Prolactin blood concentration relies on the scalability of the TIDA neurons' network efficiency in vivo.

Our understanding and management of reproductive health and related disorders such as infertility, menstrual irregularities, and pituitary disorders depend on understanding the intricate sex-specific mechanisms governing prolactin secretion. Using ex vivo experiments in acute slices, in parallel with in vivo calcium imaging (GRIN lens technology), we found that dopamine neurons inhibiting PRL secretion (TIDA), organize as functional networks both in and ex vivo. We defined an index of efficiency of networking (Ieff) using the duration of calcium events and the ability to form plastic economic networks. It determined TIDA neurons' ability to inhibit PRL secretion in vivo. Ieff variations in both sexes demonstrated TIDA neurons' adaptability to physiological changes. A variation in the number of active neurons contributing to the network explains the sexual dimorphism in basal [PRL]blood secretion patterns. These sex-specific differences in neuronal activity and network organization contribute to the understanding of hormone regulation.

TSH Pulses Finely Tune Thyroid Hormone Release and TSH Receptor Transduction.

Detection of circulating TSH is a first-line test of thyroid dysfunction, a major health problem (affecting about 5% of the population) that, if untreated, can lead to a significant deterioration of quality of life and adverse effects on multiple organ systems. Human TSH levels display both pulsatile and (nonpulsatile) basal TSH secretion patterns; however, the importance of these in regulating thyroid function and their decoding by the thyroid is unknown. Here, we developed a novel ultra-sensitive ELISA that allows precise detection of TSH secretion patterns with minute resolution in mouse models of health and disease. We characterized the patterns of ultradian TSH pulses in healthy, freely behaving mice over the day-night cycle. Challenge of the thyroid axis with primary hypothyroidism because of iodine deficiency, a major cause of thyroid dysfunction worldwide, results in alterations of TSH pulsatility. Induction in mouse models of sequential TSH pulses that mimic ultradian TSH profiles in periods of minutes were more efficient than sustained rises in basal TSH levels at increasing both thyroid follicle cAMP levels, as monitored with a genetically encoded cAMP sensor, and circulating thyroid hormone. Hence, this mouse TSH assay provides a powerful tool to decipher how ultradian TSH pulses encode thyroid outcomes and to uncover hidden parameters in the TSH-thyroid hormone set-point in health and disease.

Median eminence blood flow influences food intake by regulating ghrelin access to the metabolic brain.

Central integration of peripheral appetite-regulating signals ensures maintenance of energy homeostasis. Thus, plasticity of circulating molecule access to neuronal circuits involved in feeding behavior plays a key role in the adaptive response to metabolic changes. However, the mechanisms involved remain poorly understood despite their relevance for therapeutic development. Here, we investigated the role of median eminence mural cells, including smooth muscle cells and pericytes, in modulating gut hormone effects on orexigenic/anorexigenic circuits. We found that conditional activation of median eminence vascular cells impinged on local blood flow velocity and altered ghrelin-stimulated food intake by delaying ghrelin access to target neurons. Thus, activation of median eminence vascular cells modulates food intake in response to peripheral ghrelin by reducing local blood flow velocity and access to the metabolic brain.

Cell Networks in Endocrine/Neuroendocrine Gland Function.

Reproduction, growth, stress, and metabolism are determined by endocrine/neuroendocrine systems that regulate circulating hormone concentrations. All these systems generate rhythms and changes in hormone pulsatility observed in a variety of pathophysiological states. Thus, the output of endocrine/neuroendocrine systems must be regulated within a narrow window of effective hormone concentrations but must also maintain a capacity for plasticity to respond to changing physiological demands. Remarkably most endocrinologists still have a "textbook" view of endocrine gland organization which has emanated from 20th century histological studies on thin 2D tissue sections. However, 21st -century technological advances, including in-depth 3D imaging of specific cell types have vastly changed our knowledge. We now know that various levels of multicellular organization can be found across different glands, that organizational motifs can vary between species and can be modified to enhance or decrease hormonal release. This article focuses on how the organization of cells regulates hormone output using three endocrine/neuroendocrine glands that present different levels of organization and complexity: the adrenal medulla, with a single neuroendocrine cell type; the anterior pituitary, with multiple intermingled cell types; and the pancreas with multiple intermingled cell types organized into distinct functional units. We give an overview of recent methodologies that allow the study of the different components within endocrine systems, particularly their temporal and spatial relationships. We believe the emerging findings about network organization, and its impact on hormone secretion, are crucial to understanding how homeostatic regulation of endocrine axes is carried out within endocrine organs themselves. © 2022 American Physiological Society. Compr Physiol 12:3371-3415, 2022.

Innervation of GnRH Neuron Distal Projections and Activation by Kisspeptin in a New GnRH-Cre Rat Model.

The neural mechanisms generating pulsatile GnRH release from the median eminence (ME) remain unclear. Studies undertaken in the mouse demonstrate that GnRH neurons extend projections to the ME that have properties of both dendrites and axons, termed "dendrons," and that the kisspeptin neuron pulse generator targets these distal dendrons to drive pulsatile GnRH secretion. It presently remains unknown whether the GnRH neuron dendron exists in other species. We report here the generation of a knock-in Gnrh1-Ires-Cre rat line with near-perfect targeting of Cre recombinase to the GnRH neuronal phenotype. More than 90% of adult male and female GnRH neurons express Cre with no ectopic expression. Adeno-associated viruses were used in adult female Gnrh1-Ires-Cre rats to target mCherry or GCAMP6 to rostral preoptic area GnRH neurons. The mCherry tracer revealed the known unipolar and bipolar morphology of GnRH neurons and their principal projection pathways to the external zone of the ME. Synaptophysin-labeling of presynaptic nerve terminals revealed that GnRH neuron distal projections received numerous close appositions as they passed through the arcuate nucleus and into the median eminence. Confocal GCaMP6 imaging in acute horizontal brain slices demonstrated that GnRH neuron distal projections lateral to the median eminence were activated by kisspeptin. These studies indicate the presence of a dendron-like arrangement in the rat with GnRH neuron distal projections receiving synaptic input and responding to kisspeptin.

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.

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.

Pulse and Surge Profiles of Luteinizing Hormone Secretion in the Mouse.

Using a new tail-tip bleeding procedure and a sensitive ELISA, we describe here the patterns of LH secretion throughout the mouse estrous cycle; in ovariectomized mice; in ovariectomized, estradiol-treated mice that model estrogen-negative and -positive feedback; and in transgenic GNR23 mice that exhibit allele-dependent reductions in GnRH neuron number. Pulsatile LH secretion was evident at all stages of the estrous cycle, with LH pulse frequency being approximately one pulse per hour in metestrous, diestrous, and proestrous mice but much less frequent at estrus (less than one pulse per 4 h). Ovariectomy resulted in substantial increases in basal and pulsatile LH secretion with pulses occurring approximately every 21 minutes. Chronic treatment with negative-feedback, estradiol-filled capsules returned LH pulse frequency to intact follicular phase levels, although pulse amplitude remained elevated. On the afternoon of proestrus, the LH surge was found to begin in a highly variable manner over a 4-hour range, lasting for more than 3 hours. In contrast, ovariectomized, estradiol-treated, positive-feedback mice exhibited a relatively uniform surge onset at approximately 0.5 hour prior to lights out. Gonadectomized wild-type and heterozygous GNR23 (∼200 GnRH neurons) male mice exhibited an LH pulse every 60 minutes. Homozygous GNR23 mice (∼80 GnRH neurons) had very low basal LH concentrations but continued to exhibit small amplitude LH pulses every 90 minutes. These studies provide the first characterization in mice of pulse and surge modes of LH secretion across the estrous cycle and demonstrate that very few GnRH neurons are required for pulsatile LH secretion.

Optogenetic activation of GnRH neurons reveals minimal requirements for pulsatile luteinizing hormone secretion.

The mechanisms responsible for generating the pulsatile release of gonadotropins from the pituitary gland are unknown. We develop here a methodology in mice for controlling the activity of the gonadotropin-releasing hormone (GnRH) neurons in vivo to establish the minimal parameters of activation required to evoke a pulse of luteinizing hormone (LH) secretion. Injections of Cre-dependent channelrhodopsin (ChR2)-bearing adeno-associated virus into the median eminence of adult GnRH-Cre mice resulted in the selective expression of ChR2 in hypophysiotropic GnRH neurons. Acute brain slice experiments demonstrated that ChR2-expressing GnRH neurons could be driven to fire with high spike fidelity with blue-light stimulation frequencies up to 40 Hz for periods of seconds and up to 10 Hz for minutes. Anesthetized, ovariectomized mice had optical fibers implanted in the vicinity of GnRH neurons within the rostral preoptic area. Optogenetic activation of GnRH neurons for 30-s to 5-min time periods over a range of different frequencies revealed that 10 Hz stimulation for 2 min was the minimum required to generate a pulse-like increment of LH. The same result was found for optical activation of GnRH projections in the median eminence. Increases in LH secretion were compared with endogenous LH pulse parameters measured from ovariectomized mice. Driving GnRH neurons to exhibit simultaneous burst firing was ineffective at altering LH secretion. These observations provide an insight into how GnRH neurons generate pulsatile LH secretion in vivo.

Dependence of fertility on kisspeptin-Gpr54 signaling at the GnRH neuron.

Signaling between kisspeptin and its receptor, G-protein-coupled receptor 54 (Gpr54), is now recognized as being essential for normal fertility. However, the key cellular location of kisspeptin-Gpr54 signaling is unknown. Here we create a mouse with a GnRH neuron-specific deletion of Gpr54 to assess the role of gonadotropin-releasing hormone (GnRH) neurons. Mutant mice are infertile, fail to go through puberty and exhibit markedly reduced gonadal size and follicle-stimulating hormone levels alongside GnRH neurons that are unresponsive to kisspeptin. In an attempt to rescue the infertile phenotype of global Gpr54⁻/⁻ mutants, we use BAC transgenesis to target Gpr54 to the GnRH neurons. This results in mice with normal puberty onset, estrous cyclicity, fecundity and a recovery of kisspeptin's stimulatory action upon GnRH neurons. Using complimentary cell-specific knockout and knockin approaches we demonstrate here that the GnRH neuron is the key site of kisspeptin-Gpr54 signaling for fertility.