Evidence of a Role for Three Neuropeptides that Mediate Steroid Negative Feedback on Gonadotropin Releasing Hormone/Luteinizing Hormone Secretion in the Ewe: Kisspeptin, Neurokinin B and Orphanin FQ

Gonadotropin-releasing hormone (GnRH) is the final common output from the central nervous system for controlling luteinizing hormone (LH) secretion from the anterior pituitary. GnRH, and subsequently LH, secretion for most of the menstrual/estrous cycle occurs in an episodic pattern that is controlled by the inhibitory actions of estradiol and progesterone. However, GnRH neurons are devoid of the appropriate steroid receptors, therefore, estradiol and progesterone must mediate their effects on GnRH/LH secretion through interneurons. Three likely candidates are neurons containing kisspeptin, neurokinin B (NKB) and orphanin FQ (OFQ).;Puberty in the ewe is marked by an increase in GnRH/LH secretion which in part results from a lessening of estradiol negative feedback. Kisspeptin and NKB are coexpressed in the same subsets of neurons in the arcuate nucleus (ARC) of the hypothalamus and are proposed to be critical for normal timing of puberty. Given that greater than 95% of kisspeptin/NKB neurons colocalize with estrogen receptor-alpha (ERalpha) and progesterone receptor in the ewe, we examined changes in kisspeptin- and NKB-positive neurons in the ARC of pre- and postpubertal ewes in the presence and absence of sex steroids. We observed a greater number of kisspeptin, but not NKB, positive neurons in the ARC of postpubertal ewes compared to prepubertal ewes, which corresponded with changes in LH secretion. Also, we showed that an intravenous injection of senktide, an NKB receptor agonist, stimulates LH secretion in prepubertal ewes. These results support a role for kisspeptin and NKB in ovine puberty.;OFQ when given centrally has been shown to inhibit LH secretion in rats and ewes and OFQ localizes to the external zone of the median eminence. Given its effect on LH secretion and its location, we investigated whether OFQ acts at the pituitary to inhibit LH release and/or at the hypothalamus to inhibit GnRH secretion. OFQ had no direct influence on ovine anterior pituitary cells in cell culture. However, we did observe a decrease in GnRH secretion in hypophyseal portal blood samples following infusion of an OFQ agonist into the third cerebroventricle of ovariectomized ewes. From this we conclude that OFQ acts centrally at the hypothalamus to inhibit GnRH secretion.;Given its central inhibitory action on GnRH secretion in the ewe, we examined if OFQ neurons in the preoptic area (POA) and hypothalamus contain steroid receptors. Furthermore, we investigated a role for endogenous OFQ by infusing an OFQ receptor antagonist into steroid-treated ewes. POA OFQ neurons did not contain steroid receptors, while a majority of OFQ neurons in the ARC contained both ERalpha and progesterone receptor. Furthermore, ovariectomized (OVX) ewes with luteal phase concentrations of progesterone and estradiol, but not OVX ewes implanted with only estradiol or ovary-intact anestrous ewes, showed a significant increase in LH pulse frequency during infusion of an OFQ receptor antagonist. Therefore, we conclude that OFQ mediates, at least in part, the negative feedback action of progesterone on GnRH/LH secretion in the ewe

Evidence that dopamine acts via Kisspeptin to Hold GnRH pulse frequency in check in Anestrous Ewes

Recent work has implicated stimulatory kisspeptin neurons in the arcuate nucleus (ARC) as important for seasonal changes in reproductive function in sheep, but earlier studies support a role for inhibitory A15 dopaminergic (DA) neurons in the suppression of GnRH (and LH) pulse frequency in the nonbreeding (anestrous) season. Because A15 neurons project to the ARC, we performed three experiments to test the hypothesis that A15 neurons act via ARC kisspeptin neurons to inhibit LH in anestrus: 1) we used dual immunocytochemistry to determine whether these ARC neurons contain D2 dopamine receptor (D2-R), the receptor responsible for inhibition of LH in anestrus; 2) wetested the ability of local administration of sulpiride, a D2-R antagonist, into theARCto increase LH secretion in anestrus; and 3) we determined whether an antagonist to the kisspeptin receptor could block the increase in LH secretion induced by sulpiride in anestrus. In experiment 1, 40% of this ARC neuronal subpopulation contained D2-R in breeding season ewes, but this increased to approximately 80% in anestrus. In experiment 2, local microinjection of the two highest doses (10 and 50 nmol) of sulpiride into the ARC significantly increased LH pulse frequency to levels 3 times that seen with vehicle injections. Finally, intracerebroventricular infusion of a kisspeptin receptor antagonist completely blocked the increase in LH pulse frequency induced by systemic administration of sulpiride to anestrous ewes. These results support the hypothesis that DA acts to inhibit GnRH (and LH) secretion in anestrus by suppressing the activity of ARC kisspeptin neurons.We thank Heather Bungard and Jennifer Lydon (West Virginia University Food Animal Research Facility) for the care of animals and Paul Harton for his technical assistance in sectioning tissue. We also thank Dr. Al Parlow and the National Hormone and Peptide Program (Torrance, CA) for the reagents used to measure LH and prolactin.http://endo.endojournals.org/am201

Kisspeptin, neurokinin B, and dynorphin cct in the arcuate nucleus to control activity of the GnRH pulse generator in Ewes

Recent work has led to the hypothesis that kisspeptin/neurokinin B/dynorphin (KNDy) neurons in the arcuate nucleus play a key role in GnRH pulse generation, with kisspeptin driving GnRH release and neurokinin B (NKB) and dynorphin acting as start and stop signals, respectively. In this study, we tested this hypothesis by determining the actions, if any, of four neurotransmitters found in KNDy neurons (kisspeptin, NKB, dynorphin, and glutamate) on episodic LH secretion using local administration of agonists and antagonists to receptors for these transmitters in ovariectomized ewes. We also obtained evidence that GnRH-containing afferents contact KNDy neurons, so we tested the role of two components of these afferents: GnRH and orphanin-FQ. Microimplants of a Kiss1r antagonist briefly inhibited LH pulses and microinjections of 2 nmol of this antagonist produced a modest transitory decrease in LH pulse frequency. An antagonist to the NKB receptor also decreased LH pulse frequency, whereas NKB and an antagonist to the receptor for dynorphin both increased pulse frequency. In contrast, antagonists toGnRHreceptors, orphanin-FQ receptors, and the N-methyl-D-aspartate glutamate receptor had no effect on episodic LH secretion.Wethus conclude that the KNDy neuropeptides act in the arcuate nucleus to control episodic GnRH secretion in the ewe, but afferent input from GnRH neurons to this area does not. These data support the proposed roles forNKBand dynorphin within theKNDyneural network and raise the possibility that kisspeptin contributes to the control ofGnRHpulse frequency in addition to its established role as an output signal from KNDy neurons that drives GnRH pulses.National Institutes of Health Grants R01-HD039916 and RO1-HD017864.http://press.endocrine.org/journal/endoam201

Narrowband imaging of Escaping Lyman-Continuum Emission in the SSA22 Field

We present the results of an ultradeep, narrowband imaging survey for Lyman-continuum (LyC) emission at z~3 in the SSA22a field. We employ a custom narrowband filter centered at 3640A (NB3640), which probes the LyC region for galaxies at z>3.06. We also analyze new and archival NB4980 imaging tuned to the wavelength of the Lya emission line at z=3.09, and archival broadband B, V, and R images of the non-ionizing UV continuum. Our NB3640 images contain 26 z>3.06 Lyman Break Galaxies (LBGs) as well as a set of 130 Lya emitters (LAEs), identified by their excess NB4980 flux relative to the BV continuum. Six LBGs and 28 LAEs are detected in the NB3640 image. LBGs appear to span a range of NB3640-R colors, while LAEs appear bimodal in their NB3640-R properties. We estimate average UV to LyC flux density ratios, corrected for foreground contamination and intergalactic medium absorption, finding ^{LBG} = 11.3^{+10.3}_{-5.4}, which implies a LBG LyC escape fraction f_{esc}^{LyC} ~ 0.1, and ^{LAE} = 2.2^{+0.9}_{-0.6}. The strikingly blue LAE flux density ratios defy interpretation in terms of standard stellar population models. Assuming ^{LBG} applies down to L=0.1L*, we estimate a galaxy contribution to the intergalactic hydrogen ionization rate that is consistent with independent estimates based on the Lya forest opacity at z~3. If we assume that ^{LAE} holds at the faintest luminosities, the galaxy contribution significantly exceeds that inferred from the Lya forest. Further follow-up study of these faint LAEs is crucial, given the potentially important contribution similar objects make to the process of reionization. (Abridged)Comment: 30 pages, 15 figures; Accepted for publication by Ap

Agouti-related peptide neural circuits mediate adaptive behaviors in the starved state

In the face of starvation, animals will engage in high-risk behaviors that would normally be considered maladaptive. Starving rodents, for example, will forage in areas that are more susceptible to predators and will also modulate aggressive behavior within a territory of limited or depleted nutrients. The neural basis of these adaptive behaviors likely involves circuits that link innate feeding, aggression and fear. Hypothalamic agouti-related peptide (AgRP)-expressing neurons are critically important for driving feeding and project axons to brain regions implicated in aggression and fear. Using circuit-mapping techniques in mice, we define a disynaptic network originating from a subset of AgRP neurons that project to the medial nucleus of the amygdala and then to the principal bed nucleus of the stria terminalis, which suppresses territorial aggression and reduces contextual fear. We propose that AgRP neurons serve as a master switch capable of coordinating behavioral decisions relative to internal state and environmental cues

Evidence That Agouti-Related Peptide May Directly Regulate Kisspeptin Neurons in Male Sheep

Agouti-related peptide (AgRP) neurons, which relay information from peripheral metabolic signals, may constitute a key central regulator of reproduction. Given that AgRP inhibits luteinizing hormone (LH) secretion and that nutritional suppression of LH elicits an increase in AgRP while suppressing kisspeptin expression in the arcuate nucleus (ARC) of the hypothalamus, we sought to examine the degree to which AgRP could directly regulate ARC kisspeptin neurons. Hypothalamic tissue was collected from four castrated male sheep (10 months of age) and processed for the detection of protein (AgRP input to kisspeptin neurons) using immunohistochemistry and mRNA for melanocortin 3 and 4 receptors (MC3R; MC4R) in kisspeptin neurons using RNAscope. Immunohistochemical analysis revealed that the majority of ARC kisspeptin neurons are contacted by presumptive AgRP terminals. RNAscope analysis revealed that nearly two thirds of the ARC kisspeptin neurons express mRNA for MC3R, while a small percentage (<10%) colocalize MC4R. Taken together, this data provides neuroanatomical evidence for a direct link between orexigenic AgRP neurons and reproductively critical kisspeptin neurons in the sheep, and builds upon our current understanding of the central link between energy balance and reproduction

Kisspeptin, Neurokinin B, and Dynorphin Expression during Pubertal Development in Female Sheep

The neural mechanisms underlying increases in gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion that drive puberty onset are unknown. Neurons coexpressing kisspeptin, neurokinin B (NKB), and dynorphin, i.e., KNDy neurons, are important as kisspeptin and NKB are stimulatory, and dynorphin inhibitory, to GnRH secretion. Given this, we hypothesized that kisspeptin and NKB expression would increase, but that dynorphin expression would decrease, with puberty. We collected blood and hypothalamic tissue from ovariectomized lambs implanted with estradiol at five, six, seven, eight (puberty), and ten months of age. Mean LH values and LH pulse frequency were the lowest at five to seven months, intermediate at eight months, and highest at ten months. Kisspeptin and NKB immunopositive cell numbers did not change with age. Numbers of cells expressing mRNA for kisspeptin, NKB, or dynorphin were similar at five, eight, and ten months of age. Age did not affect mRNA expression per cell for kisspeptin or NKB, but dynorphin mRNA expression per cell was elevated at ten months versus five months. Thus, neither KNDy protein nor mRNA expression changed in a predictable manner during pubertal development. These data raise the possibility that KNDy neurons, while critical, may await other inputs for the initiation of puberty

Agouti-related peptide neural circuits mediate adaptive behaviors in the starved state

In the face of starvation, animals will engage in high-risk behaviors that would normally be considered maladaptive. Starving rodents, for example, will forage in areas that are more susceptible to predators and will also modulate aggressive behavior within a territory of limited or depleted nutrients. The neural basis of these adaptive behaviors likely involves circuits that link innate feeding, aggression and fear. Hypothalamic agouti-related peptide (AgRP)-expressing neurons are critically important for driving feeding and project axons to brain regions implicated in aggression and fear. Using circuit-mapping techniques in mice, we define a disynaptic network originating from a subset of AgRP neurons that project to the medial nucleus of the amygdala and then to the principal bed nucleus of the stria terminalis, which suppresses territorial aggression and reduces contextual fear. We propose that AgRP neurons serve as a master switch capable of coordinating behavioral decisions relative to internal state and environmental cues