@article{chronic inflammation decreases arcuate kisspeptin expression in male sheep._2024, url={https://www.sciencedirect.com/science/article/pii/S0739724024000316}, journal={Domestic Animal Encocrinology}, year={2024}, month={Jun} }
 @article{ahern_martins_florez_ross_huisman_cushman_shuping_nestor_desaulniers_white_et al._2024, title={Development of KISS1 knockout pigs is characterized by hypogonadotropic hypogonadism, normal growth, and reduced skatole}, ISSN={["1529-7268"]}, DOI={10.1093/biolre/ioae140}, abstractNote={Abstract Kisspeptin is a major regulator of gonadotropin secretion in pigs. Previously, CRISPR/Cas9 knockout of KISS1 was used to develop a mosaic parental line of pigs to generate offspring that would not need castration due to loss of kisspeptin. The current goal was to characterize growth and reproductive development of F1 pigs from this parental line. Body weights, gonadotropin concentrations and gonadal development were measured from birth through development (boars to 220 d of age, n = 42; gilts to 160 d of age, n = 36). Testosterone, skatole, and androstenone were also measured in boars. Blood samples were collected by jugular venipuncture for quantification of serum hormones, gonadal tissues collected for gross morphology and histology, and a fat biopsy collected (boars) for skatole and androstenone analysis. Body weight did not differ with genotype. There were no differences between KISS1+/+ and heterozygote KISS1+/− animals for most parameters measured. Gonadotropin concentrations were reduced in KISS1−/− boars and gilts compared with KISS1+/+ and KISS1+/− animals (P < 0.05). Concentrations of testosterone in serum and both androstenone and skatole in adipose were less in KISS1−/− boars than in KISS1+/+ and KISS1+/− boars (P < 0.05). Hypogonadism was in all KISS1−/− gilts and boars. These data indicate that knocking out KISS1 causes hypogonadotropic hypogonadism but does not negatively affect growth in pigs. Only one KISS1 allele is needed for normal gonadotropin secretion and gonadal development, and accumulation of compounds in adipose leading to boar taint.}, journal={BIOLOGY OF REPRODUCTION}, author={Ahern, Daniel F. and Martins, Kyra and Florez, Julio M. and Ross, Caitlin E. and Huisman, Abe and Cushman, Robert A. and Shuping, Sydney L. and Nestor, Casey C. and Desaulniers, Amy T. and White, Brett R. and et al.}, year={2024}, month={Oct} }
 @article{nestor_2023, title={Central Regulation of Kndy Neurons During Undernutrition in Sheep}, volume={101}, ISSN={["1525-3163"]}, DOI={10.1093/jas/skad281.279}, abstractNote={Abstract
               Adequate energy intake is essential for reproduction. While we have known for decades that feed restriction inhibits gonadotropin-releasing hormone/luteinizing hormone (GnRH/LH) secretion, the central mechanisms that govern this reduction during undernutrition remain to be fully elucidated. The past few years, we have focused on examining central mechanisms whereby chronic feed restriction in young sheep results in the inhibition of LH secretion. This presentation will focus on our current understanding of how feed restriction impacts kisspeptin/neurokinin B/dynorphin (KNDy) neurons in young sheep and will give a glimpse into our next few years as we examine how input to KNDy neurons could play a key role in regulating reproduction during undernutrition.}, journal={JOURNAL OF ANIMAL SCIENCE}, author={Nestor, Casey}, year={2023}, month={Nov}, pages={230–231} }
 @article{nestor_merkley_lehman_hileman_goodman_2023, title={KNDy neurons as the GnRH pulse generator: Recent studies in ruminants}, volume={164}, ISSN={["1873-5169"]}, DOI={10.1016/j.peptides.2023.171005}, abstractNote={This review considers three aspects of recent work on the role of KNDy neurons in GnRH pulse generation in ruminants. First, work on basic mechanisms of pulse generation includes several tests of this hypothesis, all of which support it, and evidence that Kiss1r-containing neurons form a positive feedback circuit with the KNDy neural network that strengthen the activity of this network. The second section on pathways mediating external inputs focuses on the influence of nutrition and photoperiod, and describes the evidence supporting roles for proopiomelanocortin (POMC) and agouti-related peptide (AgRP) afferents to KNDy cells in each of these. Finally, we review studies exploring the potential applications of manipulating signaling by kisspeptin, and the other KNDy peptides, to control reproductive function in domestic animals and conclude that, although these approaches show some promise, they do not have major advantages over current practices at this time.}, journal={PEPTIDES}, author={Nestor, Casey C. and Merkley, Christina M. and Lehman, Michael N. and Hileman, Stanley M. and Goodman, Robert L.}, year={2023}, month={Jun} }
 @article{griesgraber_onslow_shuping_bowdridge_hardy_aerts_coolen_nestor_hileman_lehman_et al._2023, title={Role of Kndy and Arcuate Kiss1r-Containing Neurons in the Preovulatory Luteinizing Hormone Surge and Puberty Onset of Female sheep}, volume={101}, ISSN={["1525-3163"]}, DOI={10.1093/jas/skad281.284}, abstractNote={Abstract
               Neurons within the arcuate nucleus (ARC) of the hypothalamus containing kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons) have an important role in regulating the pulsatile secretion of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH). In sheep, kisspeptin neurons also contribute to the LH surge, as kisspeptin receptor (Kiss1r) antagonist administration reduces surge amplitude by 50% and KNDy neurons are likely involved, based on increased Fos expression at the time of the surge. However, the extent to which kisspeptin acts within the ARC regulate the GnRH/LH surge remains unclear. Thus, herein we tested the hypothesis that deletion of KNDy or ARC Kiss1r-containing neurons would impair the LH surge. Adult female sheep received bilateral injections targeting the ARC of NKB-saporin (NKB-SAP, n = 8), kisspeptin-saporin (Kiss-SAP, n = 10), or blank-saporin (Blank-SAP, n = 7) as a control. In other work, NKB-SAP lesioned over 90% of ovine KNDy neurons, while Kiss-SAP lesioned 67% of Kiss1r-containing cells without affecting KNDy or GnRH cell number. Ewes were also ovariectomized and a subcutaneous silastic estradiol (E2) implant was inserted at the time of neurosurgery. Two artificial luteal phases were simulated with progesterone-containing CIDRs, immediately followed by E2 treatment via implants to induce an LH surge. Blood samples were collected every two to four hours over two days and analyzed for LH via radioimmunoassay. LH surge amplitude in six of eight NKB-SAP ewes (49.5 ± 11.7 ng/mL) was significantly reduced compared with Blank-SAP control ewes (156.7 ± 20.2 ng/mL, p = 0.0001), a reduction similar to that produced by treatment with a Kiss1r antagonist. Nine of ten Kiss-SAP treated ewes displayed little to no increase of LH at the time of the expected surge (16.6 ± 5.3 ng/mL, p < 0.0001). Lesion effectiveness is currently being assessed by RNAscope, however all Kiss-SAP animals examined to date have significantly reduced ARC Kiss1r cell numbers except a single ewe which exhibited a normal LH surge. Based on these data, we propose that in ewes, KNDy neurons contribute to, but are not required for, the LH surge. In contrast, ARC Kiss1r-containing cells are essential for a functional LH surge. Given these results, we are currently assessing the role of ARC Kiss1r neurons in ovine puberty using a similar approach. Our data to date shows that time to puberty onset is similar for Kiss-SAP, Blank-SAP, and non-surgical control animals as measured by an increase in progesterone (p = 0.35). Blood samples to detect LH pulses and the LH surge are currently being analyzed, as are ARC Kiss1r cell numbers.}, journal={JOURNAL OF ANIMAL SCIENCE}, author={Griesgraber, Max J. and Onslow, Kayla M. and Shuping, Sydney and Bowdridge, Elizabeth and Hardy, Steven and Aerts, Eliana and Coolen, Lique and Nestor, Casey and Hileman, Stanley and Lehman, Michael and et al.}, year={2023}, month={Nov}, pages={235–236} }
 @article{aerts_griesgraber_shuping_bowdridge_hardy_goodman_nestor_hileman_2023, title={The effect of NK3-Saporin injection within the arcuate nucleus on puberty, the LH surge, and the response to Senktide in female sheep}, volume={10}, ISSN={["1529-7268"]}, url={https://doi.org/10.1093/biolre/ioad147}, DOI={10.1093/biolre/ioad147}, abstractNote={Abstract
               The timing of puberty onset is reliant on increased gonadotropin-releasing hormone (GnRH). This elicits a corresponding increase in luteinizing hormone (LH) due to a lessening of sensitivity to the inhibitory actions of estradiol (E2). The mechanisms underlying the increase in GnRH release likely involve a subset of neurons within the arcuate (ARC) nucleus of the hypothalamus that contain kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons). We aimed to determine if KNDy neurons in female sheep are critical for: timely puberty onset; the LH surge; and the response to an intravenous injection of the neurokinin-3 receptor (NK3R) agonist, senktide. Prepubertal ewes received injections aimed at the ARC containing blank-saporin (control, n = 5) or NK3-saporin (NK3-SAP, n = 6) to ablate neurons expressing NK3R. Blood samples taken 3/week for 65 days following surgery were assessed for progesterone to determine onset of puberty. Control ewes exhibited onset of puberty at 33.2 ± 3.9 days post sampling initiation, whereas 5/6 NK3-SAP treated ewes didn’t display an increase in progesterone. After an artificial LH surge protocol, surge amplitude was lower in NK3-SAP ewes. Finally, ewes were treated with senktide to determine if an LH response was elicited. LH pulses were evident in both groups in the absence of injections, but the response to senktide vs saline was similar between groups. These results show that KNDy cells are necessary for timely puberty onset and for full expresson of the LH surge. The occurrence of LH pulses in NK3-SAP treated ewes may indicate a recovery from an apulsatile state.}, journal={BIOLOGY OF REPRODUCTION}, author={Aerts, Eliana G. and Griesgraber, Max J. and Shuping, Sydney L. and Bowdridge, Elizabeth C. and Hardy, Steven L. and Goodman, Robert L. and Nestor, Casey C. and Hileman, Stanley M.}, year={2023}, month={Oct} }
 @article{harlow_griesgraber_seman_shuping_sommer_griffith_hileman_nestor_2022, title={The impact of undernutrition on KNDy (kisspeptin/neurokinin B/dynorphin) neurons in female lambs}, volume={34}, ISSN={["1365-2826"]}, url={https://doi.org/10.1111/jne.13135}, DOI={10.1111/jne.13135}, abstractNote={AbstractUndernutrition limits reproduction through inhibition of gonadotropin‐releasing hormone (GnRH)/luteinizing hormone (LH) secretion. Because KNDy neurons coexpress neuropeptides that play stimulatory (kisspeptin and neurokinin B [NKB]) and inhibitory (dynorphin) roles in pulsatile GnRH/LH release, we hypothesized that undernutrition would inhibit kisspeptin and NKB expression at the same time as increasing dynorphin expression. Fifteen ovariectomized lambs were either fed to maintain pre‐study body weight (controls) or feed‐restricted to lose 20% of pre‐study body weight (FR) over 13 weeks. Blood samples were collected and plasma from weeks 0 and 13 were assessed for LH by radioimmunoassay. At week 13, animals were killed, and brain tissue was processed for assessment of KNDy peptide mRNA or protein expression. Mean LH and LH pulse amplitude were lower in FR lambs compared to controls. We observed lower mRNA abundance for kisspeptin within KNDy neurons of FR lambs compared to controls with no significant change in mRNA for NKB or dynorphin. We also observed that FR lambs had fewer numbers of arcuate nucleus kisspeptin and NKB perikarya compared to controls. These findings support the idea that KNDy neurons are important for regulating reproduction during undernutrition in female sheep.}, number={6}, journal={JOURNAL OF NEUROENDOCRINOLOGY}, publisher={Wiley}, author={Harlow, KaLynn and Griesgraber, Max J. and Seman, Andrew D. and Shuping, Sydney L. and Sommer, Jeffrey R. and Griffith, Emily H. and Hileman, Stanley M. and Nestor, Casey C.}, year={2022}, month={May} }
 @article{merkley_shuping_sommer_nestor_2021, title={Evidence That Agouti-Related Peptide May Directly Regulate Kisspeptin Neurons in Male Sheep}, volume={11}, ISBN={2218-1989}, DOI={10.3390/metabol1030138}, number={3}, journal={METABOLITES}, author={Merkley, Christina M. and Shuping, Sydney L. and Sommer, Jeffrey R. and Nestor, Casey C.}, year={2021}, month={Mar} }
 @article{harlow_renwick_shuping_sommer_lents_knauer_nestor_2021, title={Evidence that pubertal status impacts kisspeptin/neurokinin B/dynorphin neurons in the gilt(dagger)}, volume={105}, ISSN={["1529-7268"]}, DOI={10.1093/biolre/ioab189}, abstractNote={Abstract
               Puberty onset is a complex physiological process, which enables the capacity for reproduction through increased gonadotropin-releasing hormone and subsequently luteinizing hormone secretion. While cells that coexpress kisspeptin, neurokinin B (NKB), and dynorphin in the hypothalamic arcuate nucleus are believed to govern the timing of puberty, the degree to which kisspeptin/NKB/dynorphin (KNDy) neurons exist and are regulated by pubertal status remains to be determined in the gilt. Hypothalamic tissue from prepubertal and postpubertal, early follicular phase gilts was used to determine the expression of kisspeptin, NKB, and dynorphin within the arcuate nucleus. Fluorescent in situ hybridization revealed that the majority (>74%) of arcuate nucleus neurons that express mRNA for kisspeptin coexpressed mRNA for NKB and dynorphin. There were fewer arcuate nucleus cells that expressed mRNA for dynorphin in postpubertal gilts compared to prepubertal gilts (P < 0.05), but the number of arcuate nucleus cells expressing mRNA for kisspeptin or NKB was not different between groups. Within KNDy neurons, mRNA abundance for kisspeptin, NKB, and dynorphin of postpubertal gilts was the same as, less than, and greater than, respectively, prepubertal gilts. Immunostaining for kisspeptin did not differ between prepubertal and postpubertal gilts, but there were fewer NKB immunoreactive fibers in postpubertal gilts compared to prepubertal gilts (P < 0.05). Together, these data reveal novel information about KNDy neurons in gilts and support the idea that NKB and dynorphin play a role in puberty onset in the female pig.}, number={6}, journal={BIOLOGY OF REPRODUCTION}, author={Harlow, KaLynn and Renwick, Allison N. and Shuping, Sydney L. and Sommer, Jeffrey R. and Lents, Clay A. and Knauer, Mark T. and Nestor, Casey C.}, year={2021}, month={Dec}, pages={1533–1544} }
 @article{aerts_harlow_griesgraber_bowdridge_hardy_nestor_hileman_2021, title={Kisspeptin, Neurokinin B, and Dynorphin Expression during Pubertal Development in Female Sheep}, volume={10}, ISSN={["2079-7737"]}, url={https://doi.org/10.3390/biology10100988}, DOI={10.3390/biology10100988}, abstractNote={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.}, number={10}, journal={BIOLOGY-BASEL}, author={Aerts, Eliana G. and Harlow, KaLynn and Griesgraber, Max J. and Bowdridge, Elizabeth C. and Hardy, Steven L. and Nestor, Casey C. and Hileman, Stanley M.}, year={2021}, month={Oct} }
 @article{harlow_renwick_shuping_sommer_knauer_nestor_2020, title={Effects of genetic selection for early puberty on the hypothalamic-pituitary-ovarian axis in gilts}, volume={98}, ISSN={["1525-3163"]}, DOI={10.1093/jas/skaa054.368}, abstractNote={Abstract
               Puberty onset in gilts is an awakening of the hypothalamic-pituitary-ovarian axis that is the result of reduced estradiol-negative feedback at the level of the hypothalamus which yields elevated gonadotropin secretion from the anterior pituitary. Given the importance of hypothalamic kisspeptin and neurokinin B (NKB) signaling for the onset of puberty in other species, the objective of this study was to determine if gilts selected for early pubertal onset (SELECT) would display measurable differences within the hypothalamus (i.e. increased expression of kisspeptin and NKB) and within the ovary (i.e. increased ovarian mass) compared to age-matched and weight-matched gilts (CONTROL) that achieve puberty 20 days later than SELECT gilts. Gilts were sacrificed at three timepoints: Timepoint A, both groups were determined to be prepubertal (n=6/group), Timepoint B, SELECT gilts were determined to be pubertal and CONTROL gilts were determined to be prepubertal (n=6/group), and Timepoint C, both groups were determined to be pubertal (n=6/group). All animals were euthanized, heads were perfused with 8 L of 4% paraformaldehyde, and ovaries were harvested. Brain tissue was removed post-fixation, submerged in fixative for 24 hrs followed by 20% sucrose until sectioned for immunohistochemistry. Ovarian mass tended (p≤0.10) to be greater for SELECT gilts on the right ovary (4.34 vs. 3.67 g) and the left ovary (4.49 vs. 3.68 g) when compared to CONTROL (Timepoints A and C), and at Timepoint B right ovary mass from SELECT gilts was heavier than CONTROL gilts (p< 0.05; 7.22 vs. 4.65 g). Hypothalamic immunohistochemistry for kisspeptin and NKB revealed differences in neuronal fiber density between both groups at various timepoints. Therefore, we conclude that gilts genetically selected for early puberty do so via changes within the hypothalamus that increase gonadotropin secretion and, in turn, stimulate ovarian growth to ultimately advance the timing of puberty onset.}, journal={JOURNAL OF ANIMAL SCIENCE}, author={Harlow, KaLynn and Renwick, Allison and Shuping, Sydney and Sommer, Jeff and Knauer, Mark and Nestor, Casey}, year={2020}, month={Nov}, pages={212–212} }
 @article{merkley_shuping_nestor_2020, title={Neuronal networks that regulate gonadotropin-releasing hormone/luteinizing hormone secretion during undernutrition: evidence from sheep}, volume={3}, url={https://doi.org/10.1016/j.domaniend.2020.106469}, DOI={10.1016/j.domaniend.2020.106469}, abstractNote={Gonadotropin-releasing hormone (GnRH) neurons are the final common conduit from the central nervous system in the reproductive axis, controlling luteinizing hormone (LH) secretion from the gonadotropes of the anterior pituitary. Although it is generally accepted that undernutrition inhibits GnRH/LH secretion, the central mechanisms that underlie the link between energy balance and reproduction remain to be fully elucidated. Sheep have been a longstanding and invaluable animal model for examination of the nutritional regulation of GnRH/LH secretion, given their ability to serve a biomedical and agricultural purpose. In this review, we summarize work that has used the ovine model to examine the central mechanisms whereby undernutrition regulates GnRH/LH secretion. Specifically, we focus our attention to the arcuate nucleus of the hypothalamus and on neurons that express kisspeptin, neurokinin B, dynorphin, proopiomelanocortin, and neuropeptide y/agouti-related peptide (NPY/AgRP). We examine their roles in mediating the effects of leptin and insulin and their effects on LH during undernutrition, as well as their regulation under conditions of undernutrition. This review will also highlight the interactions between the aforementioned neuronal networks themselves, which may be important for our understanding of the roles each play in relaying information regarding energy status during times of undernutrition to ultimately regulate GnRH/LH secretion.}, journal={Domestic Animal Endocrinology}, publisher={Elsevier BV}, author={Merkley, C.M. and Shuping, S.L. and Nestor, C.C}, year={2020}, month={Oct}, pages={106469} }
 @misc{nestor_bedenbaugh_hileman_coolen_lehman_goodman_2018, title={Regulation of GnRH pulsatility in ewes}, volume={156}, ISSN={["1470-1626"]}, DOI={10.1530/REP-18-0127}, abstractNote={Early work in ewes provided a wealth of information on the physiological regulation of pulsatile gonadotropin-releasing hormone (GnRH) secretion by internal and external inputs. Identification of the neural systems involved, however, was limited by the lack of information on neural mechanisms underlying generation of GnRH pulses. Over the last decade, considerable evidence supported the hypothesis that a group of neurons in the arcuate nucleus that contain kisspeptin, neurokinin B and dynorphin (KNDy neurons) are responsible for synchronizing secretion of GnRH during each pulse in ewes. In this review, we describe our current understanding of the neural systems mediating the actions of ovarian steroids and three external inputs on GnRH pulsatility in light of the hypothesis that KNDy neurons play a key role in GnRH pulse generation. In breeding season adults, estradiol (E2) and progesterone decrease GnRH pulse amplitude and frequency, respectively, by actions on KNDy neurons, with E2decreasing kisspeptin and progesterone increasing dynorphin release onto GnRH neurons. In pre-pubertal lambs, E2inhibits GnRH pulse frequency by decreasing kisspeptin and increasing dynorphin release, actions that wane as the lamb matures to allow increased pulsatile GnRH secretion at puberty. Less is known about mediators of undernutrition and stress, although some evidence implicates kisspeptin and dynorphin, respectively, in the inhibition of GnRH pulse frequency by these factors. During the anoestrus, inhibitory photoperiod acting via melatonin activates A15 dopaminergic neurons that innervate KNDy neurons; E2increases dopamine release from these neurons to inhibit KNDy neurons and suppress the frequency of kisspeptin and GnRH release.}, number={3}, journal={REPRODUCTION}, author={Nestor, Casey C. and Bedenbaugh, Michelle N. and Hileman, Stanley M. and Coolen, Lique M. and Lehman, Michael N. and Goodman, Robert L.}, year={2018}, month={Sep}, pages={R83–R99} }
 @article{padilla_qiu_nestor_zhang_smith_whiddon_rønnekleiv_kelly_palmiter_2017, title={AgRP to Kiss1 neuron signaling links nutritional state and fertility}, volume={114}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.1621065114}, DOI={10.1073/pnas.1621065114}, abstractNote={Significance
          Women suffering from malnutrition and athletes with low body fat become infertile as a result of low gonadotropin secretion. Gonadotropin release is determined by a neural endocrine circuit; however, the metabolic cues that are responsible for attenuating this axis during starvation remain unclear. Here, we find that starvation-activated agouti-related peptide (AgRP) neurons can inhibit the reproductive neuroendocrine circuit. Furthermore, artificial activation of genetically defined AgRP neurons is sufficient to delay estrous cycle length and parturition in female mice. This work demonstrates a mechanism by which AgRP neurons can relay metabolic information to the fertility axis during starvation.}, number={9}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Padilla, Stephanie L. and Qiu, Jian and Nestor, Casey C and Zhang, Chunguang and Smith, Arik W. and Whiddon, Benjamin B. and Rønnekleiv, Oline K. and Kelly, Martin J. and Palmiter, Richard D.}, year={2017}, month={Feb}, pages={2413–2418} }
 @article{estradiol protects proopiomelanocortin neurons against insulin resistance._2018, url={http://europepmc.org/articles/PMC5774249}, DOI={10.1210/en.2017-00793}, abstractNote={Insulin resistance is at the core of the metabolic syndrome, and men exhibit a higher incidence of metabolic syndrome than women in early adult life, but this sex advantage diminishes sharply when women reach the postmenopausal state. Because 17β-estradiol (E2) augments the excitability of the anorexigenic proopiomelanocortin (POMC) neurons, we investigated the neuroprotective effects of E2 against insulin resistance in POMC neurons from diet-induced obese (DIO) female and male mice. The efficacy of insulin to activate canonical transient receptor potential 5 (TRPC5) channels and depolarize POMC neurons was significantly reduced in DIO male mice but not in DIO female mice. However, the insulin response in POMC neurons was abrogated in ovariectomized DIO females but restored with E2 replacement. E2 increased T-type calcium channel Cav3.1 messenger RNA (mRNA) expression and whole-cell currents but downregulated stromal-interaction molecule 1 mRNA, which rendered POMC neurons more excitable and responsive to insulin-mediated TRPC5 channel activation. Moreover, E2 prevented the increase in suppressor of cytokine signaling-3 mRNA expression with DIO as seen in DIO males. As proof of principle, insulin [intracerebroventricular injection into the third ventricle (ICV)] decreased food intake and increased metabolism in female but not male guinea pigs fed a high-fat diet. The uncoupling of the insulin receptor from its downstream effector system was corroborated by the reduced expression of phosphorylated protein kinase B in the arcuate nucleus of male but not female guinea pigs following insulin. Therefore, E2 protects female POMC neurons from insulin resistance by enhancing POMC neuronal excitability and the coupling of insulin receptor to TRPC5 channel activation.}, journal={Endocrinology}, year={2018}, month={Feb} }
 @article{agouti-related peptide neural circuits mediate adaptive behaviors in the starved state._2016, url={http://europepmc.org/articles/PMC4846501}, DOI={10.1038/nn.4274}, abstractNote={Starving animals are less likely to defend their home territory and more likely to engage in risky foraging behaviors. This work describes a circuit involving hypothalamic AgRP neurons projecting to neurons in the medial nucleus of the amygdala and their projections to the bed nucleus of the stria terminalis, which, when activated, mimics these behaviors in mice that are well fed. 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.}, journal={Nature neuroscience}, year={2016}, month={Mar} }
 @article{qiu_nestor_zhang_padilla_palmiter_kelly_rønnekleiv_2016, title={High-frequency stimulation-induced peptide release synchronizes arcuate kisspeptin neurons and excites GnRH neurons}, volume={5}, ISSN={2050-084X}, url={http://dx.doi.org/10.7554/eLife.16246}, DOI={10.7554/eLife.16246}, abstractNote={Kisspeptin (Kiss1) and neurokinin B (NKB) neurocircuits are essential for pubertal development and fertility. Kisspeptin neurons in the hypothalamic arcuate nucleus (Kiss1ARH) co-express Kiss1, NKB, dynorphin and glutamate and are postulated to provide an episodic, excitatory drive to gonadotropin-releasing hormone 1 (GnRH) neurons, the synaptic mechanisms of which are unknown. We characterized the cellular basis for synchronized Kiss1ARH neuronal activity using optogenetics, whole-cell electrophysiology, molecular pharmacology and single cell RT-PCR in mice. High-frequency photostimulation of Kiss1ARH neurons evoked local release of excitatory (NKB) and inhibitory (dynorphin) neuropeptides, which were found to synchronize the Kiss1ARH neuronal firing. The light-evoked synchronous activity caused robust excitation of GnRH neurons by a synaptic mechanism that also involved glutamatergic input to preoptic Kiss1 neurons from Kiss1ARH neurons. We propose that Kiss1ARH neurons play a dual role of driving episodic secretion of GnRH through the differential release of peptide and amino acid neurotransmitters to coordinate reproductive function.}, journal={eLife}, publisher={eLife Sciences Publications, Ltd}, author={Qiu, Jian and Nestor, Casey C and Zhang, Chunguang and Padilla, Stephanie L and Palmiter, Richard D and Kelly, Martin J and Rønnekleiv, Oline K}, year={2016}, month={Aug} }
 @article{nestor_qiu_padilla_zhang_bosch_fan_palmiter_rønnekleiv_kelly_aicher_2016, title={Optogenetic Stimulation of Arcuate Nucleus Kiss1 Neurons Reveals a Steroid-Dependent Glutamatergic Input to POMC and AgRP Neurons in Male Mice}, volume={30}, url={http://europepmc.org/articles/PMC4884339}, DOI={10.1210/me.2016-1026}, abstractNote={Abstract
               Kisspeptin (Kiss1) neurons are essential for reproduction, but their role in the control of energy balance and other homeostatic functions remains unclear. Proopiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons, located in the arcuate nucleus (ARC) of the hypothalamus, integrate numerous excitatory and inhibitory inputs to ultimately regulate energy homeostasis. Given that POMC and AgRP neurons are contacted by Kiss1 neurons in the ARC (Kiss1ARC) and they express androgen receptors, Kiss1ARC neurons may mediate the orexigenic action of testosterone via POMC and/or AgRP neurons. Quantitative PCR analysis of pooled Kiss1ARC neurons revealed that mRNA levels for Kiss1 and vesicular glutamate transporter 2 were higher in castrated male mice compared with gonad-intact males. Single-cell RT-PCR analysis of yellow fluorescent protein (YFP) ARC neurons harvested from males injected with AAV1-EF1α-DIO-ChR2:YFP revealed that 100% and 88% expressed mRNAs for Kiss1 and vesicular glutamate transporter 2, respectively. Whole-cell, voltage-clamp recordings from nonfluorescent postsynaptic ARC neurons showed that low frequency photo-stimulation (0.5 Hz) of Kiss1-ChR2:YFP neurons elicited a fast glutamatergic inward current in POMC and AgRP neurons. Paired-pulse, photo-stimulation revealed paired-pulse depression, which is indicative of greater glutamate release, in the castrated male mice compared with gonad-intact male mice. Group I and group II metabotropic glutamate receptor agonists depolarized and hyperpolarized POMC and AgRP neurons, respectively, which was mimicked by high frequency photo-stimulation (20 Hz) of Kiss1ARC neurons. Therefore, POMC and AgRP neurons receive direct steroid- and frequency-dependent glutamatergic synaptic input from Kiss1ARC neurons in male mice, which may be a critical pathway for Kiss1 neurons to help coordinate energy homeostasis and reproduction.}, number={6}, journal={Molecular Endocrinology}, author={Nestor, CC and Qiu, J and Padilla, SL and Zhang, C and Bosch, MA and Fan, W and Palmiter, RD and Rønnekleiv, OK and Kelly, MJ and Aicher, SA}, year={2016}, pages={630–644} }
 @article{qiu_zhang_borgquist_nestor_smith_bosch_ku_wagner_rønnekleiv_kelly_2014, title={Insulin Excites Anorexigenic Proopiomelanocortin Neurons via Activation of Canonical Transient Receptor Potential Channels}, volume={19}, ISSN={1550-4131}, url={http://dx.doi.org/10.1016/j.cmet.2014.03.004}, DOI={10.1016/j.cmet.2014.03.004}, abstractNote={<h2>Summary</h2> Proopiomelanocortin (POMC) neurons within the hypothalamic arcuate nucleus are vital anorexigenic neurons. Although both the leptin and insulin receptors are coupled to the activation of phosphatidylinositide 3 kinase (PI3K) in POMC neurons, they are thought to have disparate actions on POMC excitability. Using whole-cell recording and selective pharmacological tools, we have found that, similar to leptin, purified insulin depolarized POMC and adjacent kisspeptin neurons via activation of TRPC5 channels, which are highly expressed in these neurons. In contrast, insulin hyperpolarized and inhibited NPY/AgRP neurons via activation of K<sub>ATP</sub> channels. Moreover, Zn<sup>2+</sup>, which is found in insulin formulations at nanomolar concentrations, inhibited POMC neurons via activation of K<sub>ATP</sub> channels. Finally, as predicted, insulin given intracerebroventrically robustly inhibited food intake and activated c-<i>fos</i> expression in arcuate POMC neurons. Our results show that purified insulin excites POMC neurons in the arcuate nucleus, which we propose is a major mechanism by which insulin regulates energy homeostasis.}, number={4}, journal={Cell Metabolism}, publisher={Elsevier BV}, author={Qiu, Jian and Zhang, Chunguang and Borgquist, Amanda and Nestor, Casey C and Smith, Arik W. and Bosch, Martha A. and Ku, Stephen and Wagner, Edward J. and Rønnekleiv, Oline K. and Kelly, Martin J.}, year={2014}, month={Apr}, pages={682–693} }
 @article{porter_hileman_hardy_nestor_lehman_goodman_2014, title={Neurokinin-3 Receptor Activation in the Retrochiasmatic Area is Essential for the Full Pre-Ovulatory Luteinising Hormone Surge in Ewes}, volume={26}, ISSN={0953-8194}, url={http://dx.doi.org/10.1111/jne.12180}, DOI={10.1111/jne.12180}, abstractNote={Neurokinin B (NKB) is essential for human reproduction and has been shown to stimulate luteinising hormone (LH) secretion in several species, including sheep. Ewes express the neurokinin‐3 receptor (NK3R) in the retrochiasmatic area (RCh) and there is one report that placement of senktide, an NK3R agonist, therein stimulates LH secretion that resembles an LH surge in ewes. In the present study, we first confirmed that local administration of senktide to the RCh produced a surge‐like increase in LH secretion, and then tested the effects of this agonist in two other areas implicated in the control of LH secretion and where NK3R is found in high abundance: the preoptic area (POA) and arcuate nucleus (ARC). Bilateral microimplants containing senktide induced a dramatic surge‐like increase in LH when given in the POA similar to that seen with RCh treatment. By contrast, senktide treatment in the ARC resulted in a much smaller but significant increase in LH concentrations suggestive of an effect on tonic secretion. The possible role of POA and RCh NK3R activation in the LH surge was next tested by treating ewes with SB222200, an NK3R antagonist, in each area during an oestradiol‐induced LH surge. SB222200 in the RCh, but not in the POA, reduced the LH surge amplitude by approximately 40% compared to controls, indicating that NK3R activation in the former region is essential for full expression of the pre‐ovulatory LH surge. Based on these data, we propose that the actions of NKB in the RCh are an important component of the pre‐ovulatory LH surge in ewes.}, number={11}, journal={Journal of Neuroendocrinology}, publisher={Wiley}, author={Porter, K. L. and Hileman, S. M. and Hardy, S. L. and Nestor, C. C and Lehman, M. N. and Goodman, R. L.}, year={2014}, month={Oct}, pages={776–784} }
 @article{rønnekleiv_fang_zhang_nestor_mao_kelly_2014, title={Research Resource: Gene Profiling of G Protein–Coupled Receptors in the Arcuate Nucleus of the Female}, volume={28}, ISSN={0888-8809 1944-9917}, url={http://dx.doi.org/10.1210/me.2014-1103}, DOI={10.1210/me.2014-1103}, abstractNote={The hypothalamic arcuate nucleus controls many critical homeostatic functions including energy homeostasis, reproduction, and motivated behavior. Although G protein-coupled receptors (GPCRs) are involved in the regulation of these functions, relatively few of the GPCRs have been identified specifically within the arcuate nucleus. Here, using TaqMan low-density arrays we quantified the mRNA expression of nonolfactory GPCRs in mouse arcuate nucleus. An unprecedented number of GPCRs (total of 292) were found to be expressed, of which 183 were known and 109 were orphan GPCRs. The known GPCR genes expressed were classified into several functional clusters including hormone/neurotransmitter, growth factor, angiogenesis and vasoactivity, inflammation and immune system, and lipid messenger receptors. The plethora of orphan genes expressed in the arcuate nucleus were classified into 5 structure-related classes including class A (rhodopsin-like), class B (adhesion), class C (other GPCRs), nonsignaling 7-transmembrane chemokine-binding proteins, and other 7-transmembrane proteins. Therefore, for the first time, we provide a quantitative estimate of the numerous GPCRs expressed in the hypothalamic arcuate nucleus. Finally, as proof of principle, we documented the expression and function of one of these receptor genes, the glucagon-like peptide 1 receptor (Glp1r), which was highly expressed in the arcuate nucleus. Single-cell RT-PCR revealed that Glp1r mRNA was localized in proopiomelanocortin neurons, and using whole-cell recording we found that the glucagon-like peptide 1-selective agonist exendin-4 robustly excited proopiomelanocortin neurons. Thus, the quantitative GPCR data emphasize the complexity of the hypothalamic arcuate nucleus and furthermore provide a valuable resource for future neuroendocrine/endocrine-related experiments.}, number={8}, journal={Molecular Endocrinology}, publisher={The Endocrine Society}, author={Rønnekleiv, Oline K. and Fang, Yuan and Zhang, Chunguang and Nestor, Casey C. and Mao, Peizhong and Kelly, Martin J.}, year={2014}, month={Aug}, pages={1362–1380} }
 @article{nestor_kelly_rønnekleiv_2014, title={Cross-talk between reproduction and energy homeostasis: central impact of estrogens, leptin and kisspeptin signaling}, volume={17}, ISSN={1868-1891 1868-1883}, url={http://dx.doi.org/10.1515/hmbci-2013-0050}, DOI={10.1515/hmbci-2013-0050}, abstractNote={Abstract
               The central nervous system receives hormonal cues (e.g., estrogens and leptin, among others) that influence reproduction and energy homeostasis. 17β-estradiol (E2) is known to regulate gonadotropin-releasing hormone (GnRH) secretion via classical steroid signaling and rapid non-classical membrane-initiated signaling. Because GnRH neurons are void of leptin receptors, the actions of leptin on these neurons must be indirect. Although it is clear that the arcuate nucleus of the hypothalamus is the primary site of overlap between these two systems, it is still unclear which neural network(s) participate in the cross-talk of E2 and leptin, two hormones essential for reproductive function and metabolism. Herein we review the progress made in understanding the interactions between reproduction and energy homeostasis by focusing on the advances made to understand the cellular signaling of E2 and leptin on three neural networks: kisspeptin, pro-opiomelanocortin (POMC) and neuropeptide Y (NPY). Although critical in mediating the actions of E2 and leptin, considerable work still remains to uncover how these neural networks interact in vivo.}, number={3}, journal={Hormone Molecular Biology and Clinical Investigation}, publisher={Walter de Gruyter GmbH}, author={Nestor, Casey C. and Kelly, Martin J. and Rønnekleiv, Oline K.}, year={2014}, month={Jan} }
 @article{nestor_coolen_nesselrod_valent_connors_hileman_cheng_lehman_goodman_2013, title={Evidence that Orphanin FQ Mediates Progesterone Negative Feedback in the Ewe}, volume={154}, ISSN={0013-7227 1945-7170}, url={http://dx.doi.org/10.1210/en.2013-1274}, DOI={10.1210/en.2013-1274}, abstractNote={Orphanin FQ (OFQ), a member of the opioid family, is found in many areas of the hypothalamus and, when given centrally OFQ inhibits episodic LH secretion in rodents and sheep. Because GnRH neurons are devoid of the appropriate receptors to mediate steroid negative feedback directly, neurons that release OFQ may be involved. Using immunocytochemistry, we first determined that most OFQ neurons in the arcuate nucleus (ARC) and other hypothalamic regions of luteal phase ewes contained both estrogen receptor α and progesterone (P) receptor. Given a similar high degree of steroid receptor colocalization in other ARC subpopulations, we examined whether OFQ neurons of the ARC contained those other neuropeptides and neurotransmitters. OFQ did not colocalize with kisspeptin, tyrosine hydroxylase, or agouti-related peptide, but all ARC OFQ neurons coexpressed proopiomelanocortin. To test for a role for endogenous OFQ, we examined the effects of an OFQ receptor antagonist, [Nphe1,Arg14,Lys15]Nociceptin-NH2 (UFP-101) (30 nmol intracerebroventricular/h), on LH secretion in steroid-treated ewes in the breeding season and ovary-intact ewes in anestrus. Ovariectomized ewes with luteal phase concentrations of P and estradiol showed a significant increase in LH pulse frequency during infusion of UFP-101 (4.5 ± 0.5 pulses/6 h) compared with saline infusion (2.6 ± 0.4 pulses/6 h), whereas ewes implanted with only estradiol did not. Ovary-intact anestrous ewes displayed no significant differences in LH pulse amplitude or frequency during infusion of UFP-101. Therefore, we conclude that OFQ mediates, at least in part, the negative feedback action of P on GnRH/LH pulse frequency in sheep.}, number={11}, journal={Endocrinology}, publisher={The Endocrine Society}, author={Nestor, Casey C and Coolen, Lique M. and Nesselrod, Gail L. and Valent, Miro and Connors, John M. and Hileman, Stanley M. and Cheng, Guanliang and Lehman, Michael N. and Goodman, Robert L.}, year={2013}, month={Nov}, pages={4249–4258} }
 @article{goodman_hileman_nestor_porter_connors_hardy_millar_cernea_coolen_lehman_et al._2013, title={Kisspeptin, Neurokinin B, and Dynorphin Act in the Arcuate Nucleus to Control Activity of the GnRH Pulse Generator in Ewes}, volume={154}, ISSN={0013-7227 1945-7170}, url={http://dx.doi.org/10.1210/en.2013-1331}, DOI={10.1210/en.2013-1331}, abstractNote={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 to GnRH receptors, orphanin-FQ receptors, and the N-methyl-D-aspartate glutamate receptor had no effect on episodic LH secretion. We thus 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 for NKB and dynorphin within the KNDy neural network and raise the possibility that kisspeptin contributes to the control of GnRH pulse frequency in addition to its established role as an output signal from KNDy neurons that drives GnRH pulses.}, number={11}, journal={Endocrinology}, publisher={The Endocrine Society}, author={Goodman, Robert L. and Hileman, Stanley M. and Nestor, Casey C and Porter, Katrina L. and Connors, John M. and Hardy, Steve L. and Millar, Robert P. and Cernea, Maria and Coolen, Lique M. and Lehman, Michael N. and et al.}, year={2013}, month={Nov}, pages={4259–4269} }
 @article{goodman_maltby_millar_hileman_nestor_whited_tseng_coolen_lehman_2012, title={Evidence That Dopamine Acts via Kisspeptin to Hold GnRH Pulse Frequency in Check in Anestrous Ewes}, volume={153}, ISSN={0013-7227 1945-7170}, url={http://dx.doi.org/10.1210/en.2012-1611}, DOI={10.1210/en.2012-1611}, abstractNote={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) we tested the ability of local administration of sulpiride, a D2-R antagonist, into the ARC to 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.}, number={12}, journal={Endocrinology}, publisher={The Endocrine Society}, author={Goodman, Robert L. and Maltby, Matthew J. and Millar, Robert P. and Hileman, Stanley M. and Nestor, Casey C and Whited, Brant and Tseng, Ashlie S. and Coolen, Lique M. and Lehman, Michael N.}, year={2012}, month={Dec}, pages={5918–5927} }
 @article{nestor_briscoe_davis_valent_goodman_hileman_2012, title={Evidence of a Role for Kisspeptin and Neurokinin B in Puberty of Female Sheep}, volume={153}, ISSN={0013-7227 1945-7170}, url={http://dx.doi.org/10.1210/en.2011-2009}, DOI={10.1210/en.2011-2009}, abstractNote={Puberty onset in female sheep is marked by a decrease in estradiol-negative feedback, allowing for the increase in GnRH and LH pulses that heralds the first ovulation. Based on recent genetic studies in humans, two possible neuropeptides that could promote puberty onset are kisspeptin and neurokinin B (NKB). Our first experiment determined whether the NKB agonist, senktide, could stimulate LH secretion in prepubertal ewes. A second study used prepubertal and postpubertal ewes that were intact or ovariectomized (OVX) to test the hypothesis that expression of kisspeptin and NKB in the arcuate nucleus increased postpubertally. For comparison, kisspeptin and NKB expression in age-matched intact, and castrated males were also examined. In experiment 1, the percentage of ewes showing an LH pulse immediately after injection of senktide (100 μg, 60%; 500 μg, 100%) was greater than that for water-injected controls (experiment 1a, 25%; experiment 1b, 20%). In experiment 2, kisspeptin-positive cell numbers in the arcuate nucleus increased after puberty in intact females and were increased by OVX in prepubertal but not postpubertal ewes. Changes in kisspeptin cell numbers were paralleled by changes in kisspeptin-close contacts onto GnRH neurons in the medial preoptic area. NKB cell numbers did not differ significantly between intact prepubertal and postpubertal ewes but increased with OVX in both age groups. NKB fiber immunoreactivity was greater in postpubertal than in prepubertal intact ewes. In age-matched males, kisspeptin and NKB cell numbers increased with castration, but decreased with age. These results support the hypothesis that kisspeptin is a gatekeeper to female ovine puberty and raise the possibility that NKB may also play a role, albeit through different means.}, number={6}, journal={Endocrinology}, publisher={The Endocrine Society}, author={Nestor, Casey C and Briscoe, Amanda M.S. and Davis, Shay M. and Valent, Miro and Goodman, Robert L. and Hileman, Stanley M.}, year={2012}, month={Jun}, pages={2756–2765} }
 @article{goodman_holaskova_nestor_connors_billings_valent_lehman_hileman_2011, title={Evidence that the Arcuate Nucleus Is an Important Site of Progesterone Negative Feedback in the Ewe}, volume={152}, ISSN={0013-7227 1945-7170}, url={http://dx.doi.org/10.1210/en.2011-0195}, DOI={10.1210/en.2011-0195}, abstractNote={There is now considerable evidence that dynorphin neurons mediate the negative feedback actions of progesterone to inhibit GnRH and LH pulse frequency, but the specific neurons have yet to be identified. In ewes, dynorphin neurons in the arcuate nucleus (ARC) and preoptic area (POA) are likely candidates based on colocalization with progesterone receptors. These studies tested the hypothesis that progesterone negative feedback occurs in either the ARC or POA by determining whether microimplants of progesterone into either site would inhibit LH pulse frequency (study 1) and whether microimplants of the progesterone receptor antagonist, RU486, would disrupt the inhibitory effects of peripheral progesterone (study 2). Both studies were done in ovariectomized (OVX) and estradiol-treated OVX ewes. In study 1, no inhibitory effects of progesterone were observed during treatment in either area. In study 2, microimplants of RU486 into the ARC disrupted the negative-feedback actions of peripheral progesterone treatments on LH pulse frequency in both OVX and OVX+estradiol ewes. In contrast, microimplants of RU486 into the POA had no effect on the ability of systemic progesterone to inhibit LH pulse frequency. We thus conclude that the ARC is one important site of progesterone-negative feedback in the ewe. These data, which are the first evidence on the neural sites in which progesterone inhibits GnRH pulse frequency in any species, are consistent with the hypothesis that ARC dynorphin neurons mediate this action of progesterone.}, number={9}, journal={Endocrinology}, publisher={The Endocrine Society}, author={Goodman, Robert L. and Holaskova, Ida and Nestor, Casey C. and Connors, John M. and Billings, Heather J. and Valent, Miro and Lehman, Michael N. and Hileman, Stanley M.}, year={2011}, month={Sep}, pages={3451–3460} }
 @article{lin_huang_zhang_hollander_frisbee_martin_nestor_goodman_yu_2010, title={Inactivation of L-type calcium channel modulated by HCN2 channel}, volume={298}, ISSN={0363-6143 1522-1563}, url={http://dx.doi.org/10.1152/ajpcell.00355.2009}, DOI={10.1152/ajpcell.00355.2009}, abstractNote={ Ca2+ entry is delicately controlled by inactivation of L-type calcium channel (LTCC) composed of the pore-forming subunit α1C and the auxiliary subunits β1 and α2δ. Calmodulin is the key protein that interacts with the COOH-terminal motifs of α1C, leading to the fine control of LTCC inactivation. In this study we show evidence that a hyperpolarization-activated cyclic nucleotide-gated channel, HCN2, can act as a nonchannel regulatory protein to narrow the L-type Ca2+ channel current-voltage curve. In the absence of LTCC auxiliary subunits, HCN2 can induce α1C inactivation. Without α2δ, HCN2-induced fast inactivation of α1C requires calmodulin. With α2δ, the α1C/HCN2/α2δ channel inactivation does not require calmodulin. In contrast, β1-subunit plays a relatively minor role in the interaction of α1C with HCN2. The NH2 terminus of HCN2 and the IQ motif of α1C subunit are required for α1C/HCN2 channel interaction. Ca2+ channel inactivation is significantly slowed in hippocampus neurons (HNs) overexpressing HCN2 mutant lacking NH2 terminus and accelerated in HNs overexpressing the wild-type HCN2 compared with HN controls. Collectively, these results revealed a potentially novel protection mechanism for achieving the LTCC inactivation via interaction with HCN2. }, number={5}, journal={American Journal of Physiology-Cell Physiology}, publisher={American Physiological Society}, author={Lin, Yen-Chang and Huang, Jianying and Zhang, Qi and Hollander, John M. and Frisbee, Jefferson C. and Martin, Karen H. and Nestor, Casey and Goodman, Robert and Yu, Han-Gang}, year={2010}, month={May}, pages={C1029–C1037} }
 @article{billings_connors_altman_hileman_holaskova_lehman_mcmanus_nestor_jacobs_goodman_et al._2010, title={Neurokinin B Acts via the Neurokinin-3 Receptor in the Retrochiasmatic Area to Stimulate Luteinizing Hormone Secretion in Sheep}, volume={151}, ISSN={0013-7227 1945-7170}, url={http://dx.doi.org/10.1210/en.2010-0174}, DOI={10.1210/en.2010-0174}, abstractNote={Recent data have demonstrated that mutations in the receptor for neurokinin B (NKB), the NK-3 receptor (NK3R), produce hypogonadotropic hypogonadism in humans. These data, together with reports that NKB expression increases after ovariectomy and in postmenopausal women, have led to the hypothesis that this tachykinin is an important stimulator of GnRH secretion. However, the NK3R agonist, senktide, inhibited LH secretion in rats and mice. In this study, we report that senktide stimulates LH secretion in ewes. A dramatic increase in LH concentrations to levels close to those observed during the preovulatory LH surge was observed after injection of 1 nmol senktide into the third ventricle during the follicular, but not in the luteal, phase. Similar increases in LH secretion occurred after insertion of microimplants containing this agonist into the retrochiasmatic area (RCh) in anestrous or follicular phase ewes. A low-dose microinjection (3 pmol) of senktide into the RCh produced a smaller but significant increase in LH concentrations in anestrous ewes. Moreover, NK3R immunoreactivity was clearly evident in the RCh, although it was not found in A15 dopaminergic cell bodies in this region. These data provide evidence that NKB stimulates LH (and presumably GnRH) secretion in ewes and point to the RCh as one important site of action. Based on these data, and the effects of NK3R mutations in humans, we hypothesize that NKB plays an important stimulatory role in the control of GnRH and LH secretion in nonrodent species.}, number={8}, journal={Endocrinology}, publisher={The Endocrine Society}, author={Billings, Heather J. and Connors, John M. and Altman, Stephanie N. and Hileman, Stanley M. and Holaskova, Ida and Lehman, Michael N. and McManus, Christina J. and Nestor, Casey C and Jacobs, Britni H. and Goodman, Robert L. and et al.}, year={2010}, month={Aug}, pages={3836–3846} }
 @article{bogusz_hardy_lehman_connors_hileman_sliwowska_billings_mcmanus_valent_singh_et al._2008, title={Evidence that γ-Aminobutyric Acid Is Part of the Neural Circuit Mediating Estradiol Negative Feedback in Anestrous Ewes}, volume={149}, ISSN={0013-7227 1945-7170}, url={http://dx.doi.org/10.1210/en.2007-1362}, DOI={10.1210/en.2007-1362}, abstractNote={Seasonal anestrus in ewes is driven by an increase in response to estradiol (E2) negative feedback. Compelling evidence indicates that inhibitory A15 dopaminergic (DA) neurons mediate the increased inhibitory actions of E2 in anestrus, but these neurons do not contain estrogen receptors. Therefore, we have proposed that estrogen-responsive afferents to A15 neurons are part of the neural circuit mediating E2 negative feedback in anestrus. This study examined the possible role of afferents containing γ-aminobutyric acid (GABA) and nitric oxide (NO) in modulating the activity of A15 neurons. Local administration of NO synthase inhibitors to the A15 had no effect on LH, but GABA receptor ligands produced dramatic changes. Administration of either a GABAA or GABAB receptor agonist to the A15 increased LH secretion in ovary-intact ewes, suggesting that GABA inhibits A15 neural activity. In ovariectomized anestrous ewes, the same doses of GABA receptor agonist had no effect, but combined administration of a GABAA and GABAB receptor antagonist to the A15 inhibited LH secretion. These data are consistent with the hypothesis that endogenous GABA release within the A15 is low in ovary-intact anestrous ewes and elevated after ovariectomy. Using dual immunocytochemistry, we observed that GABAergic varicosities make close contacts on to A15 neurons and that A15 neurons contain both the GABAA-α1 and the GABAB-R1 receptor subunits. Based on these data, we propose that in anestrous ewes, E2 inhibits release of GABA from afferents to A15 DA neurons, increasing the activity of these DA neurons and thus suppressing episodic secretion of GnRH and LH.}, number={6}, journal={Endocrinology}, publisher={The Endocrine Society}, author={Bogusz, Adrienne L. and Hardy, Steven L. and Lehman, Michael N. and Connors, John M. and Hileman, Stanley M. and Sliwowska, Joanna H. and Billings, Heather J. and McManus, Christina J. and Valent, Miroslav and Singh, Sushma R. and et al.}, year={2008}, month={Jun}, pages={2762–2772} }