Suzanne M. Moenter

Sampling of Hypophyseal Portal Blood of Conscious Sheep for Direct Monitoring of Hypothalamic Neurosecretory Substances

Publisher Summary This chapter describes a procedure for remote continuous sampling of hypophyseal portal blood of sheep. The procedure offers advantages over earlier methods for portal blood collection because it allows both hypothalamic and pituitary hormones to be monitored for prolonged periods from animals that are fully conscious and apparently undisturbed. Additional exciting new applications should arise by combining the procedure with other techniques used in neuroendocrine investigation, for example, electrical stimulation and recording as well as the local delivery of regulatory substances to discrete neuroendocrine centers implicated in the control of anterior pituitary function. The procedure should be adaptable to species other than sheep, given certain anatomical features of the pituitary and its vasculature. In this regard, four features of the sheep make it especially well suited. The first is the angle of the pituitary. The anterior face of the pituitary of most adult sheep hangs nearly perpendicularly from the base of the brain such that the densest array of portal vessels is readily accessible for surgical approach.

Reproductive Refractoriness of the Ewe to Inhibitory Photoperiod Is Not Caused by Alteration of the Circadian Secretion of Melatonin

The 24-hour pattern of melatonin secretion was determined in 5 Suffolk ewes during extended exposure to a long day length to assess whether the eventual loss of response (photorefractoriness) to inhibitory long days resulted from an alteration in the circadian secretion of melatonin. Determinations of the secretory profile of melatonin were made once in short days (8 h light/day) before the switch to long days, and 11 times throughout the 250-day period of exposure to long days (16 h light/day). Samples were obtained hourly for 24 or 48 h. Reproductive state was assessed by response to estradiol-negative feedback, monitored as serum LH in ovariectomized ewes bearing estradiol implants. The characteristic secretory pattern of melatonin (low during the day, high at night), the duration of the melatonin elevation, and its phase relative to the light/dark cycle did not change as ewes became refractory to the inhibitory effects of long days. These results are consonant with the hypothesis that refractoriness of the ewe to inhibitory day length does not result from an alteration of the circadian rhythm of melatonin secretion.

Voltage-Gated Potassium Currents Are Targets of Diurnal Changes in Estradiol Feedback Regulation and Kisspeptin Action on Gonadotropin-Releasing Hormone Neurons in Mice

Estradiol has both negative and positive feedback actions upon gonadotropin-releasing hormone (GnRH) release; the latter actions trigger the preovulatory GnRH surge. Although neurobiological mechanisms of the transitions between feedback modes are becoming better understood, the roles of voltage-gated potassium currents, major contributors to neuronal excitability, are unknown. Estradiol alters two components of potassium currents in these cells: a transient current, IA, and a sustained current, IK. Kisspeptin is a potential mediator between estradiol and GnRH neurons and can act directly on GnRH neurons. We examined how estradiol, time of day, and kisspeptin interact to regulate these conductances in a mouse model exhibiting daily switches between estradiol negative (morning) and positive feedback (evening). Whole-cell voltage clamp recordings were made from GnRH neurons in brain slices from ovariectomized (OVX) mice and from OVX mice treated with estradiol (OVX+E). There were no diurnal changes in either IA or IK in GnRH neurons from OVX mice. In contrast, in GnRH neurons from OVX+E mice, IA and IK were greater during the morning when GnRH neuron activity is low and smaller in the evening when GnRH neuron activity is high. Estradiol increased IA in the morning and decreased it in the evening, relative to that in cells from OVX mice. Exogenously applied kisspeptin reduced IA regardless of time of day or estradiol status. Estradiol, interacting with time of day, and kisspeptin both depolarized IA activation. These findings extend our understanding of both the neurobiological mechanisms of estradiol negative vs. positive regulation of GnRH neurons and of kisspeptin action on these cells.

Leap of Faith: Does serum luteinizing hormone always accurately reflect central reproductive neuroendocrine activity?

The function of the central aspects of the hypothalamic-pituitary-gonadal axis has been assessed in a number of ways including direct measurements of the hypothalamic output and indirect measures using gonadotropin release from the pituitary as a bioassay for reproductive neuroendocrine activity. Here, methods for monitoring these various parameters are briefly reviewed and then examples presented of both concordance and discrepancy between central and peripheral measurements, with a focus on situations in which elevated gonadotropin-releasing hormone neurosecretion is not reflected accurately by pituitary luteinizing hormone release. Implications for the interpretation of gonadotropin data are discussed.

Long-Term recordings of arcuate nucleus kisspeptin neurons reveal patterned activity that is modulated by gonadal steroids in male mice

&NA; Pulsatile release of gonadotropin‐releasing hormone (GnRH) is key to fertility. Pulse frequency is modulated by gonadal steroids and likely arises subsequent to coordination of GnRH neuron firing activity. The source of rhythm generation and the site of steroid feedback remain critical unanswered questions. Arcuate neurons that synthesize kisspeptin, neurokinin B, and dynorphin (KNDy) may be involved in both of these processes. We tested the hypotheses that action potential firing in KNDy neurons is episodic and that gonadal steroids regulate this pattern. Targeted extracellular recordings were made of green fluorescent protein‐identified KNDy neurons in brain slices from adult male mice that were intact, castrated, or castrated and treated with estradiol or dihydrotestosterone (DHT). KNDy neurons exhibited marked peaks and nadirs in action potential firing activity during recordings lasting 1 to 3.5 hours. Peaks, identified by Cluster analysis, occurred more frequently in castrated than intact mice, and either estradiol or DHT in vivo or blocking neurokinin type 3 receptor in vitro restored peak frequency to intact levels. The frequency of peaks in firing rate and estradiol regulation of this frequency is similar to that observed for GnRH neurons, whereas DHT suppressed firing in KNDy but not GnRH neurons. We further examined the patterning of action potentials to identify bursts that may be associated with increased neuromodulator release. Burst frequency and duration are increased in castrated compared with intact and steroid‐treated mice. The observation that KNDy neurons fire in an episodic manner that is regulated by steroid feedback is consistent with a role for these neurons in GnRH pulse generation and regulation.

Contents Vol. 102, 2015

77 Abstracts of the 12th Annual ENETS Conference for the Diagnosis and Treatment of Neuroendocrine Tumor Disease March 11–13, 2015, Barcelona, Spain Guest Editors: Reed, N. (Glasgow); Couvelard, A. (Paris); Ruszniewski, P. (Clichy) 169 ENETS Newsletter Summer 2015 E-Mail karger@karger.com www.karger.com

The Preovulatory Surge of Gonadotropin Releasing Hormone

The regulation of gonadotropin releasing hormone secretion during the preovulatory surge of luteinizing hormone has been a focus of neuroendocrinological investigation for decades. Although it is clear that an increase in estradiol secretion from the developing follicle is an essential ovarian signal for the LH discharge, a fundamental question remains: Does estradiol elicit the LH surge by stimulating the secretion of GnRH? Despite the importance of this question, it is only in the laboratory rat that there has been general agreement and ample direct evidence for heightened secretion of GnRH (1–6). In the rat, however, an increased secretion of GnRH at the time of the LH surge would seem a virtual certainty, for the surge in this species is precisely timed by a circadian clock located in the brain (5–7). The circadian signal emanating from the clock would be expected to be relayed to the pituitary via a hypophyseotropic stimulus discharged into pituitary portal blood. In species in which the preovulatory LH surge is not tightly coupled to a circadian clock, for example in primates (8) and sheep (9), enhanced secretion of GnRH at the time of the LH surge is not necessarily expected, nor is there consistent evidence that such an increase actually exists. Further, a stimulatory action of estradiol upon the anterior pituitary gland is documented to be one mode by which this steroid elicits the preovulatory LH discharge (10–16), and in at least one species, the rhesus monkey, this pituitary action of estradiol may itself be sufficient for LH-surge induction (16). Therefore, the fundamental question remains: Does estradiol induce the preovulatory LH surge by enhancing the secretion of GnRH?