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.

Imaging and Manipulating Pituitary Function in the Awake Mouse.

Extensive efforts have been made to explore how the activities of multiple brain cells combine to alter physiology through imaging and cell-specific manipulation in different animal models. However, the temporal regulation of peripheral organs by the neuroendocrine factors released by the brain is poorly understood. We have established a suite of adaptable methodologies to interrogate in vivo the relationship of hypothalamic regulation with the secretory output of the pituitary gland, which has complex functional networks of multiple cell types intermingled with the vasculature. These allow imaging and optogenetic manipulation of cell activities in the pituitary gland in awake mouse models, in which both neuronal regulatory activity and hormonal output are preserved. These methodologies are now readily applicable for longitudinal studies of short-lived events (e.g., calcium signals controlling hormone exocytosis) and slowly evolving processes such as tissue remodeling in health and disease over a period of days to weeks.

The Processes of Anterior Pituitary Hormone Pulse Generation.

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

Use of a CD laser pickup head to fabricate microelectrodes in polymethylmethacrylate substrates for biosensing applications.

In this work, we report a simple fabrication method for microelectrodes on a polymethylmethacrylate substrate, using a low-cost laser platform based on a CD-DVD unit for direct rapid-prototyping. We used this laser microfabrication technique to etch any desired design on polymethylmethacrylate substrates to produce microchannels with controlled geometry, with a highly repeatable micron-scale resolution. Those shallow microchannels were then filled with a conductive paste of material of our choice that was converted into microelectrodes of desired shapes and geometries after drying. To validate our process, different geometries, sizes and materials were used as electrodes, and then tested for amperometry and impedance measurements. Development of these microelectrodes is motivated by their potential application in sensors and biosensors, such as glucose and cell counting, as demonstrated in this paper.

Castration-induced modifications of GnRH-elicited [Ca2+](i) signaling patterns in male mouse pituitary gonadotrophs in situ: studies in the acute pituitary slice preparation.

Gonadotropin-releasing hormone (GnRH) binds to pituitary gonadotroph receptors and initiates [Ca(2+)](i) signals and gonadotropin secretion. Here, we recorded GnRH-induced Ca(2+) signals in acute pituitary slices from both intact and castrated male mice 15 and 45 days after orchiectomy (GnX). Cells responding with "noncanonical" sequences of Ca(2+) signaling to increasing GnRH concentrations ([GnRH]; oscillatory responses at a given [GnRH] and transient responses at both lower and higher concentrations) were augmented significantly in the castrated mice. Also, 15 days after GnX the number and size of gonadotrophs were augmented, confirming earlier anatomical studies. Hypertrophied gonadotrophs after 15 days after GnX tended to display GnRH-induced Ca(2+) responses of greater amplitude. Furthermore, median effective dose (ED50) for GnRH decreased from 0.17 nM (control) to ~0.07 nM after GnX, suggesting increased GnRH responsiveness of the gonadotroph population. The progression of Ca(2+) response patterns reported in control male rat gonadotrophs (oscillations declining and spike-plateau responses dominating at increasing [GnRH]) was less conspicuous in mouse gonadotrophs in situ. Also, GnX-induced alterations in rat gonadotrophs (persistence of Ca(2+) oscillations even at [GnRH] >100 nM) were not mirrored by mouse gonadotrophs in situ. Contrary to observations in intact and 15-day castrated mice, after 45 days of GnX the hump component diminished and oscillations were augmented with increasing [GnRH], but Ca(2+) response patterns of gonadotrophs in situ remained virtually unchanged in response to [GnRH]s >1 nM, suggesting dose discrimination failure at high [GnRH]s. This study underscores the notion that GnRH responsiveness and the effects of testosterone deficiency may not be equal in pituitary gonadotrophs across species.