Download At the crossroads of metabolism and reproduction in the brain

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