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.hormone diffusion | in vivo imaging | median eminence | metabolism C ontinuous integration of peripheral signals by neurons belonging to the arcuate nucleus of the hypothalamus (ARH) is critical for central regulation of energy balance and neuroendocrine function (1). To dynamically report alterations to homeostasis and ensure an appropriate neuronal response, blood-borne factors such as hormones must rapidly access the central nervous system (CNS). This is particularly evident in the case of food intake, which is regulated by a plethora of circulating satiety signals (2) whose levels fluctuate in an ultradian manner. Despite this, it remains unclear how key energy status-signaling hormones such as ghrelin can be rapidly sensed by target neurons to alter feeding responses (3). Elucidation of the mechanisms underlying molecule entry into the brain is important for understanding not only normal maintenance of homeostasis but also how this is perturbed during common pathologies such as obesity and diabetes (4, 5).Although molecule transport mechanisms within the ARH are poorly characterized, they likely assume one of two forms. First, chronic feedback may be accomplished by uptake of circulating molecules into the ARH via saturable receptor-mediated transport at the level of the choroid plexus and/or bloodbrain barrier (BBB) (6-9). Second, the ARH is morphologically located in close apposition to the median eminence (ME), a circumventricular organ composed of fenestrated capillaries. Because these vessels project toward the ventromedial ARH (vmARH), they could represent a direct vascular input for passive diffusion of peripheral molecules into the hypothalamus (10-13). So far, study of the functional importance of fenestrated capillaries in molecule entry into the metabolic brain has been impeded by lack of appropriate tools.To evaluate the role of fenestrated ME/ARH capillaries in rapid detection of peripheral signals by the hypothalamus, we used a recently developed in vivo imaging approach to visualize in real time the extravasation of fluorescent molecules (14). Ghrelin was chosen as a candidate hormone because i...

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.

Both endocrine and non-endocrine cells of the pituitary gland are organized into structural and functional networks which are formed during embryonic development but which may be modified throughout life. Structural mapping of the various endocrine cell types has highlighted the existence of distinct network motifs and relationships with the vasculature which may relate to temporal differences in their output. Functional characterization of the network activity of growth hormone and prolactin cells has revealed a role for cell organization in gene regulation, the plasticity of pituitary hormone output and remarkably the ability to memorize altered demand. As such, the description of these endocrine cell networks alters the concept of the pituitary from a gland which simply responds to external regulation to that of an oscillator which may memorize information and constantly adapt its coordinated networks' responses to the flow of hypothalamic inputs.

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The pituitary gland has long been considered to be a random patchwork of hormone-producing cells. By using pituitary-scale tridimensional imaging for two of the least abundant cell lineages, the corticotropes and gonadotropes, we have now uncovered highly organized and interdigitated cell networks that reflect homotypic and heterotypic interactions between cells. Although newly differentiated corticotrope cells appear on the ventral surface of the gland, they rapidly form homotypic strands of cells that extend from the lateral tips of the anterior pituitary along its ventral surface and into the medial gland. As the corticotrope network is established away from the microvasculature, cell morphology changes from rounded, to polygonal, and finally to cells with long cytoplasmic processes or cytonemes that connect corticotropes to the perivascular space. Gonadotropes differentiate later and are positioned in close proximity to corticotropes and capillaries. Blockade of corticotrope terminal differentiation produced by knockout of the gene encoding the transcription factor Tpit results in smaller gonadotropes within an expanded cell network, particularly in the lateral gland. Thus, pituitary-scale tridimensional imaging reveals highly structured cell networks of unique topology for each pituitary lineage. The sequential development of interdigitated cell networks during organogenesis indicate that extensive cell:cell interactions lead to a highly ordered cell positioning rather than random patchwork.reproduction | stress | systems biology | pro-opiomelanocortin

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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

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.blood flow | hormone pulsatility | oxygen pressure | tissue microenvironment | extracellular space