An Updated View of Hypothalamic–Vascular–Pituitary Unit Function and Plasticity

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An updated view of hypothalamic– vascular–pituitary unit function and plasticity

Paul Le Tissier1, Pauline Campos2–4, Chrystel Lafont2–4, Nicola Romanò1, David J. Hodson5,6 and Patrice Mollard2–4 Abstract | 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.

To maximize reproductive success through the appro- increased output will recur. A mechanistic understanding priate timing of ovulation, lactation or body growth, of these alterations in hypothalamic–pituitary function the outputs of several hypothalamic–pituitary axes are is fundamental to interpret and treat defects that lead to dramatically altered. These adaptive changes occur over endocrine diseases resulting from hormone deficiencies differing timescales, with varying frequencies and lev- (for example, dwarfism) or excess (such as polycystic els of predictability. For example, the occurrence of the ovary syndrome (PCOS)). In this Review, we focus on increase in growth hormone (GH) output at puberty is three pituitary axes that have roles in driving changes in largely predictable. On a relatively short timescale (days), physiology: the gonadatropin, prolactin and GH axes. the surge in luteinizing hormone (LH) secretion that is The level of our understanding varies for each of these required for oestrus is an acute change that occurs regu- axes, and the features that might serve as general prin- larly once every reproductive cycle and, in humans, con- ciples of hypothalamic–pituitary and other endocrine tinues for years in the absence of pregnancy. On a longer organ function are highlighted in the text. timescale (months to years, depending on the species), the increase in prolactin that is required for lactation is Beyond stimulus–secretion coupling maintained for a variable time (which depends on when The path of communication between the brain and offspring are weaned) and recurs at each pregnancy, but peripheral tissues is exemplified by muscle contraction, Correspondence to P.L.T. and P.M. is unpredictable before gestation. These large changes in which requ res the transfer of electrical signals from [email protected]; the output of the various pituitary axes require modifi- axons via the neuromuscular junction. This sequence [email protected] cation of both hypothalamic and pituitary function, but of events, which is known as excitation–contraction 1 doi:10.1038/nrendo.2016.193 whether this effect is reversed on cessation of physio- coupling , takes less than 1 second in mammals and Published online 9 Dec 2016 logical demand is likely to reflect the expectation that adapts to altered functional demand. Similarly, in the

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Key points of pituitary function (FIG. 1). Specifically, the excitation of specific hypothalamic neuron populations, determined by • The activity of hypothalamic neurons is modified by inputs leading to higher brain centres and peripheral feedback, is relayed as heterogeneous activity; a small proportion of the total population can drive an unmodified series of signals to drive balanced pituitary pituitary hormone pulsatility hormone output11. The release of neurohormones and the • Neurohormone output can vary following neuron excitation according to the subsequent transportation, as well as the effects on target physiological status, which might also lead to declining neuroendocrine output cells, were considered to be passive events in the regula- with age tion of pituitary hormone secretion, with only variation • The release of hypothalamic factors into the blood is modified by alterations in the in the number of endocrine cells seeming to affect the juxtaposition of nerve terminals with the vasculature and tanycytes in the median 11,14 eminence response levels . Similarly, alterations in gene expression and cell proliferation, which support maintenance • Cells in the pituitary gland form homotypic networks, and the organization and relationship of a network with the vasculature is distinct for each endocrine axis, of hormone output, were simply considered a correlated 15 which modifies responses to regulatory factors and patterns of output in response response to hypothalamic regulation of secretion . to demand In the early twenty-first century, a series of paradigm • The reorganisation of the pituitary network can store long-term memories of shifts in our understanding of the hypothalamic–pituitary increased output and enhance function on repeated challenge system was established as a consequence of newly devel- • Understanding the importance of coordinated hypothalamic–vasculature–pituitary oped tools and techniques, including calcium imaging, function provides new understanding of a range of endocrine axes defects and fluorescent protein identification of specific cell types and targets for novel therapies two-photon microscopy, for use in genetically modified mice (for examples, see REFS 16–18). The use of these methods have shown that both the pituitary gland and the portal system can no longer be considered as static struc- Author addresses tures simply responding to neurohormonal regulation 1Centre for Integrative Physiology, University of Edinburgh, George Square, Edinburgh, (BOX 1), although they have confirmed the pre-eminence EH8 9XD, UK. of the hypothalamus as the driver of pituitary hormone 2Centre National de la Recherche Scientifique (CNRS), UMR‑5203, Institut de output. In addition, hypothalamic neuron function has Génomique Fonctionnelle, rue de la Cardonille, F-34000 Montpellier, France. been found to be more dynamic than initially thought, 3 INSERM, U661, rue de la Cardonille, F-34000 Montpellier, France. which might contribute to modifications in its regulation 4 Université de Montpellier, rue de la Cardonille, UMR‑5203, F-34000 Montpellier, France. of the pituitary under different physiological states2 (FIG. 1). 5Institute of Metabolism and Systems Research and Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham, Edgbaston, B15 2TT, UK. Gonadotroph axis 6Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners, Birmingham, B15 2TH, UK. The reproductive system is crucially dependent on pulsatile secretion of gonadotropin-releasing hormone (GnRH) and LH; however, the understanding of pulse hypothalamus, just a few thousand neurons can also send generation has been hampered by the complexity of the signals to the periphery, in this case, towards the median regulatory mechanisms, many of which will clearly be eminence (ME) via a specialized neurohaemal junction. lost in in vitro preparations. In the late 1980s, investi- In this junction, nerve terminal depolarization originat- gators using pituitary portal bleeding and microdialysis ing either from the perikarya2 or the terminals them- documented the pulsatile nature of GnRH release into selves3 enables the sufficiently rapid entry of calcium the portal vasculature of sheep, monkeys and rats19–25, ions to trigger exocytosis of neurohormones towards the and showed a strong correlation between GnRH and first loop of the portal fenestrated capillaries4,5. This rapid LH pulses22,26,27. However, the scattered distribution (<1 s) sequence of events was termed ‘stimulus–secretion and the relative paucity of GnRH cell bodies limited coupling’ owing to the clear similarities to excitation– the investigation of the cellular events that lead to pul- contraction coupling5,6. Soon after release, neurohor- satile secretion of LH in vivo. In the past few years, the mones pervade the second loop of fenestrated capillaries development of optogenetic techniques in rats and mice within the pituitary gland, before binding to cognate and of an ultrasensitive ELISA capable of measuring receptors on endocrine cells to induce pituitary hor- LH levels in whole-blood microsamples28 has allowed mone exocytosis through a second ‘stimulus–secretion the investigators to dissect the GnRH neuron excitation coupling’ event7–10 (FIG. 1). parameters that generate LH pulses29. In these studies, In the second half of the twentieth century (and the stimulation of just 60 GnRH neurons (~5% of the building on Harris’ work on the hypothalamus–pituitary total GnRH neurons) can trigger short-lived increases axis11), the analogy between excitation–contraction and in LH secretion that resemble endogenous pulses29. stimulus–secretion coupling was developed further12. Given the crucial importance of GnRH neurons in the However, important and fundamental differences exist survival of all mammalian species, a degree of functional between the two processes. Specifically, in the hypothala- redundancy within this cell population is expected. This mus, endogenous rhythms with axis-dependent frequen- finding is consistent with studies showing that just 10% cies exist, and the timescale for pituitary hormone release of the GnRH neuron population is sufficient to main- is measured in minutes to several hours13. Nevertheless, tain pulsatile LH secretion30,31. Varying the timing and the analogy with neural control of locomotor activity led frequency of stimulation has demonstrated that a brief to a generally accepted model of hypothalamic regulation (2 min) optogenetic stimulation at high frequency

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Hypothalamic neuron firing a Stimulus–secretion coupling b Adaptive stimulus–secretion coupling Hypothalamic Different patterns with different efficiency Complex changes in different physiological states Hypothalamus neuron

Burst firing is more efficient for Efficiency of neurohormone output at ME is not only neurohormone output at ME function of the firing pattern Neurohormone output at ME Pituitary gland Portal vessel Hormone secretion parallels Hormone secretion might not strictly follow Pituitary hypothalamic rhythms hypothalamic rhythms hormone output in bloodstream

Figure 1 | A dynamic interplay between hypothalamic neuron output, the vasculatureNature and the Reviews pituitary | Endocrinology response alters hormone output. a | The concept of stimulus–secretion coupling considered pituitary hormone output to result from a cascade of events that are dictated by the pattern of regulatory neuron excitation. b | Recent studies have shown that simple stimulus–secretion coupling is too simplistic to be responsible for the changes in hypothalamic–pituitary output. Alterations at the level of neuron response to excitation, release of hypothalamic factors in the median eminence (ME) and pituitary cell responses are all capable of adapting output of hormone to the periphery.

(10 Hz) evokes an LH pulse, whereas shorter periods a pulsatile and cyclic pattern of secretion38, and inhibi- and lower frequencies cannot elicit an LH output that tion of nitric oxide synthesis within the ME can disrupt resembles endogenous pulses29. A bursting pattern of reproductive cyclicity39. Conversely, in the GnRH neu- stimulation, which had been assumed to be most effec- ron perikarya, basal nitric oxide synthase activity might tive for pulse generation and which had been the focus provide the tonic inhibition of the GnRH neural system of many previous studies32, also failed to increase LH that is required to maintain nadir levels of LH40. secretion. Whether such a stimulatory signal exists Once GnRH is released into the ME, its transport to endogenously in vivo and where its origin might be is not the pituitary and the pattern of gonadotroph exposure known, although a ‘GnRH pulse driver’ might be located to the neurohormone have been largely assumed to in the mediobasal hypothalamus, specifically at the level represent a simple linear process22. However, the use of neurons co‑expressing kisspeptin, neurokinin B and of 4‑kDa dextran-based fluorescent tracking, which dynorphin A (so‑called KNDy neurons)33,34. mimics the size of most hypothalamic neurohormones, The generation of GnRH pulses requires a coordi- has shown that the diffusion processes, at the level of nated release from multiple neurons through the syn- both the ME and the pituitary capillaries, are complex chronization of xGnRH neuron population excitation. and nonlinear8. Consequently, the portal vessel net- The cell bodies of GnRH neurons are scattered through- work might function as a ‘physical integrator’, enabling out the basal forebrain, whereas their projections have neurohormones to be transferred from the ME to the dendrodendritic bundling and/or shared synapses, gonadotroph within a few seconds42. Once in the blood and become highly concentrated around the ME35. stream, the moderately rapid clearance rate (which varies Fascinatingly, these projections simultaneously receive between species) of LH generates the specific asymmetric and integrate synaptic inputs — they possess both axonal pulse shape of this hormone, which is characterized by and dendritic characteristics, leading to their descrip- a fast increase that is immediately followed by a slower tion as ‘dendrons’, before finally acquiring an axonal decrease28. Importantly, a faithful delivery of the pulsa- morphology within the ME and ramifying into numer- tile pattern of GnRH secretion to the pituitary is crucial ous terminals that appose blood vessels36. Dendrons for gonadotroph function43–46. For example, high GnRH might be an ideal location for putative afferent axons pulse frequencies (>1 pulse per h) activate LH produc- to modulate the excitability of multiple GnRH neuron tion, whereas low frequencies (<1 pulse per 2–3 h) pref- dendrites and for multiple GnRH neurons to align erentially induce follicle-stimulating hormone (FSH) their firing pattern, which thereby provides a potential synthesis and release47. Overall, the intricate relationships mechanism for their synchronized activity directly in between pulsatile GnRH release, secretory competency of the mediobasal hypothalamus36. An additional source of the pituitary gonadotrophs and regulatory mechanisms pulse synchronization is in the ME, in which hypophys- within the vasculature generate the rhythmic fluctuations iotropic GnRH neurons terminate within the external in LH secretion. zone close to endothelial cells of the portal vascula- ture37 (FIG. 2). Endothelial cells in the ME might mod- GnRH and LH surge generation. The GnRH–LH surge ulate GnRH release through nitric oxide secretion38–40 mechanism is sex specific and normally occurs only in (reviewed in REF. 41). At the ME, nitric oxide is sponta- women48,49. During the oestrous cycle, increasing con- neously released from an endothelial source and follows centrations of plasma oestrogen alter the feedback to

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Box 1 | Organization of pituitary cells into homotypic networks of surge levels, compared with that required for a pulse. Indeed, in transgenic mice with a dose-dependent Somatotrophs, corticotrophs, gonadotrophs and lactotrophs form networks with the reduction in GnRH neuron migration, 10% of the nor- 16,64,65,100,139,140 following features : mal GnRH neuronal content failed to rescue ovulation, • The lineages have distinct developmental programmes but cyclicity was restored when approximately 30% of -- The placement of each endocrine cell network occurs at distinct stages of pituitary the GnRH population was present31. organogenesis, before expansion in early postnatal life16,64 In addition to the putative change in GnRH popu- • The networks have distinct motifs and interact with the vasculature lation electrical activity, anatomical changes are found --Somatotrophs organize as clusters linked with strands16 along the capillary within the external zone of the ME, in which GnRH network139, whereas lactotrophs form a honeycomb structure100 aligned with 58,59 (FIG. 2) these vessels139. Gonadotrophs connect to one or more blood vessels via their nerve terminals are ensheathed by tanycytes . protrusions65, whereas the corticotroph network has a loose arrangement64. Cell The cellular conformation changes with fluctuating type-specific homotypic network organization and the distinct relationships of oestrogen profiles throughout the oestrous cycle. For the networks with the vasculature are likely to affect levels and timing of example, in rats, semaphorin 7A‑dependent structural hormone release8,16,70,100,141 remodelling of tanycytes occurs during the preovula- -- The mechanisms underlying the distinct endocrine cell–vascular relationship are tory surge, resulting in release of the engulfed axons unclear, although one factor that was shown to be important is PROP1, as its loss and direct access of GnRH nerve terminals to the portal leads to failure of organ vascularization142 vasculature60. By contrast, rat fenestrated endothelial • Hypothalamic and steroidal factors generate network motifs cells of hypothalamic–hypophyseal portal vessels release -- Loss of growth hormone-releasing hormone (GHRH) leads to isolated somatotrophs, semaphorin 3A, which is thought to induce GnRH neu- whereas somatotroph ablation with intact GHRH stimulation results in clusters of ron axonal growth and sprouting within the ME as a cells that are isolated from each other143. Gonadal steroids also influence function of the oestrous cycle61. These mechanisms are somatotroph cell clustering and cell movement leading to reorganisation124,125 likely to enable the generation of high concentrations of • Pituitary networks have functional relevance GnRH, which evoke the GnRH–LH surge, to be released -- Pituitary networks integrate, amplify and propagate hypothalamic signals that 62,63 arrive from the median eminence. For example, the male somatotroph network into the pituitary portal circulation . responds to GHRH input with large, coordinated, oscillatory Ca2+ increases that Within the pituitary, the distinct network organization 64 outlast the stimulus to drive large excursions in hormone secretion124 of gonadotrophs and their large-scale reorganization 65 • Endocrine and non-endocrine homotypic networks interact during puberty suggest that the communication that -- Communication between each pituitary hormonal cell type, as well as between is mediated by cell contact between gonadotrophs has hormonal cell types and non-hormonal folliculostellate cells, via gap a functional role in the regulation of gonadotropin out- junction-mediated coupling91,100 and paracrine and autocrine interactions139,144 can put (BOX 1). However, this organization has not been modify the output of each of the pituitary hormones and enable the crosstalk studied in the same detail as that of other pituitary axes between pituitary axes to date. Although the dynamic gonadotroph responses -- Loss of specific pituitary hormonal cell types influences the network organization of at the time of the pro-oestrous surge have not yet been heterotypic cells. For example, changes in the gonadotroph network occur after described in vivo, sampling of pituitaries at single time 64 alteration of corticotroph terminal differentiation in mice points in rats and sheep suggests that changes occur in gonadotropin subunit expression, granule distribution and GnRH receptor abundance66,67. Sequencing of GnRH neuronal afferent networks and gonadotrophs mRNA isolated from gonadotrophs of anterior pituitary from negative to positive to induce the gonadotropin glands from female mice reveals that genes regulating surge50–52. That the oestrogen-responsive kisspeptin neu- the secretory process, blood pressure and cell adhe- rons in the rostral periventricular area of the third ven- sion were also enriched during pro-oestrus68. Likewise, tricle have a crucial role in enabling ovulation in rodents immortalized cell lines and cells in pituitary slices by activating GnRH neurons is now well accepted53. increase their cellular movement after GnRH stimula- Importantly, the relative contribution of hypothalamic tion69, and extend cellular processes and increase their and pituitary levels to oestrogen-induced gonadotropin cellular movement at puberty65 (BOX 1; FIG. 3). These surge seems to be species dependent, with the latter being findings suggest that changes in the relationship of the the predominant mechanism in human and non-human gonadotroph network with the vasculature modify primates54. In female sheep, the GnRH surge is com- the secretory response of gonadotrophs70. posed of high-frequency pulsatile events superimposed on a constantly elevated level of GnRH release, although Clinical relevance. The mechanisms that underlie both whether the surge is driven by a fundamentally altered pulsatile secretion and surge generation of LH have pattern of GnRH secretion55 or by a simple increase in important implications for the treatment of infertility in the frequency of pulsatile secretion is unclear56. This women. For example, PCOS, the most common anovula- huge increase in GnRH secretion continues for a period tory cause of infertility71 affecting more than 100 million of 24 hours, which is considerably longer than the dura- women worldwide, is associated with a dysregulation of tion of the LH surge that it induces, before returning the normal pattern of LH secretion72. Whether the ori- to a strictly episodic pattern of release25,55,57. The firing gin of this multifactorial disorder is at the level of the pattern of GnRH neurons that is needed to generate the hypothalamic–pituitary axis is not known73, but PCOS is GnRH–LH surge is not known. However, it is reasona- characterized by increases in GnRH pulse frequency and ble to assume that the prolonged firing of an increased sensitivity of the pituitary gland to the neurohormone74,75. number of GnRH neurons is required for the secretion Consequently, potential interventions that modify the

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a Reproduction genes encoding neuropeptides (such as kisspeptin), tran- Dioestrus Pro-oestrus scription factors (such as chromodomain helicase DNA- binding 7 (CHD7)) and G protein-coupled receptors77. GnRH NO neuron To induce female fertility, hypogonadotropic hypog- axon onadism of pituitary origin can be reversed by subcu- Tanycyte taneous injections of FSH followed by human chorionic 76 GnRH gonadotropin or LH to trigger ovulation . Conversely, hypogonadotropic hypogonadism of hypothalamic origin can be treated using GnRH pumps to restore pituitary 76 Portal vesselPortal vessel hormone secretion . Pulsatile GnRH has the advantage of decreasing the risk of multiple pregnancy and ovarian hyperstimulation syndrome78. In both situations, the pulsatility of GnRH or the rhythmic secretion of LH and/or b Lactation Non-lactating Lactating FSH is required to obtain sufficient follicular maturation and proper ovulation76. Advances in understanding of Cell body ﬁring GnRH secretion and its interactions with LH are essential for designing novel, and indeed modifying existing, ther- + PRL + apies for hypogonadotropic hypogonadism. For example, DA output an estimated 22% of patients with this disease, who are yet to undergo treatment, have transient phases of nor- Opioid? mal fertility79. The underlying mechanisms and relevant DA therapeutic interventions to maintain this phenomenon might be elucidated by further investigation of pulsatility and rhythmicity.

The prolactin axis The prolactin axis is unique among the pituitary hormo- c Ageing nal systems, as in men and non-lactating women it can Young adult Old adult be considered a system that is primed for activation but Internal tonically inhibited by hypothalamic dopamine80. In this zone situation, low concentrations (<25 ng per ml) of circulating prolactin are maintained by short-loop feedback, with ME prolactin receptor-mediated stimulation of dopamine neuron firing rate leading to an increase in catecholamine production81 and output2. The timescale of the feedback External response to prolactin (~10–20 min) could be explained zone Vacuole by the coordinated release of dopamine from multiple neurons2. One possible mechanism involves coordinated changes in firing rates of a subset of tuberoinfundibular Vesicle Portal vasculature dopamine neurons over tens of minutes; these changes correlate with the episodic dopamine secretion that is 2 Figure 2 | Output of hypothalamic regulatory factorsNature to the Reviews median | Endocrinologyeminence. recorded from multiple terminals at the ME in mice . a | A simplified view of tanycyte regulation of gonadotropin-releasing hormone (GnRH) Gap junctions and local dendritic dopamine release have release is shown. Access of GnRH nerve terminals to capillaries at dioestrus is blocked by been proposed to mediate this activity82,83, and integra- tanycyte ensheathment, which retracts at pro-oestrus, possibly in response to tion of single-cell firing rates seems to be involved in the oestrogen-driven secretion of nitric oxide (NO), and leads to the formation of a direct generation of longer dopamine release output events neurohaemal junction and augmented release of GnRH. b | Feedback inhibition of (N.R. and P.M., unpublished observations). prolactin (PRL) secretion is reduced in lactation by an uncoupling of dopamine (DA) Variations in prolactin output occur in virgin female production and release from neuronal electrical activity and by a possible switch to rats as a surge at pro-oestrus, which coincides with that of production and secretion of opioids. c | Growth hormone-releasing hormone secretion is LH84. Prolactin also increases after vaginal stimulation of reduced in late adulthood, with an altered localisation of nerve terminals and 85 86 redistribution of secretory vesicles to autophagic vacuoles, both resulting in reduced both rats and mice as twice daily surges. These surges neuropeptide release to the portal circulation. ME, median evidence. are coordinated by signals from the suprachiasmatic nucleus87, probably through the actions of vasoactive intestinal peptide88. At the level of the pituitary, lactotrophs dynamics of GnRH output, its transport to the ME or its form a network of honeycomb motifs (BOX 1) that enable actions in the pituitary might have implications for the the congregation of cells along the fine pituitary capillary treatment of PCOS. This is also the case for congenital network89 (FIG. 3). This organization supports low levels of hypogonadotropic hypogonadism, which results from a cell–cell coordination, with a small proportion (~1–10%) pituitary or a hypothalamic defect with or without anos- of cells acting as coordinating nodes by functionally con- mia76. Several novel gene mutations that are associated necting distant ensembles90. In addition to synchronizing with this disorder have been identified, including those in calcium activity, cellular organization also mediates the

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a and it might contribute to hormone gene expression and Dioestrus Pro-oestrus cell proliferation92–94. Precisely how gap junctions might Blood vessel orchestrate this mechanism remains unknown.

Increased prolactin output during lactation. The long- term requirement for large increases in circulating prolactin during lactation is associated with a decrease in dopamine output, which begins in late pregnancy and Gonadotroph is coincident with a surge of pituitary prolactin secre- tion95 (FIG. 2). The dopamine tone needs to be strongly decreased throughout lactation to enable the necessary increase in circulating prolactin and is mediated by a decrease in phosphorylation of tyrosine hydroxylase, b the rate-limiting enzyme for dopamine synthesis95. This Nullipara Lactation Weaning mechanism is not the result of a reduced feedback of prolactin on dopamine neurons, which remain electrically responsive at the level of the cell body, but, rather, neuronal firing becomes uncoupled from dopamine secre- tion2. Remarkably, the reduction in dopamine tone is accompanied by the production of opioids, which might Lactotroph enable these neurons to stimulate prolactin secretion96,97. In concert with changes in the hypothalamic inhibition of prolactin secretion, substantial alterations occur Hub in the pituitary to support the 10–50‑fold increase in pro- lactotroph lactin secretion that is required for milk production in mammals98. In humans and rats, this increased hormone secretion is generally accompanied by proliferation and c hypertrophy of lactotrophs, although the reports describ- Juvenile Young adult ing hypertrophy are based on 2D histological studies98. By contrast, in lineage tracing and fluorescence-activated cell sorting (FACS) studies in mice, lactotrophs become hypertrophied during lactation and increase their volume threefold without the accompanying increase in number99. Other investigators have confirmed these findings and also showed that the lactotroph network Somatotroph in situ becomes highly connected during lactation, which is associated with the strength of the suckling stimu- lus100. This increase in structural connectivity leads to an ~100% increase in the proportion of the subpopulation of lactotrophs that function as coordinating nodes and orchestrate increased output of prolactin100.

Figure 3 | Pituitary cells form homotypic networks with distinct organizational Nature Reviews | Endocrinology Memory of prolactin demand after weaning. At weaning, motifs and relationships with the vasculature, which alter with the physiological a rapid decrease in prolactin secretion occurs as a result status. a | Gonadotroph cells are arranged as strings of cells that align with capillaries of a return of dopamine inhibition101. In rodents101,102 and and make direct contact with the vasculature through protrusions at pro-oestrus. 103 b | The organization of lactotrophs into honeycomb structures in virgin mice becomes humans , basal prolactin secretion is reduced below that more pronounced in animals that are lactating, with alteration of cell–cell contacts, of virgin animals, which might reflect an increased pitu- increase in cell size and appearance of cells that act as hubs of functional connectivity itary response to dopamine inhibition104. Strikingly, and (darker orange). This change in structural and functional organization is maintained for despite this reduction in basal prolactin secretion, lacto months after cessation of physiological demand at weaning. c | The homotypic network trophs remain enlarged and well connected with each of somatotrophs changes dramatically at puberty, with the formation of large clusters other at both structural and functional levels, with a two- that are linked by strands of cells in young males. The strands of cells that are found in fold increased number of nodes, which persists for many juvenile and mature animals are closely associated with capillaries, which also line the months after lactation has ceased100 (FIG. 3). Such hard- post-pubertal somatotroph clusters. wiring, or ‘memory’, of previous stimuli, which was previously thought to only exist for neurons and immune coordination of gene transcription, with gap-junction sig- cells, leads to augmented network-mediated lactotroph nalling enabling local correlation of bursts of transcrip- calcium activity during suckling of subsequent litters, tional activity that are otherwise randomly timed91,92. This which drives even higher concentrations of prolactin100. mechanism resembles quorum-sensing in which appar- This mechanism is independent of reproductive experi- ently random systems display complex activity as long ence per se, as it can be prevented by reducing the suckling as the components (in this case, the cells) can interact, demand100.

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Clinical relevance. Dysregulation of the prolactin axis, and electrical properties over the first 6 weeks of postnatal owing to either pituitary adenomas105 or to an adverse life correlates with, and is likely to drive, at least in part, effect of treatment with antipsychotic drugs106, leads to increased pituitary GH output and sexual dimorphism122. impaired fertility. Hyperprolactinaemia has a prevalence The intrinsic hourly rhythms of GHRH neuronal activ- of ~10 and ~30 per 100,000 in men and women, respec- ity that are predicted by simulation studies of in vivo GH tively, and is the second most common cause of infertility pulsatility have not been identified119,123; however, soma- in women after PCOS107. Whereas commonly used dopa- tostatin can generate GHRH neuron pulsatile output mine receptor agonists represent an effective treatment by delaying oscillations of action potential firing via a for most patients with hyperprolactinaemia, the adverse recurring inhibition of inhibitory GABAergic interneu- effects of these drugs, including nausea, headaches and rons (that is, inhibition of inhibition)123. Consequently, postural hypotension, lead to compliance problems108. somatostatin can acutely inhibit the excitability of GHRH Clearly, an understanding of the interactions that lead to neurons and can also promote their patterned output altered dopamine output and of the response of the pitu- together with more-sustained GHRH neuron stimulation itary might help to identify novel treatment strategies for in response to other stimuli in the brain (for example, ace- this disease. In rodent studies, prolactin seems to affect tylcholine122) and peripheral tissues (such as, ghrelin117). multiple neuroendocrine axes, including those regulating fertility, body weight, appetite, stress and maternal Modification of pituitary somatotroph output. No full behaviour80, and these processes warrant further study to description of the in vivo dynamics of GHRH and soma- determine the potential effects of prolactin oversecretion tostatin neurons and of their regulation of pituitary soma- in humans. For example, hyperprolactinaemia might lead totrophs exists. This event can be viewed as a three-step to changes in GnRH neuron activity via interactions with process: delivery of neurohormone to target cells; cellular the G protein-coupled receptor 54 (GPR54; also known secretory responses to regulation; and entry of pituitary as KISS1R)–kisspeptin pathway in mice109, and GnRH hormone into the peripheral circulation8. In vivo imaging pulsatility has been reinstated in mice with physiological of the mouse portal system and somatotroph network have hyperprolactinaemia by administration of kisspeptin110. provided insights into the first step of this process and into In contrast with the study in mice in which an acute high the role of the vasculature in shaping the pattern of expo- dose of prolactin was delivered peripherally109, in studies sure of the pituitary to hypothalamic neuropeptides8. In treating sheep with a chronic, centrally administered low this study, delivery of neuropeptides such as GHRH to (10,000‑fold lower) prolactin dose, no effects of prolactin the somatotroph network, which extends throughout the on hypothalamic kisspeptin expression have been seen111. pituitary gland, follows specific vascular–capillary routes An improved understanding of these pathways could aid and results in a specific temporal patterned regional regu- the development of treatments for women with hyper- lation rather than in a homogenous exposure of the whole prolactinaemia that is resistant to dopamine agonists108. pituitary to the secretagogue. In addition, the initial stimulation by GHRH evokes a coordinated enhancement of The GH axis oxygen supply to the stimulated somatotroph network via In humans and animals, such as cattle and horses, in increased capillary blood flow, which provides fuel for which the measurements can be taken, pulsatile GH energy-depleting secretory responses. Indeed, this study output is present from birth112–114. However, the output also demonstrated that capillaries closely line the clusters is markedly increased at puberty when sexually dimor- and strings of the cells that form the GH network, which phic body growth occurs115. Since the discoveries of suggests an important role of local oxygen regulation in GH-releasing hormone (GHRH) and somatostatin that GH release (FIG. 3). The dynamic association of gonado control GH secretion from pituitary somatotrophs115–117, trophs with the vasculature, which varies through the a remarkable advancement of our understanding of GH ovarian cycle65, suggests that this situation is also the case pulse generation during critical physiological windows for other pituitary axes. The second step in pituitary reg- has taken place. ulation has been characterized using ex vivo data from acute pituitary slices in which the somatotroph network Pulsatile GHRH output. Using genetically modified organization is preserved; in this step, the homotypic net- mouse models with GHRH neurons marked with green work mediates the coordination of stimulation, triggering fluorescent protein118, several investigators have defined long-lasting GH secretion124 (BOX 1). These studies have the mechanisms that underlie pulse generation using also shown that network organization is likely to have a ex vivo brain slices. Before puberty, GHRH neurons are major role in the increased GH output at puberty. In par- excitable neuroendocrine neurons with complex synaptic ticular, using males, the study showed that the volume inputs119. These early stages of hypothalamic development of the GH network undergoes large changes relative to ensure appropriate regulation of the somatotroph axis, as its surface area that coincide with the onset of puberty, shown in Ames dwarf mice in which loss of GH leads before gradually returning to normal prepubertal levels to a compensatory increase in GHRH cell number120. In by day 100 in mice16. These changes occur with a timing addition, steroid exposure in young animals can have that is coincident with the increased pulse output of GH programming effects on the GHRH neuron population, that is associated with puberty in males and are blocked with testosterone exposure in neonates permanently by castration, which also prevents pubertal changes in GH increasing adult GHRH cell number and GHRH gene output16. A direct effect of sex steroids on the organization transcription121. The modification of these synaptic inputs of pituitary cells into homotypic networks is apparent

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from the rapid and dramatic increased motility of soma- GH in childhood to increase growth rate128. However, totrophs in ex vivo pituitary cultures that are treated with considerable uncertainty exists regarding the optimal oestradiol125. These findings highlight the importance of dosage or regime, and current treatments that are based somatotroph network organization and its plasticity in the on injections of GH do not fully recapitulate the physio- generation of pituitary somatotroph output. The vascu- logical pattern of GH secretion115. One potential therapy lature also has an important role in somatotroph output: involves the repopulation of the pituitary with stem cells, in vivo imaging shows that capture of secreted GH is a which have been identified in the mouse129. However, controlled event during which the perivascular space such approaches are likely to require the recapitulation acts as a gatekeeper for hormone entry into the capillary of the normal cellular organization to achieve normal lumen8. The relationship between cellular network organ- pulsatility and homeostatic regulation. ization and the vasculature in the pituitary is therefore Patients with acromegaly, which results from a GH central to the delivery of incoming hypothalamic signals secreting pituitary adenoma, frequently have glycaemic and to the build‑up of GH pulses within capillaries. disorders: a lack of GH pulsatility modifies lipolysis, whereas overall GH hypersecretion can induce insulin Clinical relevance. GH deficiency that results from con- resistance130. Consequently, an improved understanding genital defects or is acquired after traumatic brain injury, of the mechanisms that determine the pattern of GH out- pituitary tumours or cranial irradiation126,127 is commonly put might help to define new therapeutic options for dys- treated with a daily subcutaneous dose of recombinant lipidaemia or diabetes mellitus. In addition, GH pulsatilty

a b

Hypothalamus

Neuronal population Terminals at ME • Ensheathment by tanycytes • Anatomical redistribution Parvocellular neurons • Interaction with vessels • Modulation of inputs • Functional plasticity • Modulation of ﬁring • Changes in intracellular pathways Tanycytes • Selective barrier and carrier Tanycyte • Anatomical and/or functional c plasticity Portal vessel

Pituitary gland

Pituitary networks Somatotroph • Variations in connectivity • Changes in number and/or size Portal vessels • Changes in hormone • Modulation of blood ﬂow production • Supply of oxygen and • Experience-dependent nutrients plasticity

Figure 4 | Updated view of the hypothalamic–vascular–pituitary b | Alterations in tanycyte ensheathment and the anatomical location of unit. In the past decade, a more complex relationship between the neuron terminals modify their interactions with the vasculature, Nature Reviews | Endocrinology hypothalamus and pituitary than previously appreciated has emerged, in changing the dynamics of neurohormone release. c | In the pituitary part owing to the new imaging techniques that now enable gland, changes in blood flow in the portal circulation alter the pattern of high-resolution optical imaging of the hypothalamic–vascular–pituitary exposure of pituitary cells to neurohormones and nutrient supply to unit in living animal models. a | At the level of the parvocellular neurons, facilitate secretion. d | Cells of the pituitary gland, such as somatotrophs, complex inputs modify the excitation of neurons, which can vary the are organized into intermingled networks with distinct morphological coupling with neurohormone output at terminals of the median features, which can be altered to meet the physiological demand, and eminence (ME) through modification of intracellular pathways. different relationships with the vasculature.

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also has an important role in a subgroup of patients who The regulation of pulsatile pituitary secretion must have clinical acromegaly with increased insulin-like now be considered as an integration of hypothalamic, growth factor 1 (IGF1) but unaltered mean 24‑hour vasculature and pituitary regulation, which has fur- GH concentration compared with healthy controls131,132. ther implications for the understanding of disease. For Altered GH pulsatility might explain the clinical presenta- example, the identification of kisspeptin has provided tion of this subgroup of patients, and our new understand- an exciting new target for the treatment of infertility133. ing of the mechanisms underlying patterning of pituitary The uncoupling of neuronal excitation and hormone output might explain the abnormal GH axis function in output also has deeper implications such as in the case these individuals and warrants further investigation. of ageing. For example, the reduction in GH output with age might be due to a failure of neurohormone secretion Conclusions from GHRH neurons without any change in their excita- The examples that were described in this Review provide tion134 (FIG. 2). The human pituitary gland can be accessed new insights into the regulation of three hypothalamic– by transphenoidal surgery, which makes this structure pituitary axes and demonstrate that these mechanisms are an ideal target organ for regenerative therapy135. The not a simple relay of stimulus–secretion coupled events. pituitary cell networks and their relationship with the The disconnection or the modulation of hypothalamic vasculature must be considered for such therapy, and excitation and neurohormone release, as well as the the microenvironment clearly has an important role in active role of the vasculature and pituitary in the network- the regulation of the pituitary gland, which might also mediated modification of responses, demonstrates that affect the development of tumours136. Finally, as pitui- the previous stimulus–secretion coupled view of the hypo- tary networks are sensitive to peripheral regulation and thalamic–pituitary system is oversimplistic. Advances in their modification can persist for extended periods, they imaging technologies are allowing us to understand more are a potential target for endocrine-disrupting chemi- about the organization and function of this axis (FIG. 4). cals. Indeed, the identification of bisphenol A‑mediated Given that the hypothalamus contains no more than a few reduction of expression of intercellular adhesion mole- thousand parvocellular neurons, the rapid development cule 5 (ICAM5) in the pituitary137 has led us to speculate of techniques for interrogating neuronal function should that some effects of endocrine-disrupting chemicals enable the characterization of the regulation and output might be mediated by changes in pituitary organiza- of this structure. Such studies will be invaluable for the tion138. These possibilities require further investigation deeper understanding of mammalian physiology, as the for the understanding of both the aetiology and the hypothalamus controls a much larger panel of known treatment of diseases that are associated with pituitary body functions than any other brain region does. hormones.

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