Maternal hypothyroidism in mice influences glucose metabolism in adult offspring.

AIMS/HYPOTHESIS: During pregnancy, maternal metabolic disease and hormonal imbalance may alter fetal beta cell development and/or proliferation, thus leading to an increased risk for developing type 2 diabetes in adulthood. Although thyroid hormones play an important role in fetal endocrine pancreas development, the impact of maternal hypothyroidism on glucose homeostasis in adult offspring remains poorly understood. METHODS: We investigated this using a mouse model of hypothyroidism, induced by administration of an iodine-deficient diet supplemented with propylthiouracil during gestation. RESULTS: Here, we show that, when fed normal chow, adult mice born to hypothyroid mothers were more glucose-tolerant due to beta cell hyperproliferation (two- to threefold increase in Ki67-positive beta cells) and increased insulin sensitivity. However, following 8 weeks of high-fat feeding, these offspring gained 20% more body weight, became profoundly hyperinsulinaemic (with a 50% increase in fasting insulin concentration), insulin-resistant and glucose-intolerant compared with controls from euthyroid mothers. Furthermore, altered glucose metabolism was maintained in a second generation of animals. CONCLUSIONS/INTERPRETATION: Therefore, gestational hypothyroidism induces long-term alterations in endocrine pancreas function, which may have implications for type 2 diabetes prevention in affected individuals.

Diving into the brain: deep-brain imaging techniques in conscious animals.

In most species, survival relies on the hypothalamic control of endocrine axes that regulate critical functions such as reproduction, growth, and metabolism. For decades, the complexity and inaccessibility of the hypothalamic-pituitary axis has prevented researchers from elucidating the relationship between the activity of endocrine hypothalamic neurons and pituitary hormone secretion. Indeed, the study of central control of endocrine function has been largely dominated by 'traditional' techniques that consist of studying in vitro or ex vivo isolated cell types without taking into account the complexity of regulatory mechanisms at the level of the brain, pituitary and periphery. Nowadays, by exploiting modern neuronal transfection and imaging techniques, it is possible to study hypothalamic neuron activity in situ, in real time, and in conscious animals. Deep-brain imaging of calcium activity can be performed through gradient-index lenses that are chronically implanted and offer a 'window into the brain' to image multiple neurons at single-cell resolution. With this review, we aim to highlight deep-brain imaging techniques that enable the study of neuroendocrine neurons in awake animals whilst maintaining the integrity of regulatory loops between the brain, pituitary and peripheral glands. Furthermore, to assist researchers in setting up these techniques, we discuss the equipment required and include a practical step-by-step guide to performing these deep-brain imaging studies.

Elevated Prolactin during Pregnancy Drives a Phenotypic Switch in Mouse Hypothalamic Dopaminergic Neurons.

Altered physiological states require neuronal adaptation. In late pregnancy and lactation, a sub-population of the mouse hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons alters their behavior to synthesize and release met-enkephalin rather than dopamine. These neurons normally release dopamine to inhibit prolactin secretion and are activated by prolactin in a short-loop feedback manner. In lactation, dopamine synthesis is suppressed in an opioid-dependent (naloxone-reversible) manner, meaning that prolactin secretion is disinhibited. Conditional deletion of the prolactin receptor in neurons reveals that this change in phenotype appears to be driven by prolactin itself, apparently through an alteration in intracellular signaling downstream of the prolactin receptor that favors enkephalin production instead of dopamine. Thus, prolactin effectively facilitates its own secretion, which is essential for lactation and maternal behavior. These studies provide evidence of a physiologically important, reversible alteration in the behavior of a specific population of hypothalamic neurons in the adult brain.

The Processes of Anterior Pituitary Hormone Pulse Generation.

More than 60 years ago, Geoffrey Harris described his "neurohumoral theory," in which the regulation of pituitary hormone secretion was a "simple" hierarchal relationship, with the hypothalamus as the controller. In models based on this theory, the electrical activity of hypothalamic neurons determines the release of hypophysiotropic hormones into the portal circulation, and the pituitary simply responds with secretion of a pulse of hormone into the bloodstream. The development of methodologies allowing the monitoring of the activities of members of the hypothalamic-vascular-pituitary unit is increasingly allowing dissection of the mechanisms generating hypothalamic and pituitary pulses. These have revealed that whereas hypothalamic input is required, its role as a driver of pulsatile pituitary hormone secretion varies between pituitary axes. The organization of pituitary cells has a key role in the modification of their response to hypophysiotropic factors that can lead to a memory of previous demand and enhanced function. Feedback can lead to oscillatory hormone output that is independent of pulses of hypophysiotropic factors and instead, results from the temporal relationship between pituitary output and target organ response. Thus, the mechanisms underlying the generation of pulses cannot be generalized, and the circularity of feedforward and feedback interactions must be considered to understand both normal physiological function and pathology. We describe some examples of the clinical implications of recognizing the importance of the pituitary and target organs in pulse generation and suggest avenues for future research in both the short and long term.

Complementary actions of dopamine D2 receptor agonist and anti-vegf therapy on tumoral vessel normalization in a transgenic mouse model.

Angiogenesis contributes in multiple ways to disease progression in tumors and reduces treatment efficiency. Molecular therapies targeting Vegf signaling combined with chemotherapy or other drugs exhibit promising results to improve efficacy of treatment. Dopamine has been recently proposed to be a novel safe anti-angiogenic drug that stabilizes abnormal blood vessels and increases therapeutic efficacy. Here, we aimed to identify a treatment to normalize tumoral vessels and restore normal blood perfusion in tumor tissue with a Vegf receptor inhibitor and/or a ligand of dopamine G protein-coupled receptor D2 (D2R). Dopamine, via its action on D2R, is an endogenous effector of the pituitary gland, and we took advantage of this system to address this question. We have used a previously described Hmga2/T mouse model developing haemorrhagic prolactin-secreting adenomas. In mutant mice, blood vessels are profoundly altered in tumors, and an aberrant arterial vascularization develops leading to the loss of dopamine supply. D2R agonist treatment blocks tumor growth, induces regression of the aberrant blood supply and normalizes blood vessels. A chronic treatment is able to restore the altered balance between pro- and anti-angiogenic factors. Remarkably, an acute treatment induces an upregulation of the stabilizing factor Angiopoietin 1. An anti-Vegf therapy is also effective to restrain tumor growth and improves vascular remodeling. Importantly, only the combination treatment suppresses intratumoral hemorrhage and restores blood vessel perfusion, suggesting that it might represent an attractive therapy targeting tumor vasculature. Similar strategies targeting other ligands of GPCRs involved in angiogenesis may identify novel therapeutic opportunities for cancer.

Multiple-scale neuroendocrine signals connect brain and pituitary hormone rhythms.

Small assemblies of hypothalamic "parvocellular" neurons release their neuroendocrine signals at the median eminence (ME) to control long-lasting pituitary hormone rhythms essential for homeostasis. How such rapid hypothalamic neurotransmission leads to slowly evolving hormonal signals remains unknown. Here, we show that the temporal organization of dopamine (DA) release events in freely behaving animals relies on a set of characteristic features that are adapted to the dynamic dopaminergic control of pituitary prolactin secretion, a key reproductive hormone. First, locally generated DA release signals are organized over more than four orders of magnitude (0.001 Hz-10 Hz). Second, these DA events are finely tuned within and between frequency domains as building blocks that recur over days to weeks. Third, an integration time window is detected across the ME and consists of high-frequency DA discharges that are coordinated within the minutes range. Thus, a hierarchical combination of time-scaled neuroendocrine signals displays local-global integration to connect brain-pituitary rhythms and pace hormone secretion.

An updated view of hypothalamic-vascular-pituitary unit function and plasticity.

The discoveries of novel functional adaptations of the hypothalamus and anterior pituitary gland for physiological regulation have transformed our understanding of their interaction. The activity of a small proportion of hypothalamic neurons can control complex hormonal signalling, which is disconnected from a simple stimulus and the subsequent hormone secretion relationship and is dependent on physiological status. The interrelationship of the terminals of hypothalamic neurons and pituitary cells with the vasculature has an important role in determining the pattern of neurohormone exposure. Cells in the pituitary gland form networks with distinct organizational motifs that are related to the duration and pattern of output, and modifications of these networks occur in different physiological states, can persist after cessation of demand and result in enhanced function. Consequently, the hypothalamus and pituitary can no longer be considered as having a simple stratified relationship: with the vasculature they form a tripartite system, which must function in concert for appropriate hypothalamic regulation of physiological processes, such as reproduction. An improved understanding of the mechanisms underlying these regulatory features has implications for current and future therapies that correct defects in hypothalamic-pituitary axes. In addition, recapitulating proper network organization will be an important challenge for regenerative stem cell treatment.

Anatomical and functional gonadotrope networks in the teleost pituitary.

Mammalian pituitaries exhibit a high degree of intercellular coordination; this enables them to mount large-scale coordinated responses to various physiological stimuli. This type of communication has not been adequately demonstrated in teleost pituitaries, which exhibit direct hypothalamic innervation and expression of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in distinct cell types. We found that in two fish species, namely tilapia and zebrafish, LH cells exhibit close cell-cell contacts and form a continuous network throughout the gland. FSH cells were more loosely distributed but maintained some degree of cell-cell contact by virtue of cytoplasmic processes. These anatomical differences also manifest themselves at the functional level as evidenced by the effect of gap-junction uncouplers on gonadotropin release. These substances abolished the LH response to gonadotropin-releasing hormone stimulation but did not affect the FSH response to the same stimuli. Dye transfer between neighboring LH cells provides further evidence for functional coupling. The two gonadotropins were also found to be differently packaged within their corresponding cell types. Our findings highlight the evolutionary origin of pituitary cell networks and demonstrate how the different levels of cell-cell coordination within the LH and FSH cell populations are reflected in their distinct secretion patterns.

Somatostatin triggers rhythmic electrical firing in hypothalamic GHRH neurons.

Hypothalamic growth hormone-releasing hormone (GHRH) neurons orchestrate body growth/maturation and have been implicated in feeding responses and ageing. However, the electrical patterns that dictate GHRH neuron functions have remained elusive. Since the inhibitory neuropeptide somatostatin (SST) is considered to be a primary oscillator of the GH axis, we examined its acute effects on GHRH neurons in brain slices from male and female GHRH-GFP mice. At the cellular level, SST irregularly suppressed GHRH neuron electrical activity, leading to slow oscillations at the population level. This resulted from an initial inhibitory action at the GHRH neuron level via K(+) channel activation, followed by a delayed, sst1/sst2 receptor-dependent unbalancing of glutamatergic and GABAergic synaptic inputs. The oscillation patterns induced by SST were sexually dimorphic, and could be explained by differential actions of SST on both GABAergic and glutamatergic currents. Thus, a tripartite neuronal circuit involving a fast hyperpolarization and a dual regulation of synaptic inputs appeared sufficient in pacing the activity of the GHRH neuronal population. These "feed-forward loops" may represent basic building blocks involved in the regulation of GHRH release and its downstream sexual specific functions.

Assessment of lactotroph axis functionality in mice: longitudinal monitoring of PRL secretion by ultrasensitive-ELISA.

The pattern of prolactin (PRL) secretion depends on the physiological state. Due to insufficient detection sensitivity of existing assays, the precise description of these patterns in mice is lacking. We described an ultrasensitive ELISA assay that can detect mouse PRL in small fractions of whole blood, allowing longitudinal studies of PRL secretion profiles in freely moving mice. Over a 24-hour period, males displayed no oscillation in PRL levels, whereas virgin and lactating females showed large pulses. Peaks of PRL secretion reached 30-40 ng/mL in lactating female mice and rarely exceeded 10 ng/mL in virgin females. These pulses of PRL in lactating females were associated with suckling. The return of pups after an experimental 12-hour weaning induced a pulse of PRL release, reaching 100 ng/mL. This approach also enabled us to assess the inhibitory tone from hypothalamic dopamine neurons on PRL secretion. We used a dopamine D2 receptor antagonist to relieve pituitary lactotrophs from the tuberoinfundibular dopaminergic inhibitory tone and demonstrate a D2-induced PRL rise that can be used to evaluate both the secretory capacity of lactotrophs and the magnitude of the inhibitory tone on pituitary PRL release. We demonstrate that, although lactotroph function is altered to enhance chronic PRL output, their secretory response to acute stimulus is not modified during lactation and that chronic hyperprolactinemia is linked to a lower inhibitory tone. The combination of a sensitive PRL ELISA and administration of D2 receptor antagonist provide a unique opportunity to investigate the function and plasticity of the lactotroph axis in freely moving mice.

Sustained alterations of hypothalamic tanycytes during posttraumatic hypopituitarism in male mice.

Traumatic brain injury is a leading cause of hypopituitarism, which compromises patients' recovery, quality of life, and life span. To date, there are no means other than standardized animal studies to provide insights into the mechanisms of posttraumatic hypopituitarism. We have found that GH levels were impaired after inducing a controlled cortical impact (CCI) in mice. Furthermore, GHRH stimulation enhanced GH to lower level in injured than in control or sham mice. Because many characteristics were unchanged in the pituitary glands of CCI mice, we looked for changes at the hypothalamic level. Hypertrophied astrocytes were seen both within the arcuate nucleus and the median eminence, two pivotal structures of the GH axis, spatially remote to the injury site. In the arcuate nucleus, GHRH neurons were unaltered. In the median eminence, injured mice exhibited unexpected alterations. First, the distributions of claudin-1 and zonula occludens-1 between tanycytes were disorganized, suggesting tight junction disruptions. Second, endogenous IgG was increased in the vicinity of the third ventricle, suggesting abnormal barrier properties after CCI. Third, intracerebroventricular injection of a fluorescent-dextran derivative highly stained the hypothalamic parenchyma only after CCI, demonstrating an increased permeability of the third ventricle edges. This alteration of the third ventricle might jeopardize the communication between the hypothalamus and the pituitary gland. In conclusion, the phenotype of CCI mice had similarities to the posttraumatic hypopituitarism seen in humans with intact pituitary gland and pituitary stalk. It is the first report of a pathological status in which tanycyte dysfunctions appear as a major acquired syndrome.

Rapid sensing of circulating ghrelin by hypothalamic appetite-modifying neurons.

To maintain homeostasis, hypothalamic neurons in the arcuate nucleus must dynamically sense and integrate a multitude of peripheral signals. Blood-borne molecules must therefore be able to circumvent the tightly sealed vasculature of the blood-brain barrier to rapidly access their target neurons. However, how information encoded by circulating appetite-modifying hormones is conveyed to central hypothalamic neurons remains largely unexplored. Using in vivo multiphoton microscopy together with fluorescently labeled ligands, we demonstrate that circulating ghrelin, a versatile regulator of energy expenditure and feeding behavior, rapidly binds neurons in the vicinity of fenestrated capillaries, and that the number of labeled cell bodies varies with feeding status. Thus, by virtue of its vascular connections, the hypothalamus is able to directly sense peripheral signals, modifying energy status accordingly.

Influence of estrogens on GH-cell network dynamics in females: a live in situ imaging approach.

The secretion of endocrine hormones from pituitary cells finely regulates a multitude of homeostatic processes. To dynamically adapt to changing physiological status and environmental stimuli, the pituitary gland must undergo marked structural and functional plasticity. Endocrine cell plasticity is thought to primarily rely on variations in cell proliferation and size. However, cell motility, a process commonly observed in a variety of tissues during development, may represent an additional mechanism to promote plasticity within the adult pituitary gland. To investigate this, we used multiphoton time-lapse imaging methods, GH-enhanced green fluorescent protein transgenic mice and sexual dimorphism of the GH axis as a model of divergent tissue demand. Using these methods to acutely (12 h) track cell dynamics, we report that ovariectomy induces a dramatic and dynamic increase in cell motility, which is associated with gross GH-cell network remodeling. These changes can be prevented by estradiol supplementation and are associated with enhanced network connectivity as evidenced by increased coordinated GH-cell activity during multicellular calcium recordings. Furthermore, cell motility appears to be sex-specific, because reciprocal alterations are not detected in males after castration. Therefore, GH-cell motility appears to play an important role in the structural and functional pituitary plasticity, which is evoked in response to changing estradiol concentrations in the female.

The comparison between circadian oscillators in mouse liver and pituitary gland reveals different integration of feeding and light schedules.

The mammalian circadian system is composed of multiple peripheral clocks that are synchronized by a central pacemaker in the suprachiasmatic nuclei of the hypothalamus. This system keeps track of the external world rhythms through entrainment by various time cues, such as the light-dark cycle and the feeding schedule. Alterations of photoperiod and meal time modulate the phase coupling between central and peripheral oscillators. In this study, we used real-time quantitative PCR to assess circadian clock gene expression in the liver and pituitary gland from mice raised under various photoperiods, or under a temporal restricted feeding protocol. Our results revealed unexpected differences between both organs. Whereas the liver oscillator always tracked meal time, the pituitary circadian clockwork showed an intermediate response, in between entrainment by the light regimen and the feeding-fasting rhythm. The same composite response was also observed in the pituitary gland from adrenalectomized mice under daytime restricted feeding, suggesting that circulating glucocorticoids do not inhibit full entrainment of the pituitary clockwork by meal time. Altogether our results reveal further aspects in the complexity of phase entrainment in the circadian system, and suggest that the pituitary may host oscillators able to integrate multiple time cues.