Naomi E Rance

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• Kambhampati, M., Panditharatna, E., Yadavilli, S., Saoud, K., Lee, S., Eze, A., Almira-Suarez, M. I., Hancock, L., Bonner, E. R., Gittens, J., Stampar, M., Gaonkar, K., Resnick, A. C., Kline, C., Ho, C. Y., Waanders, A. J., Georgescu, M. M., Rance, N. E., Kim, Y., , Johnson, C., et al. (2020). Harmonization of postmortem donations for pediatric brain tumors and molecular characterization of diffuse midline gliomas. Scientific reports, 10(1), 10954.
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  Children diagnosed with brain tumors have the lowest overall survival of all pediatric cancers. Recent molecular studies have resulted in the discovery of recurrent driver mutations in many pediatric brain tumors. However, despite these molecular advances, the clinical outcomes of high grade tumors, including H3K27M diffuse midline glioma (H3K27M DMG), remain poor. To address the paucity of tissue for biological studies, we have established a comprehensive protocol for the coordination and processing of donated specimens at postmortem. Since 2010, 60 postmortem pediatric brain tumor donations from 26 institutions were coordinated and collected. Patient derived xenograft models and cell cultures were successfully created (76% and 44% of attempts respectively), irrespective of postmortem processing time. Histological analysis of mid-sagittal whole brain sections revealed evidence of treatment response, immune cell infiltration and the migratory path of infiltrating H3K27M DMG cells into other midline structures and cerebral lobes. Sequencing of primary and disseminated tumors confirmed the presence of oncogenic driver mutations and their obligate partners. Our findings highlight the importance of postmortem tissue donations as an invaluable resource to accelerate research, potentially leading to improved outcomes for children with aggressive brain tumors.
• Coborn, J. E., Lessie, R. E., Sinton, C. M., Rance, N. E., Perez-Leighton, C. E., & Teske, J. A. (2019). Noise-induced sleep disruption increases weight gain and decreases energy metabolism in female rats. International journal of obesity (2005), 43(9), 1759-1768.
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  Inadequate sleep increases obesity and environmental noise contributes to poor sleep. However, women may be more vulnerable to noise and hence more susceptible to sleep disruption-induced weight gain than men. In male rats, exposure to environmental (i.e. ambient) noise disrupts sleep and increases feeding and weight gain. However, the effects of environmental noise on sleep and weight gain in female rats are unknown. Thus, this study was designed to determine whether noise exposure would disturb sleep, increase feeding and weight gain and alter the length of the estrous cycle in female rats.
• Krajewski-Hall, S. J., Miranda Dos Santos, F., McMullen, N. T., Blackmore, E. M., & Rance, N. E. (2019). Glutamatergic Neurokinin 3 Receptor Neurons in the Median Preoptic Nucleus Modulate Heat-Defense Pathways in Female Mice. Endocrinology, 160(4), 803-816.
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  We have proposed that arcuate neurons coexpressing kisspeptin, neurokinin B, and dynorphin (KNDy neurons) contribute to hot flushes via projections to neurokinin 3 receptor (NK3R)-expressing neurons in the median preoptic nucleus (MnPO). To characterize the thermoregulatory role of MnPO NK3R neurons in female mice, we ablated these neurons using injections of saporin toxin conjugated to a selective NK3R agonist. Loss of MnPO NK3R neurons increased the core temperature (TCORE) during the light phase, with the frequency distributions indicating a regulated shift in the balance point. The increase in TCORE in the ablated mice occurred despite changes in the ambient temperature and regardless of estrogen status. We next determined whether an acute increase in ambient temperature or higher TCORE would induce Fos in preoptic enhanced green fluorescent protein (EGFP)-immunoreactive neurons in Tacr3-EGFP mice. Fos activation was increased in the MnPO but no induction of Fos was found in NK3R (EGFP-immunoreactive) neurons. Thus, MnPO NK3R neurons are not activated by warm thermosensors in the skin or viscera and are not warm-sensitive neurons. Finally, RNAscope was used to determine whether Tacr3 (NK3R) mRNA was coexpressed with vesicular glutamate transporter 2 or vesicular γ-aminobutyric acid (GABA) transporter mRNA, markers of glutamatergic and GABAergic neurotransmission, respectively. In the MnPO, 94% of NK3R neurons were glutamatergic, but in the adjacent medial preoptic area, 97% of NK3R neurons were GABAergic. Thus, NK3R neurons in the MnPO are glutamatergic and play a role in reducing TCORE but are not activated by warm thermal stimuli (internal or external). These findings suggest that KNDy neurons modulate thermosensory pathways for heat defense indirectly via a subpopulation of glutamatergic MnPO neurons that express NK3R.
• Coborn, J., Rebecca, L., Christopher, S., Rance, N. E., Claudio, P., & Jennifer, T. (2018). Noise-induced sleep disruption increases weight gain and decreases energy metabolism in female rats. International Journal of Obesity.
• Krajewski-Hall, S. J., Blackmore, E. M., McMinn, J. R., & Rance, N. E. (2018). Estradiol alters body temperature regulation in the female mouse. Temperature (Austin, Tex.), 5(1), 56-69.
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  Hot flushes are due to estrogen withdrawal and characterized by the episodic activation of heat dissipation effectors. Recent studies (in humans and rats) have implicated neurokinin 3 (NK) receptor signaling in the genesis of hot flushes. Although transgenic mice are increasingly used for biomedical research, there is limited information on how 17β-estradiol and NK receptor signaling alters thermoregulation in the mouse. In this study, a method was developed to measure tail skin temperature (T) using a small data-logger attached to the surface of the tail, which, when combined with a telemetry probe for core temperature (T), allowed us to monitor thermoregulation in freely-moving mice over long durations. We report that estradiol treatment of ovariectomized mice reduced T during the light phase (but not the dark phase) while having no effect on T or activity. Estradiol also lowered T in mice exposed to ambient temperatures ranging from 20 to 36°C. Unlike previous studies in the rat, estradiol treatment of ovariectomized mice did not reduce T during the dark phase. Subcutaneous injections of an NK receptor agonist (senktide) in ovariectomized mice caused an acute increase in T and a reduction in T, consistent with the activation of heat dissipation effectors. These changes were reduced by estradiol, suggesting that estradiol lowers the sensitivity of central thermoregulatory pathways to NK receptor activation. Overall, we show that estradiol treatment of ovariectomized mice decreases T during the light phase, reduces the thermoregulatory effects of senktide and modulates thermoregulation differently than previously described in the rat.
• Mittelman-Smith, M. A., Krajewski-Hall, S. J., McMullen, N. T., & Rance, N. E. (2016). Ablation of KNDy Neurons Results in Hypogonadotropic Hypogonadism and Amplifies the Steroid-Induced LH Surge in Female Rats. Endocrinology, 157(5), 2015-27.
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  In the human infundibular (arcuate) nucleus, a subpopulation of neurons coexpress kisspeptin and neurokinin B (NKB), 2 peptides required for normal reproductive function. A homologous group of neurons exists in the arcuate nucleus of rodents, termed KNDy neurons based on the coexpression of kisspeptin, NKB, and dynorphin. To study their function, we recently developed a method to selectively ablate KNDy neurons using NK3-SAP, a neurokinin 3 receptor agonist conjugated to saporin (SAP). Here, we ablated KNDy neurons in female rats to determine whether these neurons are required for estrous cyclicity and the steroid induced LH surge. NK3-SAP or Blank-SAP (control) was microinjected into the arcuate nucleus using stereotaxic surgery. After monitoring vaginal smears for 3-4 weeks, rats were ovariectomized and given 17β-estradiol and progesterone in a regimen that induced an afternoon LH surge. Rats were killed at the time of peak LH levels, and brains were harvested for NKB and dual labeled GnRH/Fos immunohistochemistry. In ovary-intact rats, ablation of KNDy neurons resulted in hypogonadotropic hypogonadism, characterized by low levels of serum LH, constant diestrus, ovarian atrophy with increased follicular atresia, and uterine atrophy. Surprisingly, the 17β-estradiol and progesterone-induced LH surge was 3 times higher in KNDy-ablated rats. Despite the marked increase in the magnitude of the LH surge, the number of GnRH or anterior ventral periventricular nucleus neurons expressing Fos was not significantly different between groups. Our studies show that KNDy neurons are essential for tonic levels of serum LH and estrous cyclicity and may play a role in limiting the magnitude of the LH surge.
• Cholanian, M., Krajewski-Hall, S. J., McMullen, N. T., & Rance, N. E. (2015). Chronic oestradiol reduces the dendritic spine density of KNDy (kisspeptin/neurokinin B/dynorphin) neurones in the arcuate nucleus of ovariectomised Tac2-enhanced green fluorescent protein transgenic mice. Journal of neuroendocrinology, 27(4), 253-63.
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  Neurones in the arcuate nucleus that express neurokinin B (NKB), kisspeptin and dynorphin (KNDy) play an important role in the reproductive axis. Oestradiol modulates the gene expression and somatic size of these neurones, although there is limited information available about whether their dendritic structure, a correlate of cellular plasticity, is altered by oestrogens. In the present study, we investigated the morphology of KNDy neurones by filling fluorescent neurones in the arcuate nucleus of Tac2-enhanced green fluorescent protein (EGFP) transgenic mice with biocytin. Filled neurones from ovariectomised (OVX) or OVX plus 17β-oestradiol (E2)-treated mice were visualised with anti-biotin immunohistochemistry and reconstructed in three dimensions with computer-assisted microscopy. KNDy neurones exhibited two primary dendrites, each with a few branches confined to the arcuate nucleus. Quantitative analysis revealed that E2 treatment of OVX mice decreased the cell size and dendritic spine density of KNDy neurones. The axons of KNDy neurones originated from the cell body or proximal dendrite and gave rise to local branches that appeared to terminate within the arcuate nucleus. Numerous terminal boutons were also visualised within the ependymal layer of the third ventricle adjacent to the arcuate nucleus. Axonal branches also projected to the adjacent median eminence and exited the arcuate nucleus. Confocal microscopy revealed close apposition of EGFP and gonadotrophin-releasing hormone-immunoreactive fibres within the median eminence and confirmed the presence of KNDy axon terminals in the ependymal layer of the third ventricle. The axonal branching pattern of KNDy neurones suggests that a single KNDy neurone could influence multiple arcuate neurones, tanycytes in the wall of the third ventricle, axon terminals in the median eminence and numerous areas outside of the arcuate nucleus. In parallel with its inhibitory effects on electrical excitability, E2 treatment of OVX Tac2-EGFP mice induces structural changes in the somata and dendrites of KNDy neurones.
• Mittelman-Smith, M. A., Krajewski-Hall, S. J., McMullen, N. T., & Rance, N. E. (2015). Neurokinin 3 Receptor-Expressing Neurons in the Median Preoptic Nucleus Modulate Heat-Dissipation Effectors in the Female Rat. Endocrinology, 156(7), 2552-62.
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  KNDy neurons facilitate tail skin vasodilation and modulate the effects of estradiol on thermoregulation. We hypothesize that KNDy neurons influence cutaneous vasodilation via projections to neurons in the median preoptic nucleus (MnPO) that express the neurokinin 3 receptor (NK3R). In support of this hypothesis, focal microinjections of senktide, an NK3R agonist, into the MnPO lowers core temperature (TCORE) in the female rat. To further study the role of MnPO NK3R neurons in thermoregulation, these neurons were specifically ablated using a conjugate of a selective NK3R agonist and saporin (NK3-SAP). NK3-SAP or blank-SAP (control) was injected into the MnPO/medial septum. Tail skin temperature (TSKIN) and TCORE were measured in ovariectomized rats exposed to 3 ambient temperatures (TAMBIENT) before and after estradiol-17β (E2) treatment. Before killing, we injected senktide (sc), monitored TCORE for 70 minutes, and harvested brains for Fos immunohistochemistry. Ablation of MnPO NK3R neurons lowered TSKIN at neutral and subneutral TAMBIENT regardless of E2 treatment. However, ablation did not prevent the effects of E2 on TCORE and TSKIN. In control rats, senktide injections induced hypothermia with numerous Fos-immunoreactive cells in the MnPO. In contrast, in NK3-SAP rats, senktide did not alter TCORE and minimal Fos-immunoreactive neurons were identified in the MnPO. These data show that NK3R neurons in the MnPO are required for the hypothermic effects of senktide but not for the E2 modulation of thermoregulation. The lower TSKIN in NK3-SAP-injected rats suggests that MnPO NK3R neurons, like KNDy neurons, facilitate cutaneous vasodilation, an important heat-dissipation effector.
• Cholanian, M., Krajewski-Hall, S. J., Levine, R. B., McMullen, N. T., & Rance, N. E. (2014). Electrophysiology of arcuate neurokinin B neurons in female Tac2-EGFP transgenic mice. Endocrinology, 155(7), 2555-65.
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  Neurons in the arcuate nucleus that coexpress kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons) play an important role in the modulation of reproduction by estrogens. Here, we study the anatomical and electrophysiological properties of arcuate NKB neurons in heterozygous female transgenic mice with enhanced green fluorescent protein (EGFP) under the control of the Tac2 (NKB) promoter (Tac2-EGFP mice). The onset of puberty, estrous cyclicity, and serum LH were comparable between Tac2-EGFP and wild-type mice. The location of EGFP-immunoreactive neurons was consistent with previous descriptions of Tac2 mRNA-expressing neurons in the rodent. In the arcuate nucleus, nearly 80% of EGFP neurons expressed pro-NKB-immunoreactivity. Moreover, EGFP fluorescent intensity in arcuate neurons was increased by ovariectomy and reduced by 17β-estradiol (E2) treatment. Electrophysiology of single cells in tissue slices was used to examine the effects of chronic E2 treatment on Tac2-EGFP neurons in the arcuate nucleus of ovariectomized mice. Whole-cell recordings revealed arcuate NKB neurons to be either spontaneously active or silent in both groups. E2 had no significant effect on the basic electrophysiological properties or spontaneous firing frequencies. Arcuate NKB neurons exhibited either tonic or phasic firing patterns in response to a series of square-pulse current injections. Notably, E2 reduced the number of action potentials evoked by depolarizing current injections. This study demonstrates the utility of the Tac2-EGFP mouse for electrophysiological and morphological studies of KNDy neurons in tissue slices. In parallel to E2 negative feedback on LH secretion, E2 decreased the intensity of the EGFP signal and reduced the excitability of NKB neurons in the arcuate nucleus of ovariectomized Tac2-EGFP mice.
• Rance, N. E., Dacks, P. A., Mittelman-Smith, M. A., Romanovsky, A. A., & Krajewski-Hall, S. J. (2013). Modulation of body temperature and LH secretion by hypothalamic KNDy (kisspeptin, neurokinin B and dynorphin) neurons: a novel hypothesis on the mechanism of hot flushes. Frontiers in neuroendocrinology, 34(3), 211-27.
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  Despite affecting millions of individuals, the etiology of hot flushes remains unknown. Here we review the physiology of hot flushes, CNS pathways regulating heat-dissipation effectors, and effects of estrogen on thermoregulation in animal models. Based on the marked changes in hypothalamic kisspeptin, neurokinin B and dynorphin (KNDy) neurons in postmenopausal women, we hypothesize that KNDy neurons play a role in the mechanism of flushes. In the rat, KNDy neurons project to preoptic thermoregulatory areas that express the neurokinin 3 receptor (NK3R), the primary receptor for NKB. Furthermore, activation of NK₃R in the median preoptic nucleus, part of the heat-defense pathway, reduces body temperature. Finally, ablation of KNDy neurons reduces cutaneous vasodilatation and partially blocks the effects of estrogen on thermoregulation. These data suggest that arcuate KNDy neurons relay estrogen signals to preoptic structures regulating heat-dissipation effectors, supporting the hypothesis that KNDy neurons participate in the generation of flushes.
• Rance, N., Rance, N. E., Combs, F. J., Erly, W. K., & Valentino, C. M. (2011). Best cases from the AFIP: Balamuthia mandrillaris amebic meningoencephalitis. Radiographics : a review publication of the Radiological Society of North America, Inc.
• Rance, N., Mittelman-Smith, M. A., Williams, H., Krajewski-Hall, S. J., Lai, J., Ciofi, P., McMullen, N. T., & Rance, N. E. (2012). Arcuate kisspeptin/neurokinin B/dynorphin (KNDy) neurons mediate the estrogen suppression of gonadotropin secretion and body weight. Endocrinology, 153(6).
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  Estrogen withdrawal increases gonadotropin secretion and body weight, but the critical cell populations mediating these effects are not well understood. Recent studies have focused on a subpopulation of hypothalamic arcuate neurons that coexpress estrogen receptor α, neurokinin 3 receptor (NK(3)R), kisspeptin, neurokinin B, and dynorphin for the regulation of reproduction. To investigate the function of kisspeptin/neurokinin B/dynorphin (KNDy) neurons, a novel method was developed to ablate these cells using a selective NK(3)R agonist conjugated to the ribosome-inactivating toxin, saporin (NK(3)-SAP). Stereotaxic injections of NK(3)-SAP in the arcuate nucleus ablated KNDy neurons, as demonstrated by the near-complete loss of NK(3)R, NKB, and kisspeptin-immunoreactive (ir) neurons and depletion of the majority of arcuate dynorphin-ir neurons. Selectivity was demonstrated by the preservation of proopiomelanocortin, neuropeptide Y, and GnRH-ir elements in the arcuate nucleus and median eminence. In control rats, ovariectomy (OVX) markedly increased serum LH, FSH, and body weight, and these parameters were subsequently decreased by treatment with 17β-estradiol. KNDy neuron ablation prevented the rise in serum LH after OVX and attenuated the rise in serum FSH. KNDy neuron ablation did not completely block the suppressive effects of E(2) on gonadotropin secretion, a finding consistent with redundant pathways for estrogen negative feedback. However, regardless of estrogen status, KNDy-ablated rats had lower levels of serum gonadotropins compared with controls. Surprisingly, KNDy neuron ablation prevented the dramatic effects of OVX and 17β-estradiol (E(2)) replacement on body weight and abdominal girth. These data provide evidence that arcuate KNDy neurons are essential for tonic gonadotropin secretion, the rise in LH after removal of E(2), and the E(2) modulation of body weight.
• Rance, N., Mittelman-Smith, M. A., Williams, H., Krajewski-Hall, S. J., McMullen, N. T., & Rance, N. E. (2012). Role for kisspeptin/neurokinin B/dynorphin (KNDy) neurons in cutaneous vasodilatation and the estrogen modulation of body temperature. Proceedings of the National Academy of Sciences of the United States of America, 109(48).
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  Estrogen withdrawal in menopausal women leads to hot flushes, a syndrome characterized by the episodic activation of heat dissipation effectors. Despite the extraordinary number of individuals affected, the etiology of flushes remains an enigma. Because menopause is accompanied by marked alterations in hypothalamic kisspeptin/neurokinin B/dynorphin (KNDy) neurons, we hypothesized that these neurons could contribute to the generation of flushes. To determine if KNDy neurons participate in the regulation of body temperature, we evaluated the thermoregulatory effects of ablating KNDy neurons by injecting a selective toxin for neurokinin-3 expressing neurons [NK(3)-saporin (SAP)] into the rat arcuate nucleus. Remarkably, KNDy neuron ablation consistently reduced tail-skin temperature (T(SKIN)), indicating that KNDy neurons facilitate cutaneous vasodilatation, an important heat dissipation effector. Moreover, KNDy ablation blocked the reduction of T(SKIN) by 17β-estradiol (E(2)), which occurred in the environmental chamber during the light phase, but did not affect the E(2) suppression of T(SKIN) during the dark phase. At the high ambient temperature of 33 °C, the average core temperature (T(CORE)) of ovariectomized (OVX) control rats was significantly elevated, and this value was reduced by E(2) replacement. In contrast, the average T(CORE) of OVX, KNDy-ablated rats was lower than OVX control rats at 33 °C, and not altered by E(2) replacement. These data provide unique evidence that KNDy neurons promote cutaneous vasodilatation and participate in the E(2) modulation of body temperature. Because cutaneous vasodilatation is a cardinal sign of a hot flush, these results support the hypothesis that KNDy neurons could play a role in the generation of flushes.
• Rance, N., Dacks, P. A., Krajewski, S. J., & Rance, N. E. (2011). Activation of neurokinin 3 receptors in the median preoptic nucleus decreases core temperature in the rat. Endocrinology, 152(12).
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  Estrogens have pronounced effects on thermoregulation, as illustrated by the occurrence of hot flushes secondary to estrogen withdrawal in menopausal women. Because neurokinin B (NKB) gene expression is markedly increased in the infundibular (arcuate) nucleus of postmenopausal women, and is modulated by estrogen withdrawal and replacement in multiple species, we have hypothesized that NKB neurons could play a role in the generation of flushes. There is no information, however, on whether the primary NKB receptor [neurokinin 3 receptor (NK(3)R)] modulates body temperature in any species. Here, we determine the effects of microinfusion of a selective NK(3)R agonist (senktide) into the rat median preoptic nucleus (MnPO), an important site in the heat-defense pathway. Senktide microinfusion into the rat MnPO decreased core temperature in a dose-dependent manner. The hypothermia induced by senktide was similar in ovariectomized rats with and without 17β-estradiol replacement. The hypothermic effect of senktide was prolonged in rats exposed to an ambient temperature of 29.0 C, compared with 21.5 C. Senktide microinfusion also altered tail skin vasomotion in rats exposed to an ambient temperature of 29.0 but not 21.5 C. Comparisons of the effects of senktide at different ambient temperatures indicated that the hypothermia was not secondary to thermoregulatory failure or a reduction in cold-induced thermogenesis. Other than a very mild increase in drinking, senktide microinfusion did not affect behavior. Terminal fluorescent dextran microinfusion showed targeting of the MnPO and adjacent septum, and immunohistochemical studies revealed that senktide induced a marked increase in Fos-activation in the MnPO. Because MnPO neurons expressed NK(3)R-immunoreactivity, the induction of MnPO Fos by senktide is likely a direct effect. By demonstrating that NK(3)R activation in the MnPO modulates body temperature, these studies support the hypothesis that hypothalamic NKB neurons could be involved in the generation of menopausal flushes.
• Rance, N., Dacks, P. A., Krajewski, S. J., & Rance, N. E. (2011). Ambient temperature and 17β-estradiol modify Fos immunoreactivity in the median preoptic nucleus, a putative regulator of skin vasomotion. Endocrinology, 152(7).
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  Estrogen has pronounced effects on thermoregulation, but the anatomic sites of integration between the reproductive and thermoregulatory axes are unknown. In this study, we tested whether estradiol-17β (E(2)) treatment would alter the activity of thermoregulatory brain regions responding to mild changes in ambient temperature (T(AMBIENT)). Core and tail skin temperatures were recorded at the ambient temperatures of 20, 24, or 31 C in ovariectomized (OVX) rats with and without E(2). Neuronal activity was evaluated by counting the number of Fos-immunoreactive cells in the brains of rats killed 90 min after exposure to one of the three ambient temperatures. Of 14 brain areas examined, the median preoptic nucleus (MnPO) was the only site that exhibited increased Fos immunoreactivity at the high T(AMBIENT) of 31 C. At 24 C, OVX rats exhibited increased numbers of MnPO Fos-immunoreactive cells, compared with OVX + E(2) rats. Interestingly, tail skin vasomotion and MnPO Fos expression were affected in a similar manner by T(AMBIENT) and E(2) treatment. In the arcuate nucleus and anteroventral periventricular nucleus (AVPV), Fos immunoreactivity was highest at the low T(AMBIENT) of 20 C, with inhibitory (arcuate nucleus) and stimulatory (AVPV) effects of E(2). No other areas responded to both T(AMBIENT) and E(2) treatment. These results implicate the MnPO, the arcuate nucleus, and the AVPV as sites of integration between the reproductive and thermoregulatory axes. Combined with studies showing the importance of MnPO neurons in heat-defense pathways, the MnPO emerges as a likely site for E(2) modulation of thermoregulatory vasomotion.
• Rance, N., Dacks, P. A., & Rance, N. E. (2010). Effects of estradiol on the thermoneutral zone and core temperature in ovariectomized rats. Endocrinology, 151(3).
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  Hot flushes represent a disorder of central thermoregulation characterized by the episodic activation of heat loss mechanisms. Although flushes are associated with estrogen withdrawal, there is little understanding of the effects of estrogen on thermoregulation in any species. It has been proposed that hormone withdrawal increases the sensitivity of hypothalamic neural pathways that control heat dissipation effectors. If so, we predicted that ovariectomized rats without estradiol treatment would activate tail skin vasodilatation (a major heat loss effector) at lower ambient temperatures and thereby lower the thermoneutral zone. The thermoneutral zone, defined as the range of ambient temperatures in which thermoregulation is achieved only by sensible (dry) heat loss, was evaluated based on properties of skin vasomotion. Core and tail skin temperatures were recorded in ovariectomized rats (with and without estradiol-17beta) exposed to ambient temperatures from 13 to 34 C in an environmental chamber. Rats without estradiol exhibited increased skin vasodilatation and a shift in the thermoneutral zone to lower ambient temperatures. Moreover, the ambient temperature threshold for skin vasodilatation was significantly lower in rats without estradiol treatment. At most ambient temperatures, average core temperature was unaffected by estradiol. However, at ambient temperatures of 32.5 C and above, untreated ovariectomized rats exhibited higher core temperatures compared with estradiol-treated rats. Thus, estradiol-17beta treatment enhanced the maintenance of core temperature during heat exposure. These findings support the hypothesis that estrogen withdrawal increases the sensitivity of thermoregulatory neural pathways and modifies the activation of heat loss mechanisms.
• Rance, N., Williams, H., Dacks, P. A., & Rance, N. E. (2010). An improved method for recording tail skin temperature in the rat reveals changes during the estrous cycle and effects of ovarian steroids. Endocrinology, 151(11).
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  In the rat, tail skin vasomotion is a primary heat loss mechanism that can be monitored by changes in tail skin temperature (T(SKIN)). Previous studies showed that ovariectomy and estrogen replacement modify T(SKIN) in the rat. Based on these findings, the ovariectomized (OVX) rat has been used as a model to study the mechanisms and treatment of menopausal hot flushes. It is not known, however, if T(SKIN) changes across the estrous cycle in intact rats. Here, we describe an improved method for monitoring T(SKIN) in freely moving rats using a SubCue Mini datalogger mounted on the ventral surface of the tail. This method is noninvasive, cost-effective, and does not require restraints or tethering. We observed a distinct pattern of T(SKIN) across the estrous cycle characterized by low T(SKIN) on proestrous night. To determine whether this pattern was secondary to secretion of ovarian steroids, we monitored the thermoregulatory effects of 17β-estradiol (E(2)) and E(2) plus progesterone, administered via SILASTIC capsules to OVX rats. E(2) treatment of OVX rats significantly reduced T(SKIN) in the dark phase from 2 to 21 d after hormone treatment. The T(SKIN) of E(2)-treated OVX animals was not significantly different from OVX rats receiving E(2) plus progesterone. These data provide evidence that the reduction in T(SKIN) on proestrous night was secondary to elevated levels of ovarian estrogens. This study provides the first description of T(SKIN) changes with the estrous cycle and supports the role of estrogens in normal thermoregulation in the rat.
• Rance, N., & Rance, N. E. (2009). Menopause and the human hypothalamus: evidence for the role of kisspeptin/neurokinin B neurons in the regulation of estrogen negative feedback. Peptides, 30(1).
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  Menopause is characterized by depletion of ovarian follicles, a reduction of ovarian hormones to castrate levels and elevated levels of serum gonadotropins. Rather than degenerating, the reproductive neuroendocrine axis in postmenopausal women is intact and responds robustly to the removal of ovarian hormones. Studies in both human and non-human primates provide evidence that the gonadotropin hypersecretion in postmenopausal women is secondary to increased gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus. In addition, menopause is accompanied by hypertrophy of neurons in the infundibular (arcuate) nucleus expressing KiSS-1, neurokinin B (NKB), substance P, dynorphin and estrogen receptor alpha (ERalpha) mRNA. Ovariectomy in experimental animals induces nearly identical findings, providing evidence that these changes are a compensatory response to ovarian failure. The anatomical site of the hypertrophied neurons, as well as the extensive data implicating kisspeptin, NKB and dynorphin in the regulation of GnRH secretion, provide compelling evidence that these neurons are part of the neural network responsible for the increased levels of serum gonadotropins in postmenopausal women. We propose that neurons expressing KiSS-1, NKB, substance P, dynorphin and ERalpha mRNA in the infundibular nucleus play an important role in sex-steroid feedback on gonadotropin secretion in the human.
• Rance, N., Rometo, A. M., Krajewski, S. J., Voytko, M. L., & Rance, N. E. (2007). Hypertrophy and increased kisspeptin gene expression in the hypothalamic infundibular nucleus of postmenopausal women and ovariectomized monkeys. The Journal of clinical endocrinology and metabolism, 92(7).
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  Human menopause is characterized by ovarian failure, gonadotropin hypersecretion, and neuronal hypertrophy in the hypothalamic infundibular (arcuate) nucleus. Recent studies have demonstrated a critical role for kisspeptins in reproductive regulation, but it is not known whether menopause is accompanied by changes in hypothalamic kisspeptin neurons.
• Rance, N., Burke, M. C., Letts, P. A., Krajewski, S. J., & Rance, N. E. (2006). Coexpression of dynorphin and neurokinin B immunoreactivity in the rat hypothalamus: Morphologic evidence of interrelated function within the arcuate nucleus. The Journal of comparative neurology, 498(5).
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  Considerable evidence suggests that dynorphin and neurokinin B (NKB) neurons in the hypothalamic arcuate nucleus participate in the sex-steroid regulation of reproduction. In the present study, we used dual-label immunofluorescence to explore the distribution of prodynorphin and proNKB immunoreactivity in the rat hypothalamus. Additionally, we investigated whether arcuate prodynorphin-ir (immunoreactive) neurons expressed the neurokinin 3 receptor (NK3R) or nuclear estrogen receptor-alpha (ERalpha). We found that the majority of prodynorphin-ir neurons in the rat arcuate nucleus expressed proNKB, whereas nearly all (99%) of the proNKB neurons were immunoreactive for prodynorphin. The arcuate nucleus was the only site in the hypothalamus where neuronal somata coexpressing prodynorphin and proNKB-immunoreactivity were identified. A dense plexus of double-labeled prodynorphin/proNKB-ir fibers was found within the arcuate nucleus extending to the median eminence and throughout the periventricular zone of the hypothalamus. Prodynorphin/proNKB fibers were also identified in the paraventricular nucleus, anterior hypothalamic area, medial preoptic area, median preoptic nucleus, anteroventral periventricular nucleus, and bed nucleus of the stria terminalis in a distribution consistent with previously described arcuate nucleus projections. Interestingly, the majority of prodynorphin-ir neurons in the arcuate nucleus expressed NK3R, and nearly 100% of the prodynorphin-ir neurons contained nuclear ERalpha. Our results suggest that there is a close functional relationship between dynorphin and NKB peptides within the arcuate nucleus of the rat, which may include an autofeedback loop mediated through NK3R. The diverse hypothalamic projections of fibers expressing both prodynorphin and proNKB provide evidence that these neurons may participate in a variety of homeostatic and neuroendocrine processes.
• Rance, N., Krajewski, S. J., Anderson, M. J., Iles-Shih, L., Chen, K. J., Urbanski, H. F., & Rance, N. E. (2005). Morphologic evidence that neurokinin B modulates gonadotropin-releasing hormone secretion via neurokinin 3 receptors in the rat median eminence. The Journal of comparative neurology, 489(3).
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  Recent studies suggest that arcuate neurokinin B (NKB) neurons play a role in the regulation of gonadotropin secretion, but there is little information on the relationship between these neurons and the hypothalamic reproductive axis. In the present study, dual-label fluorescent immunohistochemistry was used to visualize the relationship between gonadotropin-releasing hormone (GnRH) neurons and either proNKB or NK3 receptor (NK3R) immunoreactivity. Immunocytochemistry was also combined with i.p. injections of the fluorescent retrograde tracer aminostilbamidine to determine whether arcuate neuroendocrine neurons expressed either proNKB or NK3R. A dense interweaving and close apposition of GnRH and proNKB-immunoreactive (ir) fibers was observed within the rat median eminence, where GnRH axons expressed NK3R immunoreactivity. These data provide morphological evidence that NKB neurons could influence GnRH secretion via interaction with NK3R in the rat median eminence. Colocalization of GnRH and NK3R was also identified in fiber tracts converging within the organum vasculosum of the lamina terminalis. In contrast, only a small number (16%) of GnRH-ir somata exhibited NK3R staining. ProNKB and NK3R-ir somata were identified within the arcuate nucleus, but none of these neurons were labeled by aminostilbamidine. Thus, we found no evidence that arcuate NKB neurons project to the primary capillary plexus of the portal system. Arcuate neuroendocrine neurons, however, were surrounded and closely apposed by proNKB-ir puncta and fibers. These data suggest that NKB neurons could indirectly influence anterior pituitary function by inputs to arcuate neuroendocrine neurons, but through a receptor other than NK3R. Our results provide an anatomic framework for putative interactions between NKB neurons and the hypothalamic reproductive axis.
• Rance, N., Escobar, C. M., Krajewski, S. J., Sandoval-Guzmán, T., Voytko, M. L., & Rance, N. E. (2004). Neuropeptide Y gene expression is increased in the hypothalamus of older women. The Journal of clinical endocrinology and metabolism, 89(5).
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  Neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons in the infundibular (arcuate) nucleus of the hypothalamus are part of a reciprocal circuit regulating reproduction and energy balance. Based on studies showing an age-related decrease in POMC mRNA, we hypothesized that NPY gene expression would increase in older women. In situ hybridization was used to compare NPY mRNA levels between young (premenopausal) and older (postmenopausal) women. We also measured NPY gene expression in intact and ovariectomized young cynomolgus monkeys. We report a significant increase ( approximately 100%) in the numbers of autoradiographic grains/NPY neuron in the retrochiasmatic area and infundibular nucleus of older women. NPY mRNA was correlated with subject age and inversely proportional to the number of POMC neurons previously counted in the same subjects. In contrast, there was no difference in hypothalamic NPY mRNA in intact vs. ovariectomized monkeys. These data show that aging in women is associated with increased NPY gene expression and suggest that the functional relationship between NPY and POMC neurons demonstrated in other species also exists in the human. Our studies of intact and ovariectomized monkeys suggest that the increase in NPY mRNA in older women is due to factors other than the ovarian failure of menopause.
• Rance, N., Sandoval-Guzmán, T., & Rance, N. E. (2004). Central injection of senktide, an NK3 receptor agonist, or neuropeptide Y inhibits LH secretion and induces different patterns of Fos expression in the rat hypothalamus. Brain research, 1026(2).
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  Arcuate neurokinin B (NKB) neurons express estrogen receptor-alpha and are strongly modulated by gonadal steroids. Although numerous studies suggest that NKB neurons participate in the reproductive axis, there is no information on the regulation of luteinizing hormone (LH) secretion by NKB or its receptor, NK3. In the present study, we determined if central injection of senktide, a selective NK3 receptor agonist, would alter serum LH in ovariectomized, estrogen-primed rats. The effects of senktide were compared to neuropeptide Y (NPY), a well-characterized modulator of LH secretion. Saline, senktide, or NPY was injected into the lateral ventricle of unanesthetized rats and serial blood samples were collected for LH radioimmunoassay. The rats were sacrificed 90 min after injection and the brains were removed and processed for Fos immunocytochemistry. A significant inhibition of serum LH was observed from 30 to 90 min after injection of senktide relative to saline controls. In the senktide-injected rats, the inhibition of serum LH was accompanied by increased Fos expression in the medial preoptic area and arcuate nucleus--two reproductive control centers. Senktide also induced Fos in the paraventricular nuclei (PVN) and supraoptic nuclei (SON). Injection of NPY also inhibited serum LH but increased Fos expression only in the PVN and SON. This study provides the first demonstration of alterations in LH secretion by an NK3 receptor agonist. These data, combined with the induction of Fos in medial preoptic and arcuate neurons, strongly support the hypothesis that NKB neurons play a role in the regulation of gonadotropin secretion.
• Rance, N., Krajewski, S. J., Abel, T. W., Voytko, M. L., & Rance, N. E. (2003). Ovarian steroids differentially modulate the gene expression of gonadotropin-releasing hormone neuronal subtypes in the ovariectomized cynomolgus monkey. The Journal of clinical endocrinology and metabolism, 88(2).
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  In the present study, we compared the morphology and distribution of neurons expressing GnRH gene transcripts in the hypothalamus and forebrain of the cynomolgus monkey to that of the human. As in the human, three subtypes of GnRH neurons were identified. Type I GnRH neurons were small, oval cells with high levels of gene expression and were located within the basal hypothalamus. Type II GnRH neurons were small and sparsely labeled and were widely scattered in the hypothalamus, midline nuclei of the thalamus, and extended amygdala. Type III neurons displayed magnocellular morphology and intermediate labeling intensity and were located in the nucleus basalis of Meynert, caudate, and amygdala. In a second experiment, we determined the effect of estrogen or estrogen plus progesterone on the gene expression of GnRH neurons in the brains of young, ovariectomized cynomolgus monkeys. We report that hormone treatment resulted in a significant decrease in GnRH mRNA in type I neurons within the basal hypothalamus of ovariectomized monkeys. In contrast, there was no effect of hormone treatment on the gene expression of type III GnRH neurons in the nucleus basalis of Meynert. The present findings provide evidence that the increase in gene expression of type I GnRH neurons in postmenopausal women is secondary to the ovarian failure of menopause. The differential responses of type I and III GnRH neurons to hormone treatment provide additional evidence that distinct subpopulations of neurons expressing GnRH mRNA exist in the primate hypothalamus.

Presentations

• Rance, N. E. (2018, August). The science underlying the treatment of hot flushes with neurokinin 3 receptor antagonists.. Australasian Menopause Society Congress. Brisbane Australia: Australasian Menopause Society.
• Rance, N. E. (2018, July). Menopause and the human hypothalamus: From LH pulses to hot flushes. International Congress of Neuroendocrinology.. Toronto, Canada: International Congress of Neuroendocrinology..
• Rance, N. E. (2018, March). KNDy (kisspeptin, neurokinin B and dynorphin) neurons, thermoregulatory circuits and the etiology of hot flushes. Annual Meeting of the Endocrine Society. Chicago: Endocrine Society.
• Rance, N. E. (2018, October). KNDy neurons, neurokinin 3 receptor signaling and the etiology of hot flushes.. North American Menopause Society Annual Meeting. San Diego: North American Menopause Society.

Poster Presentations

• Miranda Dos Santos, F., Krajewski-Hall, S. J., McMullen, N. T., & Rance, N. E. (2018, July). Preoptic neurokinin 3 receptor-expressing neurons modulate thermoregulatory pathways in the mouse.. International Congress of Neuroendocrinology.. Toronto, Ontario, Canada: International Congress of Neuroendocrinology..

Reviews

• Rance, N. E. (2016. Neuroendocrine Aging: Hypothalamic–Pituitary–Gonadal Axis in Women. Reference Module in Neuroscience and Biobehavioral Psychology.