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The ventral premammillary nucleus: At the crossroads of metabolism and reproduction in the brain Supervisor: Christian Broberger Department: Dept. of Neuroscience BACKGROUND Reproduction and energy metabolism are intimately connected. There is an evolutionary logic to this: the body cannot commit to pregnancy without resources sufficient to carry it to full term. Clinically, this relationship is illustrated by the well-established infertility associated with anorexia and starvation, wherein the neuroendocrine gonadotrophic axis shuts down. Curiously, infertility is also common in obesity, suggesting that there is a finite metabolic interval wherein successful reproduction is possible (Frisch & Revelle, 1970). How does this coordination come about? An instructive clue is offered by rare human cases of leptin deficiency, who lack the adipose tissue-derived hormone that signals energy affluence. These patients, morbidly obese due to the absence of the satiety signal provided by leptin, yet in a state of perceived fatal starvation, exhibit a failure to go into puberty, a condition that can be reversed by leptin supplement therapy (Farooqi et al., 1999). The same is observed in leptin-deficient ob/ob mice. Through elegant gene deletion-replacement experiments, Donato et al. (2011) showed that expressing functional leptin receptors exclusively in a little-studied part of the brain – the hypothalamic ventral premammillary nucleus (PMv) – is sufficient to mediate the hormone’s powerful stimulatory effect on puberty (thus anatomically distinct from the satiety-inducing actions of leptin, which are relayed through other hypothalamic nuclei). The neuronal properties, network behaviour and transmitter identity of the reproduction-regulating PMv neurons remain elusive, however. Furthermore, it is not known if neuronal activity in the PMv acts as a “switch” that needs to be timed appropriately or if the cells remain continuously sensitive to metabolic cues throughout adult reproduction. In the present proposal, the postdoctoral fellow will use state-of-the-art neuroscientific strategies to address these and other issues, with the ultimate goal of providing insight that can help develop novel therapeutic strategies for the infertility associated with obesity and malnourishment. OBJECTIVES (AIMS) 1) Characterize the action of leptin and other metabolic signals upon DAT-PMv neurons 2) Determine how puberty and adult reproductive state is regulated by DAT-PMv neurons 3) Identify the effect of dopamine-modulating drugs upon puberty and fertility METHODOLOGY Animals: Our colony of DAT-Cre mice will either be crossed with reporter lines or injected stereotactically with viral (AAV) constructs to ensure anatomical specificity. The reporter lines and constructs will drive expression of fluorescent markers (e.g. tdTomato) for visualization, optogenetic proteins (ChR2 for excitation and NphR3 for inhibition), pharmacogenetic proteins, as well as proteins for irreversible silencing (incl. diphtheria toxin and caspase). Optogenetic manipulation is performed by light delivered through stereotactically implanted optical fibers; selective pharmacogenetic manipu-lation of is accomplished by systemic (ip) administration of ligands in animals expressing activating (hM3D) or inactivating (hM4D) receptors selectively expressed in DAT-PMv cells. In vitro electrophysiology will be performed on acutely cut slices maintained at physiological temperature in artificial cerebrospinal fluid, with whole-cell patch clamp recordings performed in current and voltage clamp modes as previously described (Stagkourakis et al., 2016). Pharmacological compounds are applied in the bath or locally by pressure-pulse ejection. Determining reproductive stage: Puberty is monitored as the time of vaginal opening and first estrus (in females), preputial separation (males) and the age of successful mating with a sexually experienced partner (both sexes). Sexual development and fertility are determined through weight and histology of uterus, ovarian and testicular histology, serum LH/FSH and sex steroid levels, successful mating and number of offspring. Estrous cycle period and rhythmicity are determined through vaginal smears. WORK PLAN Aim 1: Electrophysiology of DAT-PMv neurons and its regulation through sexual development and by metabolic signals. What are the passive and active membrane properties of DAT-PMv cells in vitro? Do they exhibit rhythmic network properties? How are cellular and network properties modulated by leptin? Does leptin affect incoming synaptic impulses? Are there differences across sex and/or age? How do DAT-PMv neurons in mouse models of altered leptin signalling (ob/ob and db/db mice) behave? Do circulating metabolic signals directly impact upon the cellular and network electrophysiology of DAT-PMv cells? These experiments will provide a comprehensive understanding of the electrical behaviour of DAT-PMv cells, their susceptibility to metabolic cues and how this changes with reproductive development. Aim 2. Role of DAT-PMv neurons in puberty and in adult reproduction. How does DATPMv activity influence reproduction in vivo? What happens to reproductive status or the onset of puberty when the neurons are reversibly or terminally silenced? What is the effect when the neurons are activated? Are there sex differences? Aim 3. Modulation of reproductive status by dopaminergic agents. Can pharmacological manipulation of dopamine signalling affect reproductive status? What is the effect of commonly used dopamine-modulating drugs? Are here sex differences? Do effects differ at different ages? ENVIRONMENT AND QUALIFICATIONS: The Broberger laboratory has in recent years described the electrophysiology and neuromodulation of hypothalamic neuroendocrine dopamine neurons that regulate reproduction (e.g. Lyons et al., 2010; 2016; Briffaud et al., 2015, Stagkourakis et al., 2016). Other interests of the group include the role of Ca2+-binding proteins in the nervous and endocrine systems and the crosstalk between the hypothalamus and the cerebral cortex. A broad repertoire of state-ofthe-art neuroscience techniques are used in the lab, including patch clamp electrophysiology, neuroanatomical reconstruction and tracing, fast-scan cyclic voltammetry, optogenetics, behavioural monitoring and cell culture. The group is part of the vibrant international environment at the Dept of Neuroscience, which holds leading expertise in the study of neuronal networks. The close association with the KI Strategic Research Programme in Diabetes allows for multidisciplinary approaches to the central control of metabolism. The ideal candidate will have a background in neuroscience. Experience of electrophysiological techniques is an advantage but not a requirement. Proficiency in written and spoken English is required. REFERENCES: Briffaud V, Williams P, Courty J, Broberger C, 2015 Excitation of Tuberoinfundibular Dopamine (TIDA) Neurons by Oxytocin: Cross-talk in the Control of Lactation. J. Neurosci., 35(10):4229-37. Donato J Jr, Cravo RM, Frazão R, Gautron L, Scott MM, Lachey J, Castro IA, Margatho LO, Lee S, Lee C, Richardson JA, Friedman J, Chua S Jr, Coppari R, Zigman JM, Elmquist JK, Elias CF, 2011. Leptin's effect on puberty in mice is relayed by the ventral premammillary nucleus and does not require signaling in Kiss1 neurons. J Clin Invest.; 121(1):355-68. Farooqi IS, Jebb SA, Langmack G, Lawrence E, Cheetham CH, Prentice AM, Hughes IA, McCamish MA, O'Rahilly S, 1999. Effects of recombinant leptin therapy in a child with congenital leptin deficiency. N Engl J Med; 341(12):879-84 Frisch RE, Revelle R, 1970. Height and weight at menarche and a hypothesis of critical body weights and adolescent events. Science; 169(3943):397-9. Lyons DJ, Horjales E, Broberger C, 2010. A slow oscillation in tuberoinfundibular dopamine (TIDA) neurons: Switch to tonic firing via thyrotropin-releasing hormone (TRH). Neuron; 65(2): 217-29. Lyons DJ, Ammari R, Hellysaz A, Broberger C, 2016. Serotonin and Antidepressant SSRIs Inhibit Rat Neuroendocrine Dopamine Neurons: Parallel Actions in the Lactotrophic Axis. J. Neurosci., 36(28):7392-406 Stagkourakis S, Kim H, Lyons DJ, Broberger C, 2016. Dopamine Autoreceptor Regulation of a Hypothalamic Dopaminergic Network. Cell Reports; pii: S2211-1247(16)30333-3 Contact details Christian Broberger, MD, PhD [email protected] +46 70 22 69 327 http://ki.se/en/neuro/broberger-laboratory