DEVELOPING BRAIN AS AN ENDOCRINE ORGAN : A PARADOXI С AL REALITY

1. 2nd International Conference on Endocrinology October 20-22, 2014, Chicago, USA DEVELOPING BRAIN AS AN ENDOCRINE ORGAN : A PARADOXIСAL REALITY M.V. Ugrumov Institute of Developmental Biology RAS, Moscow

2. 2nd International Conference on Endocrinology October 20-22, 2014, Chicago, USA Developing brain as an endocrine organ: a paradoxical reality Endocrine functions of the brain in adulthood Current concept of neuroendocrine regulations in ontogenesis and contradictions. Revised concept of neuroendocrine regulations in ontogenesis. Summary and prospect

3. Extrahypothalamic centers Blood vessels Magnocellular nuclei (vasopressin, oxytocin) Endocrine functions of the brain in adulthood Brain Pituitary Endocrine glands Parvocellular nuclei (releasing- / inhibiting-hormones) Posterior hypophysis Portal circulation Hypothalamus Target organs Direct regulation Adenohypophysis • Criteria of the brain functioning as an endocrine organ : • Existence of the neurons, secreting neurohormones; • Delivery of neurohormones into the blood vessels; • Maintaining of the physiologically active concentration • of neurohormones in blood; • Delivery of neurohormones from secretory neurons • to the distant target cells via circulation. Feedback regulation General circulation

4. 2nd International Conference on Endocrinology October 20-22, 2014, Chicago, USA Developing brain as an endocrine organ: a paradoxical reality Endocrine functions of the brain in adulthood Current concept of neuroendocrine regulations in ontogenesis and contradictions Revised concept of neuroendocrine regulations in ontogenesis Summary and prospect

5. Development of the neuroendocrine regulations in ontogenesis. Current concept II. Postnatal close-looped neuroendocrine system Brain Direct regulation Feedback regulation Endocrine glands Pituitary I. Prenatal and neonatal open-looped neuroendocrine system Placenta Brain Endocrine gland Pituitary • Milestones of the concept: • The development of peripheral endocrine glands precedesthat of the “endocrine” hypothalamusand • the brain as a whole; • The hypothalamic neurons begin to secrete the adenohypophysiotropicneurohormonesafter the axon • sprouting to the hypophysial portal circulation; • Neurohormones secreted by the differentiating neurons contribute, first, to theautocrine and paracrine • regulation of the neuron development and, significantly later, to the endocrine control • of the adenohypophysial functions. Dörner (1981); Daikoku et al. (1981); Gorski (1988); Jacobson (1991);Ugrumov (1991), Int. Rev. Cytol. 129, 207-267; Ugrumov (2002) Microsc Res Tech 56, 164-171.

6. 2010. 35, 837-850. Neurochem. Res. 2010. 35, 837-850. Birth Postnatal day Embryonic day E12 P15 P1 E15 E20 Adult (rat) ‗ ‗ Age Neuronogenesis Expression of specific phenotype Development of neuronal networks, synaptogenesis Brain blood barrier Period of the neuron functioning as a secretory cell and the brain operation as an endocrine organ • If the hypothesis is valid: • The concentration of the brain-derived neurohormonesin general circulation before the closing • of the blood brain barrier in ontogenesis should be as large as that in the hypophysial portal circulation • in adulthood; • The concentration of the brain-derived neurohormones in peripheral blood should drop under inhibition • of their synthesis in the brain; • Circulating brain-derived neurohormones should contribute to the endocrine regulation of the developing • peripheral target organs and the brain itself(autoregulation)

7. 2nd International Conference on Endocrinology October 20-22, 2014, Chicago, USA Developing brain as an endocrine organ: a paradoxical reality Endocrine functions of the brain in adulthood Current concept of neuroendocrine regulations in ontogenesis and contradictions Revised concept of neuroendocrine regulations in ontogenesis Summary and prospect

8. GnRH system Serotonin Olfactory bulbs Optic chiasma Median eminence Dopamine Dopaminergic system Dopaminergic system GnRH 3 ventricle III ventricle V А11 А11 A8 A8 А13 А13 A9 A9 A10 A10 Portal circulation Olfactory bulbs A14 A14 Median eminence A12 A12 Optic chiasma Serotoninergic system General circulation Pituitary Rahe nuclei GnRH, dopamine and serotonin as markers of brain endocrine activity in ontogenesis

9. Whether the concentration of neurohormones in peripheral blood in ontogenesis before the blood brain barrier closing is sufficient to provide an endocrine control of the target organs ?

10. Male & female; Male; Female; Concentration of neurohormones in general circulation in rats in ontogenesis Dopamine GnRH 3.5 * * 14 * * 3.0 * * Concentration in portal blood of adult rats 12 2.5 10 2.0 Concentration in plasma (ng / ml) Concentration in plasma (pg / ml) 8 1.5 6 1.0 4 0.5 2 0 0 E21 P3 E18 P30 E18 P3 P36 E21 Age (days) Age (days) Blood brain barrier Blood brain barrier E, embryonic day; P, postnatal day Ugrumov et al. (2005) Comp. Biochem. Physiol. A 141, 271-279; Ugrumov (2010) Neurochem. Res. 35, 837-850; Ugrumov et al. (2012) Mol. Cell. Endocrinol. 348, 78-86.

11. Whether the developing brain is a principal source of circulating neurohormones ?

12. E18 E21 Corpus collosum Olfactory bulb Caudate, putamen Lateral septum Anterior congulate cortex Prefrontal cortex Pituitary Retina A17 Median eminence Nucleus accumbens Amigdala Entorhinal cortex Pyriform cortex Delivery of brain-derived dopamine into the general circulation in rats in ontogenesis I. Dopamine in plasma of encephalectomized rat fetuses Control Encephalectomy Encephalectomy 3.5 3.0 * 2.5 2.0 Dopamine in plasma (ng / ml) 1.5 1.0 0.5 0 Females Males II. Dopamine in the brain and plasma following an inhibition of its synthesis in the brain Control α-МPТ 100 Animals:rats at the 3rd postnatal day; Inhibitor:α-methyl-p-tyrosine (α-МPТ) Administration: lateral ventricle 80 * 60 Dopamine concentration (%) 40 * 20 Ugrumov (2010) Neurochem. Res., 35, 837-850; Ugrumov et al. (2012). Mol. Cell. Endocrinol. 348, 78-86. 0 Brain Plasma

13. Control Encephalectomy Delivery of brain-derived GnRH into the general circulation in rats in ontogenesis Microsurgical lesion of GnRH neurons in fetal brain Fetus 14 12 Encephalectomy at E18; GnRH assay at E21 10 * * GnRH concentration in plasma (pg / ml) 8 6 Inhibition of GnRH synthesis in the brain 4 2 0 Males Females Neonates: intracerebral administration of GnRH si-RNA (in progress) Ugrumov et al. (2005) Comp. Biochem.Physiol. A 141, 271-279; Ugrumov (2010) Neurochem. Res., 35, 837-850.

14. Delivery of brain-derived serotonin into the general circulation in rats in ontogenesis 140 I. Serotonin (5-HT) in blood of perinatal rats * 120 25 * * 100 20 80 5-HT concentration in plasma, ng / ml 15 60 Rate of increase of 5-HT content in plasma, ng/day 40 10 20 5 0 E21 P4 P16 P30 0 Blood brain barrier Blood brain barrier P16-P30 E21-P4 P4-P16 II. Serotonin in the brain and blood following inhibition of its synthesis in the brain 300 E, embryonic day Control 800 5-HT P, postnatal day Brain Duodenum 100 µg p-chloro- phenylalanine 250 250 700 600 200 * 500 5-HT content in blood, ng 5-HT content per brain, ng 150 400 300 100 General circulation 50 * * 50 Zubova et al. (2014) Molecular and Cellular Endocrinol. 393, 92-98. 25 0 0 Plasma Platelets Brain

15. Whether the brain-derived neurohormones provide a direct endocrine action on peripheral targets and the brain itself ?

16. Potential targets for circulating brain-derived GnRH and monoamines in the developing organism

17. Endocrine regulation of prolactin secretion by brain-derived dopamine in neonatal rats In vivo study x 102 NaCl 6-OHDA Brain 16 2,5 Dopamine 14 * Hypothalamus 2,0 Dopamine 12 10 Dopamine concentration, ng / g tissue 1,5 8 General circulation Prolactin concentration, ng / ml Portal circulation 1,0 6 4 * 0,5 Pituitary 2 Prolactin 0 0 Plasma Hypothalamus Whole brain Zubova et al, in preparation

18. 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Endocrine regulation of prolactin secretion by brain-derived dopamine in neonatal rats In vitro study Plasma Krebs-Ringer buffer Pituitary Incubation of the pituitaries of 3-day-old rats D2 Lactotropes, prolactin Plasma of rats Krebs-Ringer buffer Control (pure medium) Dopamine Control (plasma) Dopamine D2 Dopamine (1.5 ng/ml) D2 Plasma& haloperidol Dopamine (1.5 ng/ml) & haloperidol Haloperidol * 3.0 * 2.5 2.0 Prolactin, ng / mg pituitary /300 μlmedium * Prolactin, ng / mg pituitary /300 μlplasma D2, dopamine receptors 1.5 1.0 0.5 0 Zubova et al, in preparation

19. Endocrine regulation of testosterone secretion by brain-derived GnRH in neonatal rats In vitro study Krebs-Ringer buffer Plasma Incubation of the testes of 3-day-old rats Testis Krebs-Ringer buffer Plasma Control (plasma) Control (medium) GnRH (30 pg / ml) Plasma& cetrorelix Leydig cells, testosterone GnRH (30 pg / ml) &cetrorelix 12 2,5 * * Cetrorelix 2 9 * Testosterone, ng / mg testis /300 µlmedium 1,5 Testosterone, ng / mgtestis /300 µlmedium 6 GnRH GnRH 1 3 0,5 0 0 Bondarenko et al., in preparation

20. Congenital diseases          Norm    Brain  Neurons Fetus Adult    Cranium Brain  Neurons  Kallmann syndrome : Inability of GnRH neurons for migration Brain Brain Neuron Cranium Infertility Pronina T., Ugrumov M. et al. (2003a,b) J. Neuroendocrinol. 15, 549-558; J. Neuroendocrinol. 15, 925-932; Fechner A. et al. (2008) Obstet. Gynecol. Surv.63,189-194. Developing brain as a multipotent endocrine Brain Blood brain barrier General circulation Target organs Periphery

21. III. Reciprocal regulatory system in ontogenesis, before the blood brain barrier closure (Ugrumov, 2010) II. Postnatal close-loopedregulatory system Brain Direct regulation Feedback regulation Placenta Brain Endocrine glands Endocrine glands Pituitary Pituitary Neuroendocrine regulations in ontogenesis:current and revised concepts I. Prenatal open-looped regulatory system Placenta Brain Endocrine glands Pituitary

22. 2nd International Conference on Endocrinology October 20-22, 2014, Chicago, USA Developing brain as an endocrine organ: a paradoxical reality Endocrine functions of the brain in adulthood Current concept of neuroendocrine regulations in ontogenesis and contradictions Revised concept of neuroendocrine regulations in ontogenesis Summary and prospect

23. The concept on the role of the brain in neuroendocrine regulations in ontogenesis: in the past, at present and in the future • In the past.The brain does not participate in neuroendocrine regulations up to its complete • “maturation”, in rodents at prepuberty. • At present.The developing brain provides the paraadenohypophysial endocrine regulations • of peripheral target organs and the brain itself in ontogenesis, over a period from the neuron origin • till the establishment of synaptic interneuronal relations and the blood brain barrier closing. • In the future. • It should be ascertain: • Whether the brain-derived neurohormones contribute to the paradenohypophysial endocrine • regulation either of functioning or morphogenesis of the target organs; • What is the functional significance of a temporal combination of paracrine and endocrine • regulations of the target cells by the same neurohormones; • What is a role of impairing metabolism of the brain-derived neurohormones in the development • of congenital diseases

24. Thanks for your attention Michael V. UGRUMOV e-mail: michael.ugrumov@mail.ru; Tel. +7 499 135 88 42 Laboratory of Hormonal Regulations Yu. Saifityarova (PhD student) A. Sapronova (PhD) A. Sapronova (PhD), E. Volina (PhD) T. Pronina (PhD) L. Dilmuchametova (PhD student) V. Khaindrava (PhD) E. Kozina (PhD student) G. Chakimova (PhD student) E. Degtyareva (PhD student) V. Kirillova (technician)