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.

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.

Existence of long-lasting experience-dependent plasticity in endocrine cell networks.

Experience-dependent plasticity of cell and tissue function is critical for survival by allowing organisms to dynamically adjust physiological processes in response to changing or harsh environmental conditions. Despite the conferred evolutionary advantage, it remains unknown whether emergent experience-dependent properties are present in cell populations organized as networks within endocrine tissues involved in regulating body-wide homeostasis. Here we show, using lactation to repeatedly activate a specific endocrine cell network in situ in the mammalian pituitary, that templates of prior demand are permanently stored through stimulus-evoked alterations to the extent and strength of cell-cell connectivity. Strikingly, following repeat stimulation, evolved population behaviour leads to improved tissue output. As such, long-lasting experience-dependent plasticity is an important feature of endocrine cell networks and underlies functional adaptation of hormone release.

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.

Pituitary growth hormone network responses are sexually dimorphic and regulated by gonadal steroids in adulthood.

There are well-recognized sex differences in many pituitary endocrine axes, usually thought to be generated by gonadal steroid imprinting of the neuroendocrine hypothalamus. However, the recognition that growth hormone (GH) cells are arranged in functionally organized networks raises the possibility that the responses of the network are different in males and females. We studied this by directly monitoring the calcium responses to an identical GH-releasing hormone (GHRH) stimulus in populations of individual GH cells in slices taken from male and female murine GH-eGFP pituitary glands. We found that the GH cell network responses are sexually dimorphic, with a higher proportion of responding cells in males than in females, correlated with greater GH release from male slices. Repetitive waves of calcium spiking activity were triggered by GHRH in some males, but were never observed in females. This was not due to a permanent difference in the network architecture between male and female mice; rather, the sex difference in the proportions of GH cells responding to GHRH were switched by postpubertal gonadectomy and reversed with hormone replacements, suggesting that the network responses are dynamically regulated in adulthood by gonadal steroids. Thus, the pituitary gland contributes to the sexually dimorphic patterns of GH secretion that play an important role in differences in growth and metabolism between the sexes.

Cellular in vivo imaging reveals coordinated regulation of pituitary microcirculation and GH cell network function.

Growth hormone (GH) exerts its actions via coordinated pulsatile secretion from a GH cell network into the bloodstream. Practically nothing is known about how the network receives its inputs in vivo and releases hormones into pituitary capillaries to shape GH pulses. Here we have developed in vivo approaches to measure local blood flow, oxygen partial pressure, and cell activity at single-cell resolution in mouse pituitary glands in situ. When secretagogue (GHRH) distribution was modeled with fluorescent markers injected into either the bloodstream or the nearby intercapillary space, a restricted distribution gradient evolved within the pituitary parenchyma. Injection of GHRH led to stimulation of both GH cell network activities and GH secretion, which was temporally associated with increases in blood flow rates and oxygen supply by capillaries, as well as oxygen consumption. Moreover, we observed a time-limiting step for hormone output at the perivascular level; macromolecules injected into the extracellular parenchyma moved rapidly to the perivascular space, but were then cleared more slowly in a size-dependent manner into capillary blood. Our findings suggest that GH pulse generation is not simply a GH cell network response, but is shaped by a tissue microenvironment context involving a functional association between the GH cell network activity and fluid microcirculation.

Ghrelin stimulation of growth hormone-releasing hormone neurons is direct in the arcuate nucleus.

BACKGROUND: Ghrelin targets the arcuate nucleus, from where growth hormone releasing hormone (GHRH) neurones trigger GH secretion. This hypothalamic nucleus also contains neuropeptide Y (NPY) neurons which play a master role in the effect of ghrelin on feeding. Interestingly, connections between NPY and GHRH neurons have been reported, leading to the hypothesis that the GH axis and the feeding circuits might be co-regulated by ghrelin. PRINCIPAL FINDINGS: Here, we show that ghrelin stimulates the firing rate of identified GHRH neurons, in transgenic GHRH-GFP mice. This stimulation is prevented by growth hormone secretagogue receptor-1 antagonism as well as by U-73122, a phospholipase C inhibitor and by calcium channels blockers. The effect of ghrelin does not require synaptic transmission, as it is not antagonized by gamma-aminobutyric acid, glutamate and NPY receptor antagonists. In addition, this hypothalamic effect of ghrelin is independent of somatostatin, the inhibitor of the GH axis, since it is also found in somatostatin knockout mice. Indeed, ghrelin does not modify synaptic currents of GHRH neurons. However, ghrelin exerts a strong and direct depolarizing effect on GHRH neurons, which supports their increased firing rate. CONCLUSION: Thus, GHRH neurons are a specific target for ghrelin within the brain, and not activated secondary to altered activity in feeding circuits. These results support the view that ghrelin related therapeutic approaches could be directed separately towards GH deficiency or feeding disorders.

Different degrees of somatotroph ablation compromise pituitary growth hormone cell network structure and other pituitary endocrine cell types.

We have generated transgenic mice with somatotroph-specific expression of a modified influenza virus ion channel, (H37A)M2, leading to ablation of GH cells with three levels of severity, dependent on transgene copy number. GH-M2(low) mice grow normally and have normal-size pituitaries but 40-50% reduction in pituitary GH content in adult animals. GH-M2(med) mice have male-specific transient growth retardation and a reduction in pituitary GH content by 75% at 42 d and 97% by 100 d. GH-M2(high) mice are severely dwarfed with undetectable pituitary GH. The GH secretory response of GH-M2(low) and GH-M2(med) mice to GH-releasing peptide-6 and GHRH was markedly attenuated. The content of other pituitary hormones was affected depending on transgene copy number: no effect in GH-M2(low) mice, prolactin and TSH reduced in GH-M2(med) mice, and all hormones reduced in GH-M2(high) mice. The effect on non-GH hormone content was associated with increased macrophage invasion of the pituitary. Somatotroph ablation affected GH cell network organization with limited disruption in GH-M2(low) mice but more severe disruption in GH-M2(med) mice. The remaining somatotrophs formed tight clusters after puberty, which contrasts with GHRH-M2 mice with a secondary reduction in somatotrophs that do not form clusters. A reduction in pituitary beta-catenin staining was correlated with GH-M2 transgene copy number, suggesting M2 expression has an effect on cell-cell communication in somatotrophs and other pituitary cell types. GH-M2 transgenic mice demonstrate that differing degrees of somatotroph ablation lead to correlated secondary effects on cell populations and cellular network organization.

Ribosomal protein S6 kinase 1 signaling regulates mammalian life span.

Caloric restriction (CR) protects against aging and disease, but the mechanisms by which this affects mammalian life span are unclear. We show in mice that deletion of ribosomal S6 protein kinase 1 (S6K1), a component of the nutrient-responsive mTOR (mammalian target of rapamycin) signaling pathway, led to increased life span and resistance to age-related pathologies, such as bone, immune, and motor dysfunction and loss of insulin sensitivity. Deletion of S6K1 induced gene expression patterns similar to those seen in CR or with pharmacological activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a conserved regulator of the metabolic response to CR. Our results demonstrate that S6K1 influences healthy mammalian life-span and suggest that therapeutic manipulation of S6K1 and AMPK might mimic CR and could provide broad protection against diseases of aging.

The circadian clock components CRY1 and CRY2 are necessary to sustain sex dimorphism in mouse liver metabolism.

In mammals, males and females exhibit anatomical, hormonal, and metabolic differences. A major example of such sex dimorphism in mouse involves hepatic drug metabolism, which is also a noticeable target of circadian timekeeping. However, whether the circadian clock itself contributes to sex-biased metabolism has remained unknown, although several daily output parameters differ between sexes in a number of species, including humans. Here we show that dimorphic liver metabolism is altered when the circadian regulators Cryptochromes, Cry1 and Cry2, are inactivated. Indeed, double mutant Cry1(-/-) Cry2(-/-) male mice that lack a functional circadian clock express a number of sex-specific liver products, including several cytochrome P450 enzymes, at levels close to those measured in females. In addition, body growth of Cry-deficient mice is impaired, also in a sex-biased manner, and this phenotype goes along with an altered pattern of circulating growth hormone (GH) in mutant males, specifically. It is noteworthy that hormonal injections able to mimic male GH pulses reversed the feminized gene expression profile in the liver of Cry1(-/-) Cry2(-/-) males. Altogether, our observations suggest that the 24-h clock paces the dimorphic ultradian pulsatility of GH that is responsible for sex-dependent liver activity. We thus conclude that circadian timing, sex dimorphism, and liver metabolism are finely interconnected.

Somatostatin secreted by islet delta-cells fulfills multiple roles as a paracrine regulator of islet function.

OBJECTIVE: Somatostatin (SST) is secreted by islet delta-cells and by extraislet neuroendocrine cells. SST receptors have been identified on alpha- and beta-cells, and exogenous SST inhibits insulin and glucagon secretion, consistent with a role for SST in regulating alpha- and beta-cell function. However, the specific intraislet function of delta-cell SST remains uncertain. We have used Sst(-/-) mice to investigate the role of delta-cell SST in the regulation of insulin and glucagon secretion in vitro and in vivo. RESEARCH DESIGN AND METHODS: Islet morphology was assessed by histological analysis. Hormone levels were measured by radioimmunoassay in control and Sst(-/-) mice in vivo and from isolated islets in vitro. RESULTS: Islet size and organization did not differ between Sst(-/-) and control islets, nor did islet glucagon or insulin content. Sst(-/-) mice showed enhanced insulin and glucagon secretory responses in vivo. In vitro stimulus-induced insulin and glucagon secretion was enhanced from perifused Sst(-/-) islets compared with control islets and was inhibited by exogenous SST in Sst(-/-) but not control islets. No difference in the switch-off rate of glucose-stimulated insulin secretion was observed between genotypes, but the cholinergic agonist carbamylcholine enhanced glucose-induced insulin secretion to a lesser extent in Sst(-/-) islets compared with controls. Glucose suppressed glucagon secretion from control but not Sst(-/-) islets. CONCLUSIONS: We suggest that delta-cell SST exerts a tonic inhibitory influence on insulin and glucagon secretion, which may facilitate the islet response to cholinergic activation. In addition, delta-cell SST is implicated in the nutrient-induced suppression of glucagon secretion.

Evidence for lifespan extension and delayed age-related biomarkers in insulin receptor substrate 1 null mice.

Recent evidence suggests that alterations in insulin/insulin-like growth factor 1 (IGF1) signaling (IIS) can increase mammalian life span. For example, in several mouse mutants, impairment of the growth hormone (GH)/IGF1 axis increases life span and also insulin sensitivity. However, the intracellular signaling route to altered mammalian aging remains unclear. We therefore measured the life span of mice lacking either insulin receptor substrate (IRS) 1 or 2, the major intracellular effectors of the IIS receptors. Our provisional results indicate that female Irs1-/- mice are long-lived. Furthermore, they displayed resistance to a range of age-sensitive markers of aging including skin, bone, immune, and motor dysfunction. These improvements in health were seen despite mild, lifelong insulin resistance. Thus, enhanced insulin sensitivity is not a prerequisite for IIS mutant longevity. Irs1-/- female mice also displayed normal anterior pituitary function, distinguishing them from long-lived somatotrophic axis mutants. In contrast, Irs2-/- mice were short-lived, whereas Irs1+/- and Irs2+/- mice of both sexes showed normal life spans. Our results therefore suggest that IRS1 signaling is an evolutionarily conserved pathway regulating mammalian life span and may be a point of intervention for therapies with the potential to delay age-related processes.

Selective alteration at the growth-hormone- releasing-hormone nerve terminals during aging in GHRH-green fluorescent protein mice.

Growth hormone (GH) secretion decreases spontaneously during lifespan, and the resulting GH deficiency participates in aging-related morbidity. This deficiency appears to involve a defect in the activity of hypothalamic GH-releasing hormone (GHRH) neurons. Here, we investigated this hypothesis, as well as the underlying mechanisms, in identified GHRH neurons from adult ( approximately 13 weeks old) and aged ( approximately 100 weeks old) transgenic GHRH-green fluorescent protein mice, using morphological, biochemical and electrophysiological methods. Surprisingly, the spontaneous action potential frequency was similar in adult and aged GHRH neurons studied in brain slices. This was explained by a lack of change in the intrinsic excitability, and simultaneous increases in both stimulatory glutamatergic- and inhibitory GABAergic-synaptic currents of aged GHRH neurons. Aging did not decrease GHRH and enhanced green fluorescent protein contents, GHRH neuronal number or GHRH-fibre distribution, but we found a striking enlargement of GHRH-positive axons, suggesting neuropeptide accumulation. Unlike in adults, autophagic vacuoles were evident in aged GHRH-axonal profiles using electron microscopy. Thus, GHRH neurons are involved in aging of the GH axis. Aging had a subtle effect at the nerve terminal level in GHRH neurons, contrasting with the view that neuronal aging is accompanied by more widespread damage.

Mutations within Sox2/SOX2 are associated with abnormalities in the hypothalamo-pituitary-gonadal axis in mice and humans.

The transcription factor SOX2 is expressed most notably in the developing CNS and placodes, where it plays critical roles in embryogenesis. Heterozygous de novo mutations in SOX2 have previously been associated with bilateral anophthalmia/microphthalmia, developmental delay, short stature, and male genital tract abnormalities. Here we investigated the role of Sox2 in murine pituitary development. Mice heterozygous for a targeted disruption of Sox2 did not manifest eye defects, but showed abnormal anterior pituitary development with reduced levels of growth hormone, luteinizing hormone, and thyroid-stimulating hormone. Consequently, we identified 8 individuals (from a cohort of 235 patients) with heterozygous sequence variations in SOX2. Six of these were de novo mutations, predicted to result in truncated protein products, that exhibited partial or complete loss of function (DNA binding, nuclear translocation, or transactivation). Clinical evaluation revealed that, in addition to bilateral eye defects, SOX2 mutations were associated with anterior pituitary hypoplasia and hypogonadotropic hypogonadism, variable defects affecting the corpus callosum and mesial temporal structures, hypothalamic hamartoma, sensorineural hearing loss, and esophageal atresia. Our data show that SOX2 is necessary for the normal development and function of the hypothalamo-pituitary and reproductive axes in both humans and mice.

Revealing the large-scale network organization of growth hormone-secreting cells.

Pituitary growth hormone (GH)-secreting cells regulate growth and metabolism in animals and humans. To secrete highly ordered GH pulses (up to 1,000-fold rise in hormone levels in vivo), the pituitary GH cell population needs to mount coordinated responses to GH secretagogues, yet GH cells display an apparently heterogeneous scattered distribution in 2D histological studies. To address this paradox, we analyzed in 3D both positioning and signaling of GH cells using reconstructive, two-photon excitation microscopy to image the entire pituitary in GH-EGFP transgenic mice. Our results unveiled a homologous continuum of GH cells connected by adherens junctions that wired the whole gland and exhibited the three primary features of biological networks: robustness of architecture across lifespan, modularity correlated with pituitary GH contents and body growth, and connectivity with spatially stereotyped motifs of cell synchronization coordinating cell activity. These findings change our view of GH cells, from a collection of dispersed cells to a geometrically connected homotypic network of cells whose local morphology and connectivity can vary, to alter the timing of cellular responses to promote more coordinated pulsatile secretion. This large-scale 3D view of cell functioning provides a powerful approach to identify and understand other networks of endocrine cells that are thought to be scattered in situ. Many dispersed endocrine systems exhibit pulsatile outputs. We suggest that cell positioning and associated cell-cell connection mechanisms will be critical parameters that determine how well such systems can deliver a coordinated secretory pulse of hormone to their target tissues.

Hypothalamic growth hormone-releasing hormone (GHRH) deficiency: targeted ablation of GHRH neurons in mice using a viral ion channel transgene.

Animal and clinical models of GHRH excess suggest that GHRH provides an important trophic drive to pituitary somatotrophs. We have adopted a novel approach to silence or ablate GHRH neurons, using a modified H37A variant of the influenza virus M2 protein ((H37A)M2). In mammalian cells, (H37A)M2 forms a high conductance monovalent cation channel that can be blocked by the antiviral drug rimantadine. Transgenic mice with (H37A)M2 expression targeted to GHRH neurons developed postweaning dwarfism with hypothalamic GHRH transcripts detectable by RT-PCR but not by in situ hybridization and immunocytochemistry, suggesting that expression of (H37A)M2 had silenced or ablated virtually all the GHRH cells. GHRH-M2 mice showed marked anterior pituitary hypoplasia with GH deficiency, although GH cells were still present. GHRH-M2 mice were also deficient in prolactin but not TSH. Acute iv injections of GHRH in GHRH-M2 mice elicited a significant GH response, whereas injections of GHRP-6 did not. Twice daily injections of GHRH (100 microg/d) for 7 d in GHRH-M2 mice doubled their pituitary GH but not PRL contents. Rimantadine treatment failed to restore growth or pituitary GH contents. Our results show the importance of GHRH neurons for GH and prolactin production and normal growth.

Factors affecting the susceptibility of the mouse pituitary gland to CD8 T-cell-mediated autoimmunity.

We have previously shown, in a transgenic mouse model, that the pituitary gland is susceptible to CD8 T-cell-mediated autoimmunity, triggered by a cell-specific model autoantigen, resulting in pan-anterior pituitary hypophysitis and dwarfism. In the present study, we now demonstrate that antigen dose, the T-cell precursor frequency, the degree of lymphopenia and the context of target antigen expression, are important parameters determining the time course and extent of the pathological consequences of CD8 T-cell-mediated autoimmunity. Furthermore, our data indicate that the pituitary gland is susceptible to CD8 autoimmunity following an inflammatory insult such as a viral infection. As lymphocytic hypophysitis may be manifest in other autoimmune conditions, and the pituitary gland may be susceptible to T-cell-mediated pathology after immunization with a virus expressing soluble pituitary antigen, it is important to consider that strategies based on vaccination against soluble pituitary gonadotrophins could have other unexpected endocrine consequences.

SOX3 is required during the formation of the hypothalamo-pituitary axis.

The pituitary develops from the interaction of the infundibulum, a region of the ventral diencephalon, and Rathke's pouch, a derivative of oral ectoderm. Postnatally, its secretory functions are controlled by hypothalamic neurons, which also derive from the ventral diencephalon. In humans, mutations affecting the X-linked transcription factor SOX3 are associated with hypopituitarism and mental retardation, but nothing is known of their etiology. We find that deletion of Sox3 in mice leads to defects of pituitary function and of specific central nervous system (CNS) midline structures. Cells in the ventral diencephalon, where Sox3 is usually highly expressed, have altered properties in mutant embryos, leading to abnormal development of Rathke's pouch, which does not express the gene. Pituitary and hypothalamic defects persist postnatally, and SOX3 may also function in a subset of hypothalamic neurons. This study shows how sensitive the pituitary is to subtle developmental defects and how one gene can act at several levels in the hypothalamic-pituitary axis.

Physiological studies of transgenic mice overexpressing growth hormone (GH) secretagogue receptor 1A in GH-releasing hormone neurons.

The type 1A GH secretagogue (GHS) receptor (GHSR) has been proposed to mediate the effects of ghrelin on GH release, food intake, and body composition. We have overexpressed GHSR in GH-producing GC cells and GHRH neurons in an attempt to enhance signaling via this pathway selectively, in the GH axis. Constitutive overexpression of human GHSR in rat GC cell lines resulted in increased basal phosphoinositol turnover and rendered them responsive to GHS ligands. We then generated transgenic mice overexpressing human GHSR in GHRH neurons using a 38-kb rat GHRH cosmid promoter. GHRH-GHSR transgenic mice showed increased hypothalamic GHRH expression, pituitary GH contents, and postweaning growth rates. Body weights of the transgenic mice became similar in adulthood, whereas adipose mass was reduced, particularly so in female GHRH-GHSR mice. Organ and muscle weights of transgenic mice were increased despite chronic exposure to a high fat diet. These results suggest that constitutive overexpression of GHSR in GHRH neurons up-regulates basal activity in the GHRH-GH axis. However, GHRH-GHSR mice showed no evidence of increased sensitivity to acute or chronic treatment with exogenous GHS ligands. Food intake and adipose tissue responses to chronic high fat feeding and treatment with GHS ligands were unaffected, as were locomotor and anxiety behaviors, although GHRH-GHSR mice remained significantly leaner than wild-type littermates. Thus, constitutive overexpression of GHSR can up-regulate basal signaling activity in the GHRH/GH axis and reduce adiposity without affecting other GHSR-mediated signals.