of OXTR expression found in all other primate species investigated to date6-8. We found selective AVPR1a binding in the nucleus prepositus, which has been implicated in eye gaze stabilization. We found no significant correlation between the post-mortem interval of the tissue and either the specific OXTR or AVPR1a binding or the background binding for either radioligand. This indicates that postmortem interval need not be a consideration in future tissue selection for similar studies using post-mortem human brain tissue. The current study provides critical preliminary data for future studies of OXTR and AVPR1a in human brain tissue. Tissue was provided by the NICHD Brain & Tissue Bank in Baltimore, Maryland, USA. This work was supported by the NIH under Grant P51OD011107 to the California National Primate Research Center.
1. Kosfeld, M., Heinrichs, M., Zak, P. J., Fischbacher, U. & Fehr, E. Oxytocin increases trust in humans. Nature 435, 673–676 (2005).
2. Guastella, A. J., Mitchell, P. B. & Dadds, M. R. Oxytocin increases gaze to the eye region of human faces. Biological Psychiatry 63, 3–5 (2008).
3. Domes, G., Heinrichs, M., Michel, A., Berger, C. & Herpertz, S. C. Oxytocin improves ‘mind-reading’ in humans. Biological Psychiatry 61, 731–733 (2007).
4. Scheele, D. et al. Oxytocin modulates social distance between males and females. J. Neurosci. 32, 16074–16079 (2012).
5. Scheele, D. et al. Oxytocin enhances brain reward system responses in men viewing the face of their female partner. Proceedings of the National Academy of Sciences 110, 20308–20313 (2013).
6. Freeman, S. M., Inoue, K., Smith, A. L., Goodman, M. M. & Young, L. J. The neuroanatomical distribution of oxytocin receptor binding and mRNA in the male rhesus macaque (Macaca mulatta). Psychoneuroendocrinology 45, 128–141 (2014).
7. Freeman, S. M. et al. Neuroanatomical distribution of oxytocin and vasopressin 1a receptors in the socially
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monogamous coppery titi monkey (Callicebus cupreus). Neuroscience (2014). doi:10.1016/j.neuroscience.2014.04.055
8. Schorscher-Petcu, A., Dupré, A. & Tribollet, E. Distribution of vasopressin and oxytocin binding sites in the brain and upper spinal cord of the common marmoset. Neuroscience Letters 461, 217–222 (2009).
Poster 30
Chronic methamphetamine self-administration dysregulates oxytocin plasma levels and oxytocin receptor expression in the nucleus accumbens core and subthalamic nucleus of the rat
Baracz, S.J., Parker, L., Suraev, A., Everett, N.A., Goodchild, A., McGregor, I.S., Cornish, J.L.
1Department of Psychology, Macquarie University, NSW, 2Australian School of Advanced Medicine, Macquarie University, NSW, 3School of Psychology, University of Sydney, NSW, Australia
The psychostimulant methamphetamine (METH) is an addictive illicit drug. The neuropeptide oxytocin has been shown to modulate METH-related reward and abuse. Recent findings have implicated the nucleus accumbens core (NAcc) and subthalamic nucleus (STh) as key substrates involved in oxytocin modulation of acute METH-related reward and relapse to METH-seeking behaviour. Surprisingly, we have previously shown only a modest involvement of the oxytocin receptor (OTR) in both regions in oxytocin modulation of METH-primed reinstatement. Coupled with the lack of research on the role of the OTR in oxytocin attenuation of psychostimulant-induced behaviours, the aim of the current study was to investigate whether there are cellular changes to the endogenous oxytocin system in the NAcc and STh, as well as changes to oxytocin plasma levels following chronic METH intravenous self-administration (IVSA) and after behavioural extinction. Male Sprague Dawley rats underwent surgery for the implantation of a jugular vein catheter under isoflurane anaesthesia. After recovery, rats were trained to self-administer intravenous METH (0.1mg/kg/infusion) by lever press or received yoked saline injections during 2-hour sessions for 20 days. Rats then underwent behavioural extinction for 15 days. Following this, blood plasma was collected for enzyme immunoassay, and immunofluorescence was conducted on NAcc and STh coronal sections. Our results showed that rats, which self-administered METH, had higher oxytocin plasma levels, and decreased OTR-immunoreactive fibres in the NAcc than yoked controls. After extinction, oxytocin plasma levels remained elevated, OTR-immunoreactive fibre density increased in the STh, and a trend towards normalisation of OTR-immunoreactive fibre density was evident in the NAcc in rats that were previously experienced at METH IVSA compared to yoked controls. These findings demonstrate that the oxytocin system, both centrally within the NAcc and STh, and peripherally through plasma measures, is dysregulated following METH abuse. This provides important insights into the neurobiological processes of oxytocin and it’s receptor in regulating METH abuse.
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Poster 31
Manipulation of Avpr1b-expressing neuronal activity: Effects on social behavior
Williams Avram, S.K.1, Fastman, J.1, Vincent, M.1, Weischelbaum, C.1, Lee. H.-J.2, Smith, A.1, Song, J.1, Cymerblit-Sabba, A.1, Shepard, E.1, Young, W. S.1
1Section on Neural Gene Expression, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA, 2Department of Oral Microbiology and Immunology, School of Dentistry, Kyungpook National University, Daegu 700-412, Korea
Vasopressin modulates a great variety of behaviors through its activation of its receptors in the central nervous system. The vasopressin 1b receptor (Avpr1b) is exclusively expressed in the cornu ammonis 2 (CA2) subregion of the hippocampus. Genetic knockouts (KOs) of Avpr1b show deficits on male and female aggression and social memory. We have recently shown that returning Avpr1b into the CA2 in adult KO males restores typical levels of resident-intruder aggression, suggesting the critical nature of Avpr1b signaling in the expression of the aggression. Additionally, inhibition of synaptic activity in CA2 neurons was shown to inhibit social memory. However, the precise timing of Avpr1b-expressing neuronal activity in the behaviors remains unclear. In order to specifically study the role of Avpr1b-expressing neurons, we have created a transgenic knock-in mouse, in which the Avpr1b gene has been replaced by the cre recombinase gene (Avpr1b-Cre). Avpr1b-Cre mice express cre recombinase with high fidelity in CA2 pyramidal neurons. Avpr1b-cre mice have been used to manipulate CA2 neuronal activity through cre-dependent introduction of channelrhodopsin (CHR2) and designer receptors exclusively activated by designer drugs (DREADD). CHR2 studies allow for rapid (subsecond) depolarization or hyperpolarization of an anatomical subset of Avpr1b-expressing neurons. In these studies, animals express CHR2 either through virally-mediated or transgenic methods and are implanted with an optic fiber in the mid-CA2. DREADD studies allow for G-protein mediated excitation or inhibition of Avpr1b neuronal firing across the entire CA2. Using these techniques, we have begun studying intermale resident-intruder aggression and female postpartum aggression. Preliminary results suggest you cannot increase intermale aggression with direct optogenetic depolarization of CA2 pyramidal cells. Additional studies are assessing the role of Avpr1b neurons in the acquisition and retrieval of social memories. Preliminary results suggest that optogenetic depolarization cannot enhance social memory through direct stimulation of these cells. Current studies are investigating inhibition of the pyramidal cells during aggression and memory testing. These studies will define the precise timing necessary for Avpr1b-expressing neuronal activity to modulate social memory and aggression.
This research was supported by the intramural research program of the NIMH (ZIA-MH-002498-24).
Poster 32
Tonic endocannabinoid signaling in hypothalamic magnocellular neuroendocrine cells
Di, S.1, Tasker, J.G.1,2
1Department of Cell and Molecular Biology, 2Neuroscience Program, Tulane University, New Orleans, LA 70118, USA
Recent evidence suggests that different endocannabinoids (eCB) may be responsible for the effects of tonic and evoked eCB release at some synapses. Our previous findings demonstrate that anandamide is released tonically at GABA synapses on hypothalamic magnocellular neurons and is
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insensitive to glial buffering, while 2-arachidonoylglycerol is released at glutamate synapses in an activity-dependent manner, and its access to GABA synapses is regulated by astrocytes (Di et al., 2013). Based on the recent discovery that the synaptic cell adhesion molecule neuroligin is essential for tonic eCB signaling in the hippocampus (Foldy et al., 2013), we tested for the involvement of neuroligin in the mechanism of tonic eCB modulation of GABA synaptic inputs to magnocellular neurons by targeting the acetylcholinesterase (AChE) homology domain of the neuroligin protein in whole cell patch clamp recordings in hypothalamic slices. Our preliminary findings indicate that, in the presence of cholinergic receptor antagonists, bath application of the acetylcholinesterase inhibitor ambenonium (2 µM) induced a significant increase in the frequency of inhibitory postsynaptic currents (IPSCs) in the magnocellular neurons that was similar to that seen with CB1 receptor blockade, indicating a disinhibition of a tonic suppression of GABA release comparable to the tonic eCB effect. Addition of the CB1 receptor antagonist SR141716 (1 µM) failed to further increase the IPSC frequency, suggesting that inhibiting the AChE domain of neuroligin blocked the tonic eCB suppression of GABA release. This points to the neuroligins as possible transporters of anandamide across the synapse.
Poster 33
Social behaviour and nonapeptide binding in passerine species of the Tibetan plateau
Meddle S.L.1, Bishop V.R.1, Foltz S.L.2, Wang G.3, Lei F.4, Wingfield J.C.5, Davis J.E.6
1The Roslin Institute, The University of Edinburgh, Easter Bush, Midlothian, UK, 2Department of Biological Sciences, Virgina Tech, Blacksburg, VA, USA, 3Enreach International Education-Wuxi Division, Wuxi, Jiangsu Province, P. R. China, 4Chinese Academy of Sciences, Beijing, P. R. China, 5College of Biological Sciences, University of California Davis, Davis, CA, USA, 6Biology Department, Radford University, Radford, VA, USA
The Tibetan plateau presents a uniquely challenging environment for passerine birds and previous studies have demonstrated that several of these endemic species display particular social behaviour and seasonally variable hormonal profiles. Within the brain nonapeptides are strongly conserved across all vertebrate taxa and are known to influence social behaviour in a species-specific way and this is thought to be due to differences in nonapeptide receptors within the brain. Differences in nonapeptide binding have been previously reported for gregarious compared to territorial bird species birds (e.g. 1) but to date no studies have directly compared free living avian species in the non-breeding season when circulating sex-steroid levels are at their lowest. We quantified aggression and collected brain tissue from wild free living white-rumped (Montifringalla taczanowski) and rufous-necked (Montifringalla ruficollis) Pere David's (Montifringilla davidiana) snowfinches in the non-breeding season in Tibet. Using simulated territorial intrusions we found that white-rumped snowfinches were more aggressive than rufous-necked snowfinches which are facultatively social. We used the [125I] ornithine vasotocin analog (125I-OVTA) to quantify and compare nonapeptide binding in the brain. The ornithine analog 125I-OVTA is widely used as a mesotocin receptor ligand but it also binds vasotocin. We found that white-rumped snow finches had higher nonapeptide binding in the lateral septum compared to rufous-necked snowfinches; no sex differences were observed in receptor density so the sexes were combined. The lateral septum forms part of the social behaviour network in the brain with a role in regulating social behaviour such as aggression and our findings thus support mounting evidence for a role of nonapeptides in non-sexual social behaviour in birds.
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Research Supported by NSF International Research Fellowship OISE-0701325 to JED; NSF grant IOS-0750540 and the Endowed Chair in Physiology, University of California, Davis to JCW; BBSRC Strategic Roslin Institute funding to SLM.
1. Goodson, J.L., Evans, A.K., Wang Y. (2006) Neuropeptide Binding Reflects Convergent and Divergent Evolution in Species-Typical Group Sizes. Horm. Behav. 50:223–236.
Poster 34
The role of vasopressin expressing retinal ganglion cells projecting to the hypothalamic suprachiasmatic nucleus
Tsuji, T.1, Zhang, M.2, Tobin, V.A.1, Allchorne, A.J. 1, Tsuji, C.1, Pineda, R.1, Stern, J.E.2, Grinevich, V.3, Leng, G.1, Ludwig, M.1
1Centre for Integrative Physiology, University of Edinburgh, Edinburgh, UK, 2Department of Physiology, Georgia Health Sciences University, Augusta GA USA,3Schaller Research Group on Neuropeptides, German Cancer Research Center DKFZ and University of Heidelberg, Heidelberg, Germany
The retina of mammals contains intrinsically photosensitive retinal ganglion cells (RGCs) which mediate non-image-forming visual functions such as pupillary light reflex and circadian photo entrainment. Here we show a population of vasopressin (VP) expressing retinal ganglion cells (VP-RGCs) which is photosensitive, but does not express either melanopsin or PACAP. Intravitreal injections of a rAAV-expressing Venus (under the control of the VP promoter) show that the vast majority of these cells project to the ventro-lateral core part of the suprachiasmatic nucleus (SCN). Microdialysis data show an increase in intra-SCN VP concentration in response to electrical stimulation of the optic nerve or light stimulation of the eye. Furthermore, in response to these stimuli some SCN neurons showed an excitatory response which was blocked by icv injection of a VP V1 receptor antagonist. Light induced expression of the immediate early gene product c-Fos during the dark period was strongly reduced by prior administration of a VP V1 receptor antagonist or ablation of the VP-RGCs. Our data suggest that activation of VP-RGC may mediate light cues by releasing VP into the SCN to excite SCN neurons, altering SCN activity and thus regulating neuroendocrine hormonal rhythms.
Poster 35
Radioimmunological assessment of OXT in human saliva during running, sexual self-stimulation and the Trier Social Stress Test: an OXT challenge study
De Jong, T., Menon, R., Jurek, B., Biermeier, V., Bludau, A., Grund, T., Klampfl, S.M., Bosch, O.J., Landgraf, R., Neumann, I.D.
Department of Behavioural and Molecular Neurobiology, Institute for Zoology, University of Regensburg, Regensburg, Germany
Intranasal administration of synthetic OXT emerges as a treatment option for patients suffering from socio- and emotional dysfunctions. There is an urgent need to reveal subpopulations of patients with a dysfunctional endogenous OXT system, who could particularly benefit from this treatment. Therefore, we aimed to establish an OXT challenge test as a biomarker of the endogenous OXT system (re)activity, easy to perform at home or in the laboratory. Physical exercise (running), sexual
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self-stimulation and exposure to psycho-social stress, i.e. Trier Social Stress Test (TSST), are reliable biological conditions promoting OXT release from the neurohypophysis into blood in rodents and humans. Here, we radioimmunologically quantified OXT in human saliva under basal conditions and in response to those physiological challenges in order to analyse the activity and responsiveness of the OXT system.
Healthy human volunteers performed two home-based challenges: running and sexual self-stimulation both for 10 min between 6:00 and 10:00 p.m. (with at least 24h between the two challenges). Saliva was collected into pre-coded Salivettes (Sarstedt Germany) under basal conditions at least 30 min before, and 10 and 40 min after starting the stimulation. We found a 2.5-fold increase in salivary OXT as soon as 10 min after starting running which remained significantly elevated until 30 min after termination of running. Similarly, salivary OXT showed a 2-fold increase following 10 min of sexual self-stimulation compared to basal, which declined 30 min later. There were no gender-specific differences in OXT responses to running or sexual stimulation.
The TSST was performed between 1:00 and 3:30 p.m. in healthy men and women and consisted of 4 components: introduction (2 min), preparation (3 min), speech (5 min) and arithmetic task (5 min). Fifteen min after the TSST started saliva OXT concentration was doubled compared with baseline. Salivary OXT was restored to control level 30 min after the test started. At this time point salivary cortisol peaked with a 2-fold increase in females and a 4-fold increase in males indicating gender-specific differences in the response of the HPA axis.
In summary, our study demonstrates that radioimmunological quantification of OXT in saliva is a reliable method to assess the responsiveness of the endogenous OXT system to physiological challenges. Further, saliva OXT provides a non-invasive home-based collection method, which prevents stress-inducing factors of a clinical laboratory.
Poster 36
Juxtacellular recording and labelling uncovers hypertonicity-sensitive theta oscillations in hypothalamic periventricular nucleus and intracerebral axonal projections from individual magnocellular vasopressinergic neurons
Hernández, V. S.1, Marquez, M.M.1, Barrio, R.A.2, Zhang, L.1
1Departement of Physiology, Faculty of Medicine, UNAM, Mexico City, Mexico, 2Physics Institute, UNAM, Mexico City, Mexico
Since the half of the 20th century it has been proposed that neurosecretory cells of the hypothalamus send projections to intracerebral targets, however conventional immunohistochemical or in vitro electrophysiological methods fail to detect long range projections of the magnocellular neurosecretory neurons (MNN´s). Here using juxtacellular recording and labelling of identified neurons in the paraventricular (PVN) nucleus we show that MNN´s have multiple axon-like processes and axonal collaterals that express vGlut2 and innervate intrahypothalamic structures (medial preoptic area, suprachiasmatic nucleus,) integrate into main conduction systems (fornix, internal capsule, stria medularis) and innervate extra hypothalamic structures (amygdala, lateral habenula). (Fig. 1)
(Fig. 1)
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Analysis of the local field potential showed that under basal conditions a theta range (4-6) oscillation is present in the PVN. Hypertonicity induced an increase in frequency and power amplitude. Furthermore, in vivo activation of AVP-MNNs by hypertonic saline administration, induced a decrease in passive coping (freezing), and promoted an increase in active coping (escape) strategies, when rats were exposed to a live predator. Modified Fos expression patterns in lateral habenula, amygdala and thalamus, were also observed. Our results demonstrate that AVP-MNNs multiple axon-like processes and the long-range intracerebral projection is a more common feature of the MNNs, than an “occasional” phenomenon as previously thought. These magnocellular AVP-glutamatergic non-canonical pathways found here may constitute a part of the central motivational circuit activated under multifaceted stress coping. The origin, physiological significance of the theta rhythms recorded inside the PVN and its modification by hypertonicity remain to be determined.
Poster 37
Effects of maternal oxytocin administration on fetal vole pups
Kenkel, W.M.1, Perkeybile A.M.1, Connelly, J.J.2, Yee, J.R.1, Ferris, C.F3, Carter, C.S.1
1The Kinsey Institute, Indiana University, Bloomington, IN, 2Department of Psychology, University of Virginia, Charlottesville, VA, 3Center for Translational NeuroImaging, Northeastern University, Boston, MA, USA
The use of oxytocin (OT) to induce/augment labor has become common practice in the modern obstetric setting. This practice has led to dramatic improvements in infant and maternal mortality over the course of the 20th century. However, little is known about the effects of such OT administration on the central nervous system, either in the mother or the developing neonate. Given what is known about the long-term consequences of even a single OT administration at postnatal day 1 on the brain and behavior in later life, we sought to evaluate the potential for OT to produce similar effects when administered indirectly i.e. via the pregnant female as would occur in the human obstetric condition. Using timed-pregnant female prairie voles, we administered several doses of OT (0.1, 0.25 and 0.5 mg/kg) on the expected day of delivery. In one experiment, fetal heart rate was recorded while still connected to the maternal circulation via the umbilical cord. Following OT administration, fetal heart rate decreases in a OTR-specific fashion, which suggests a disruption of transplacental communication. In a parallel experiment, brains from pregnant females and fetal pups were removed 90 minutes after treatment and micro-dissected for regional analysis of epigenetic regulation of the oxytocin receptor (OTR) and vasopressin receptor 1a as well as mRNA levels of those same receptors. Preliminary results suggest that methylation of the OTR is affected in a dose-dependent manner following maternal OT administration. These results support the notion that maternally administered OT around the time of birth can influence the offspring's developing central nervous system. Future efforts will be directed at examining the long-term consequences of perinatal OT on offspring behavior and neurobiology in later life.
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Poster 38
The analgesic effect of oxytocin in humans: a double-blinded placebo controlled cross-over study using laser-evoked potentials.
Paloyelis, Y.1, Krahé, C.1, Maltezos, S.2, Williams, S.C. 1, Howard, M.A. 1, Fotopoulou, A.3
1 King’s College London, Department of Neuroimaging, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK, 2 King’s College London, Department of Forensic and Neurodevelopmental Science, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK, 3 Research Department of Clinical, Educational, and Health Psychology, University College London, Gower Street, London WC1E 6BT, UK
Oxytocin regulates social-affiliative and reproductive behavior in mammals. Despite robust preclinical evidence for oxytocin’s antinociceptive effects, human studies have produced mixed results regarding oxytocin’s analgesic role and are yet to show a specific modulation of neural processes involved in pain perception. We investigated the analgesic effects of 40IU of intranasal oxytocin in 13 healthy male volunteers using a double-blinded, placebo-controlled, cross-over design and brief radiant heat pulses generated by an infrared laser that selectively activate Aδ- and C-fiber nerve endings in the epidermis, while recording ensuing laser evoked potentials (LEPs). We predicted that oxytocin would reduce subjective pain ratings and attenuate the amplitude of the N1, N2 and P2 components. We observed that oxytocin attenuated perceived pain intensity and the local peak amplitude of the N1 and N2 LEPs, and increased the latency of the N2 component. Importantly, for the first time, this study reports an association between the analgesic effect of oxytocin and the oxytocin-induced modulation of cortical activity following noxious stimulation. These effects indicate that oxytocin modulates neural processes contributing to pain perception in humans. Our findings are consistent with robust preclinical evidence on oxytocin’s antinociceptive properties and highlight potential neural mechanisms mediating oxytocin’s analgesic effects in humans.
Poster 39
Investigating the mechanisms through which intranasal oxytocin affects brain function in humans
Paloyelis, Y.
King’s College London, Department of Neuroimaging, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK
Experimental studies and clinical trials rely almost exclusively on the intranasal administration of oxytocin to investigate its role in human behaviour and its therapeutic efficacy to treat a range of social deficits (e.g. in autistic spectrum disorder) or social and other forms of anxiety (e.g. in affective disorders). However, it is still unclear whether oxytocin enters the brain directly from the nasal cavity or whether it affects brain function by acting on peripheral receptors, while we also know very little about its pharmacodynamics in the human brain. Rodent studies have started to address these questions [e.g. 1, 2]. Given the existence of fundamental inter-species differences in the physiology of the nasal/olfactory system, blood-brain barrier permeability and the configuration of the oxytocin system in the brain, findings from animal studies cannot be immediately extrapolated to humans. We recently began to address these questions, demonstrating that we can use arterial spin labelling
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and multivariate pattern analysis in humans to measure oxytocin’s central effects and pharmacodynamics [3]. We visualized and quantified oxytocin-induced changes in regional cerebral blood flow in the living human brain unaffected by cognitive, affective, or social manipulations. In the current study we use a cross-over, double-blinded, placebo-controlled design administering oxytocin/placebo intravenously and/or intranasally in 16 healthy male volunteers. We will measure changes in regional cerebral blood flow and blood plasma oxytocin levels at baseline and for 90 minutes post-administration. We will be presenting preliminary findings regarding the dynamics of intranasal oxytocin’s central effects in blood perfusion, linking them with blood plasma levels. We will further clarify whether these effects are caused by oxytocin acting on peripheral receptors and/or penetrating the blood brain barrier to a sufficient extent to induce changes in central function. This study will help clarify the mechanisms through which intranasal oxytocin elicits behavioural and cognitive effects in humans and its pharmacodynamics in the living human brain.
1. Neumann, I.D., et al., Increased brain and plasma oxytocin after nasal and peripheral administration in rats and mice. Psychoneuroendocrinology, 2013. 38(10): p. 1985-93.
2. Ludwig, M., et al., Intranasal application of vasopressin fails to elicit changes in brain immediate early gene expression, neural activity and behavioural performance of rats. J Neuroendocrinol, 2013. 25(7): p. 655-67.
3. Paloyelis, Y., et al., A Spatiotemporal Profile of In Vivo Cerebral Blood Flow Changes Following Intranasal Oxytocin in Humans. Biol Psychiatry, 2014.
Poster 40
Oxytocin-like neuropeptide signalling in ants
Zita Liutkeviciute1, Esther Gil Mansilla1, Maria Giulia Di Giglio1, Sylvia Cremer2 and Christian W. Gruber1
1Center for Physiology and Pharmacology, Medical University of Vienna, 2IST Austria (Institute of Science and Technology Austria)
Over 25 years ago a vasopressin-like peptide has been discovered in locusts [1]. Recently it has become evident that several other insect species contain components of the oxytocin/vasopressin-like signalling system, but to date its biological function in insects has only been studied in the beetle Tribolium castaneum where it has been implicated in water retention [2; 3]. Commonly in invertebrates – similar to the vertebrate system –oxytocin/vasopressin-like signalling appears to be important for water homeostasis, reproduction, learning, memory and behaviour [4]. In recent years the genomes of different ant species have been sequenced and the putative inotocin precursor (homolog of oxytocin) and receptor were discovered [5]. It was the first evidence that oxytocin-like signalling system exists in social insects and possibly regulates individual physiology and social organization in ant colonies. Interestingly, honey bees lack the inotocin signalling system making ants a unique model system to study a role of inotocin in social insects. For our study we have chosen two ant species of the genus Lasius that are closely related genetically, but significantly differ in their ecology and colony structure [6].
Following pharmacological characterization of the ligand-receptor pair in vitro, our aim was to quantify expression levels of both the receptor and the inotocin peptide precursor in different parts of the body and developmental stages in ants using quantitative PCR. As expected, the highest expression level of precursor was found in the head of ants. In contrast to beetles where both genes exhibit the highest expression in the larvae stage [3], the expression of the receptor and precursor in early broods (eggs and larvae) of ants is about an order of magnitude smaller compared to pupae or worker. Interestingly, expression of the receptor in L. neglectus head and gaster is similar, but in L.
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niger it is about 10-fold more abundant in the abdomen as compared to the head. Next we dissected organs of L. niger worker gaster and showed that the highest expression of receptor exists in crop, nervous system and fat body. These findings indicate that the expression patterns of inotocin and its receptor are different in distinct insect species and provide further hints about diverse biological functions of this important neuropeptide signalling system. These preliminary outcomes will be important for further functional genomics studies to elucidate the role of inotocin signalling for individual and group level behaviour in ant societies.
Acknowledgements: The authors would like to thank Markus Gold-Binder, Thomas Eder, Anna Grasse, Matthias Konrad and Christopher Pull for their help and discussions. The project has been funded by the Vienna Science and Technology Fund (WWTF) through project grant LS13-017.
1. Proux JP et al., 1987; Biochem Biophys Res Commun. 149(1):180-6.
2. Aikins MJ et al., 2008; Insect Biochem Mol Biol. 38(7):740-8.
3. Stafflinger et al., 2008; Proc Natl Acad Sci USA. 105(9):3262-7
4. Gruber CW. Exp Physiol. 2014; 99(1):55-61.
5. Gruber CW, Muttenthaler M. PLoS One. 2012; 7(3):e32559.
6. Cremer S et al., 2008; PLoS One. 3(12):e3838.