Institute for Neural Signal Transduction

Hamburg, Germany

Institute for Neural Signal Transduction

Hamburg, Germany

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Hoivik E.A.,University of Bergen | Bjanesoy T.E.,University of Bergen | Mai O.,Institute for Neural Signal Transduction | Okamoto S.,National Institute of Physiological science | And 5 more authors.
Endocrinology | Year: 2011

The nuclear receptor steroidogenic factor 1/adrenal 4 binding protein (SF-1/Ad4BP) is an essential regulator of endocrine development and function, and the expression of the corresponding gene (sf-1/ad4bp) is precisely regulated in a time-and tissue-dependent manner. We previously demonstrated that the basal promoter of sf-1/ad4bp is controlled by DNA methylation and that its methylation status reflects the expression pattern of SF-1/Ad4BP. Recently, three intronic enhancers were identified in the sf-1/ad4bp gene that target SF-1/Ad4BP expression to the fetal adrenal (FAdE; fetal adrenalspecific enhancer), to pituitary gonadotropes (PGE; pituitary gonadotrope-specific enhancer), and to the ventromedial hypothalamic nucleus (VMHE; ventromedial hypothalamic nucleus-specific enhancer). Here, we demonstrate that the activity of these enhancers is correlated with their DNA methylation status. We show that they are hypomethylated in tissues where they are active and generally hypermethylated in tissues where they are not active. Furthermore, we demonstrate in transient transfection experiments that forced DNA methylation represses reporter gene activity driven by these enhancers. These data directly demonstrate a functional significance for the enhancers' methylation status. Intriguingly, further analyses of the basal promoter in gonadotropes revealed that it is methylated in these cells, in contrast to other SF-1/Ad4BP-expressing tissues. Consistent with this, sf-1/ad4bp is transcribed from an alternative promoter in gonadotropes. Taken together, our experiments show that the tissue-specific expression of SF-1/Ad4BP is epigenetically regulated and identify tissue-specific differentially methylated regions within the sf-1/ad4bp locus that are essential for its transcriptional control. Copyright © 2011 by The Endocrine Society.


Wen S.,Institute for Neural Signal Transduction | Gotze I.N.,Saarland University | Mai O.,Institute for Neural Signal Transduction | Schauer C.,Saarland University | And 2 more authors.
Endocrinology | Year: 2011

GnRH signaling regulates reproductive physiology in vertebrates via the hypothalamic-pituitary-gonadal axis. In addition, GnRH signaling has been postulated to act on the brain. However, elucidating its functional role in the central nervous system has been hampered because of the difficulty in identifying direct GnRH signaling targets in live brain tissue. Here we used a binary genetic strategy to visualize GnRH receptor (GnRHR) neurons in the mouse brain and started to characterize these cells. First, we expressed different fluorescent proteins in GnRHR neurons and mapped their precise distribution throughout the brain. Remarkably, neuronal GnRHR expression was only initiated after postnatal day 16, suggesting peri- and postpubertal functions of GnRH signaling in this organ. GnRHR neurons were found in different brain areas. Many GnRHR neurons were identified in areas influencing sexual behaviors. Furthermore, GnRHR neurons were detected in brain areas that process olfactory and pheromonal cues, revealing one efferent pathway by which the neuroendocrine hypothalamus may influence the sensitivity towards chemosensory cues. Using confocal Ca2+ imaging in brain slices, we show that GnRHR neurons respond reproducibly to extracellular application of GnRH or its analog [D-TRP6]-LH-RH, indicating that these neurons express functional GnRHR. Interestingly, the duration and shape of the Ca2+ responses were similar within and different between brain areas, suggesting that GnRH signaling may differentially influence brain functions to affect reproductive success. Our new mouse model sets the stage to analyze the next level of GnRH signaling in reproductive physiology and behavior. Copyright © 2011 by The Endocrine Society.


Kumar D.,Saarland University | Periasamy V.,Saarland University | Freese M.,Institute for Neural Signal Transduction | Voigt A.,Institute for Neural Signal Transduction | Boehm U.,Saarland University
Endocrinology | Year: 2015

The neuropeptide kisspeptin is a major regulator of the hypothalamus-pituitary-gonadal axis. Although it has long been known that kisspeptin and its receptor G protein-coupled receptor 54 (GPR54) are expressed in the developing brain well before puberty onset, the potential role of kisspeptin/GPR54 signaling in the embryonic brain has remained mysterious. Recent studies in female mice have shown that kisspeptin neurons in the arcuate nucleus of the hypothalamus (ARC) already communicate with a subset of GnRH neurons in utero. Whether this specific neural circuit is also formed in the developing male brain is not known. Here, we used a combination of different genetic strategies to analyze the ontogeny and development of the kisspeptin/GPR54 system in the male mouse brain. We demonstrate orchestrated onset of kisspeptin and GPR54 expression in the male embryonic mouse brain and find that androgen receptor and estrogen receptor-α immunoreactivity within the male brain delineate the birthplace of kisspeptin neurons in the ARC. Using conditional transsynaptic tracing from kisspeptin neurons, we find that ARC kisspeptin neurons already communicate with a subset of GnRH neurons in utero and that the neural circuits between ARC kisspeptin and GnRH neurons in the male mouse brain are established before birth. Furthermore, we also show that the connectivity between kisspeptin and GnRH neurons does not depend on the spatial position of GnRH neurons. Our data delineatethe maturing neural circuits underlying control of the reproductive axis in the male embryonic mouse brain. Copyright © 2015 by the Endocrine Society.


Voigt A.,German Institute of Human Nutrition | Voigt A.,Institute for Neural Signal Transduction | Bojahr J.,German Institute of Human Nutrition | Narukawa M.,German Institute of Human Nutrition | And 4 more authors.
Journal of Neuroscience | Year: 2015

Taste perception begins in the oral cavity by interactions of taste stimuli with specific receptors. Specific subsets of taste receptor cells (TRCs) are activated upon tastant stimulation and transmit taste signals to afferent nerve fibers and ultimately to the brain. How specific TRCs impinge on the innervating nerves and how the activation of a subset of TRCs leads to the discrimination of tastants of different qualities and intensities is incompletely understood.Toinvestigate the organization of taste circuits,weused gene targeting to express the transsynaptic tracer barley lectin (BL) in the gustatory system of mice. Because TRCs are not synaptically connected with the afferent nerve fibers, we first analyzed tracer production and transfer within the taste buds (TBs). Surprisingly, we found that BL is laterally transferred across all cell types in TBs of mice expressing the tracer under control of the endogenous Tas1r1 and Tas2r131 promotor, respectively. Furthermore, although we detected the BL tracer in both ganglia and brain, we also found local low-level Tas1r1 and Tas2r131 gene, and thus tracer expression in these tissues. Finally, we identified the Tas1r1 and Tas2r131-expressing cells in the peripheral and CNS using a binary genetic approach. Together, our data demonstrate that genetic transsynaptic tracing from bitter and umami receptor cells does not selectively label taste-specific neuronal circuits and reveal local taste receptor gene expression in the gustatory ganglia and the brain. © 2015 the authors.


Kumar D.,Saarland University | Candlish M.,Saarland University | Periasamy V.,Saarland University | Avcu N.,Institute for Neural Signal Transduction | And 2 more authors.
Endocrinology | Year: 2015

The neuropeptide kisspeptin is a potent stimulator of GnRH neurons and has been implicated as a major regulator of the hypothalamus-pituitary-gonadal axis. There are mainly two anatomically segregated populations of neurons that express kisspeptin in the female hypothalamus: one in the anteroventral periventricular nucleus (AVPV) and the other in the arcuate nucleus (ARC). Distinct roles have been proposed for AVPV and ARC kisspeptin neurons during reproductive maturation and in mediating estrogen feedback on the hypothalamus-pituitary-gonadal axis in adults. Despite their pivotal role in the regulation of reproductive physiology, little is known about kisspeptin neuron connectivity. Although previous data suggest heterogeneity within the AVPV and ARC kisspeptin neuron populations, how many and which of these potential kisspeptin neuron subpopulations are actually communicating with GnRH neurons is not known. Here we used a combinatorial genetic transsynaptic tracing strategy to start to analyze the connectivity of individual kisspeptin neurons with the GnRH neuron population in female mice with a single-cell resolution. We find that only subsets of AVPV and ARC kisspeptin neurons are synaptically connected with GnRH neurons.Wedemonstrate that the majority of kisspeptin neurons within the AVPV and ARC does not communicate with GnRH neurons. Furthermore, we show that all kisspeptin neurons within the AVPV connected to GnRH neurons are estrogen sensitive and that most of these express tyrosine hydroxylase. Our data demonstrate functional specialization within the two kisspeptin neuron populations. © 2015 by the Endocrine Society.


Sittl R.,Ludwig Maximilians University of Munich | Carr R.W.,Ludwig Maximilians University of Munich | Schwarz J.R.,Institute for Neural Signal Transduction | Grafe P.,Ludwig Maximilians University of Munich
Journal of the Peripheral Nervous System | Year: 2010

Flupirtine is an activator of Kv7 (KCNQ/M) potassium channels that has found clinical use as an analgesic with muscle relaxant properties. Kv7 potassium channels are expressed in axonal membranes and pharmacological activation of these channels may restore abnormal nerve excitability. We have examined the effect of flupirtine on the electrical excitability of myelinated axons in isolated segments of rat sural nerve. Axonal excitability was studied in vitro with the same parameters used by clinical neurophysiologists to assess peripheral nerve excitability in situ. Application of flupirtine in low micromolar concentrations resulted in an increase in threshold current, a reduction of refractoriness and an increase in post-spike superexcitability. These effects are consistent with an increase in Kv7 conductance and membrane hyperpolarization. Flupirtine also enhanced and prolonged the late, long-lasting period of axonal subexcitability that follows a short burst of action potentials. This effect was blocked by XE 991 (10 μM), an antagonist of Kv7 channels. In summary, flupirtine affects measures of excitability that are altered in the myelinated axons of patients with peripheral nerve disorders. This indicates that neuropathies with abnormal nerve excitability parameters corresponding to those affected by flupirtine may benefit from activation of axonal Kv7 potassium channels. © 2010 Peripheral Nerve Society.


Voigt A.,German Institute of Human Nutrition | Voigt A.,Institute for Neural Signal Transduction | Hubner S.,German Institute of Human Nutrition | Lossow K.,German Institute of Human Nutrition | And 4 more authors.
Chemical Senses | Year: 2012

Characterization of the peripheral taste system relies on the identification and visualization of the different taste bud cell types. So far, genetic strategies to label taste receptor cells are limited to sweet, sour, and salty detecting cells. To visualize Tas1r1 umami and Tas2r131 bitter sensing cells, we generated animals in which the Tas1r1 and Tas2r131 open reading frames are replaced by expression cassettes containing the fluorescent proteins mCherry or hrGFP, respectively. These animals enabled us to visualize and quantify the entire oral Tas1r1 and Tas2r131 cell populations. Tas1r1-mCherry cells were predominantly detected in fungiform papillae, whereas Tas2r131-hrGFP cells, which are ~4-fold more abundant, were mainly present in foliate and vallate papillae. In the palate, both cell types were similarly distributed. Mice carrying both recombinant alleles demonstrated completely segregated Tas1r1 and Tas2r131 cell populations. Only ~50% of the entire bitter cell population expressed hrGFP, indicating that bitter taste receptor cells express a subset of the bitter receptor repertoire. In extragustatory tissues, mCherry fluorescence was observed in testis and hrGFP fluorescence in testis, thymus, vomeronasal organ, and respiratory epithelium, suggesting that only few extraoral sites express Tas2r131 and Tas1r1 receptors at levels comparable to taste tissue. © The Author 2012. Published by Oxford University Press. All rights reserved.


Wen S.,Institute for Neural Signal Transduction | Ai W.,Institute for Neural Signal Transduction | Alim Z.,Institute for Neural Signal Transduction | Boehm U.,Institute for Neural Signal Transduction
Proceedings of the National Academy of Sciences of the United States of America | Year: 2010

Gonadotropin-releasing hormone (GnRH) signaling regulates reproductive physiology in mammals. GnRH is released by a subset of hypothalamic neurons and binds to GnRH receptor (GnRHR) on gonadotropes in the anterior pituitary gland to control production and secretion of gonadotropins that in turn regulate the activity of the gonads. Central control of reproduction is well understood in adult animals, but GnRH signaling has also been implicated in the development of the reproductive axis. To investigate the role of GnRH signaling during development, we selectively ablated GnRHR-expressing cells in mice. This genetic strategy permitted us to identify an essential stage in male reproductive axis development, which depends on embryonic GnRH signaling. Our experiments revealed a striking dichotomy in the gonadotrope population of the fetal anterior pituitary gland. We show that luteinizing hormone-expressing gonadotropes, but not follicle-stimulating hormone-expressing gonadotropes, express the GnRHR at embryonic day 16.75. Furthermore, we demonstrate that an embryonic increase in luteinizing hormone secretion is needed to promote development of follicle-stimulating hormone-expressing gonadotropes, which might be mediated by paracrine interactions within the pituitary. Moreover, migration of GnRH neurons into the hypothalamus appeared normal with appropriate axonal connections to the median eminence, providing genetic evidence against autocrine regulation of GnRH neurons. Surprisingly, genetic ablation of GnRHR expressing cells significantly increased the number of GnRH neurons in the anterior hypothalamus, suggesting an unexpected role of GnRH signaling in establishing the size of the GnRH neuronal population. Our experiments define a functional role of embryonic GnRH signaling.


Alim Z.,Institute for Neural Signal Transduction | Hartshorn C.,Colorado State University | Mai O.,Institute for Neural Signal Transduction | Mai O.,Saarland University | And 5 more authors.
Endocrinology | Year: 2012

Hormone-secreting cells within the anterior pituitary gland may form organized and interdigitated networks that adapt to changing endocrine conditions in different physiological contexts. For gonadotropes, this might reflect a strategy to cope with acute changes throughout different female reproductive stages. The current study examined gonadotropes in female mice at characteristically different hormonal stages: prepubertal, postpubertal, and lactating. Gonadotrope plasticity was examined at the level of the whole population and single cells at different stages by imaging both fixed and live pituitary slices. The use of a model animal providing for the identification of selectively fluorescent gonadotropes allowed the particular advantage of defining cellular plasticity specifically for gonadotropes. In vivo analyses of gonadotropes relative to vasculature showed significantly different gonadotrope distributions across physiological states. Video microscopy studies using live slices ex vivo demonstrated pituitary cell plasticity in the form of movements and protrusions in response to GnRH. As positive feedback from rising estradiol levels is important for priming the anterior pituitary gland for the LH surge, experiments provide evidence of estradiol effects on GnRH signaling in gonadotropes. The experiments presented herein provide new insight into potential plasticity of gonadotropes within the anterior pituitary glands of female mice. Copyright © 2012 by The Endocrine Society.


Kusuhara Y.,Kyushu University | Yoshida R.,Kyushu University | Ohkuri T.,Kyushu University | Yasumatsu K.,Kyushu University | And 7 more authors.
Journal of Physiology | Year: 2013

The T1R1 receptor subunit acts as an umami taste receptor in combination with its partner, T1R3. In addition, metabotropic glutamate receptors (brain and taste variants of mGluR1 and mGluR4) are thought to function as umami taste receptors. To elucidate the function of T1R1 and the contribution of mGluRs to umami taste detection in vivo, we used newly developed knock-out (T1R1-/-) mice, which lack the entire coding region of the Tas1r1 gene and express mCherry in T1R1-expressing cells. Gustatory nerve recordings demonstrated that T1R1-/- mice exhibited a serious deficit in inosine monophosphate-elicited synergy but substantial residual responses to glutamate alone in both chorda tympani and glossopharyngeal nerves. Interestingly, chorda tympani nerve responses to sweeteners were smaller in T1R1-/- mice. Taste cell recordings demonstrated that many mCherry-expressing taste cells in T1R1+/- mice responded to sweet and umami compounds, whereas those in T1R1-/- mice responded to sweet stimuli. The proportion of sweet-responsive cells was smaller in T1R1-/- than in T1R1+/- mice. Single-cell RT-PCR demonstrated that some single mCherry-expressing cells expressed all three T1R subunits. Chorda tympani and glossopharyngeal nerve responses to glutamate were significantly inhibited by addition of mGluR antagonists in both T1R1-/- and T1R1+/- mice. Conditioned taste aversion tests demonstrated that both T1R1-/- and T1R1+/- mice were equally capable of discriminating glutamate from other basic taste stimuli. Avoidance conditioned to glutamate was significantly reduced by addition of mGluR antagonists. These results suggest that T1R1-expressing cells mainly contribute to umami taste synergism and partly to sweet sensitivity and that mGluRs are involved in the detection of umami compounds. © 2013 The Authors. The Journal of Physiology © 2013 The Physiological Society.

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