ERαΔ4, an ERα splice variant missing exon4, interacts with caveolin-3 and mGluR2/3.

The two isoforms of the nuclear estrogen receptor, ERα and ERß are widely expressed in the central nervous system. Although they were first described as nuclear receptors, both isoforms have also been found at the cell membrane where they mediate cell signaling. Surface biotinylation studies using neuronal and glial primary cultures label an alternatively spliced form of ERα. The 52 kDa protein, ERαΔ4, is missing exon 4 and is highly expressed in membrane fractions derived from cultured cells. In vivo, both full-length (66 kDa) ERα and ERαΔ4 are present in membrane fractions. In response to estradiol, full-length ERα and ERαΔ4 are initially trafficked to the membrane, and then internalized in parallel. Previous studies determined that only the full-length ERα associates with metabotropic glutamate receptor-1a (mGluR1a), initiating cellular signaling. The role of ERαΔ4, remained to be elucidated. Here, we report ERαΔ4 trafficking, association with mGluR2/3, and downstream signaling in female rat arcuate nucleus (ARH). Caveolin (CAV) proteins are needed for ER transport to the cell membrane, and using co-immunoprecipitation CAV-3 was shown to associate with ERαΔ4. CAV-3 was necessary for ERαΔ4 trafficking to the membrane: in the ARH, microinjection of CAV-3 siRNA reduced CAV-3 and ERαΔ4a in membrane fractions by 50%, and 60%, respectively. Moreover, co-immunoprecipitation revealed that ERαΔ4 associated with inhibitory mGluRs, mGluR2/3. Estrogen benzoate (EB) treatment (5 µg; s.c.; every 4 days; three cycles) reduced levels of cAMP, an effect attenuated by antagonizing mGluR2/3. Following EB treatment, membrane levels of ERαΔ4 and mGluR2/3 were reduced implying ligand-induced internalization. These results implicate ERαΔ4 in an estradiol-induced inhibitory cell signaling in the ARH.

Estrogen and Progesterone Integration in an in vitro Model of RP3V Kisspeptin Neurons.

Positive feedback on gonadotropin release requires not only estrogen but also progesterone to activate neural circuits. In rodents, ovarian estradiol (E2) stimulates progesterone synthesis in hypothalamic astrocytes (neuroP), needed for the luteinizing hormone (LH) surge. Kisspeptin (kiss) neurons are the principal stimulators of gonadotropin-releasing hormone neurons, and disruption of kiss signaling abrogates the LH surge. Similarly, blocking steroid synthesis in the hypothalamus or deleting classical progesterone receptor (PGR) selectively in kiss neurons prevents the LH surge. These results suggest a synergistic action of E2 and progesterone in kiss neurons to affect gonadotropin release. The mHypoA51, immortalized kiss-expressing neuronal cell line derived from adult female mice, is a tractable model for examining integration of steroid signaling underlying estrogen positive feedback. Here, we report that kiss neurons in vitro integrate E2 and progesterone signaling to increase levels of kiss translation and release. mHypoA51 neurons expressed nonclassical membrane progesterone receptors (mPRα and mPRß) and E2-inducible PGR, required for progesterone-augmentation of E2-induced kiss expression. With astrocyte-conditioned media or in mHypoA51-astrocyte co-culture, neuroP augmented stimulatory effects of E2 on kiss protein. Progesterone activation of classical, membrane-localized PGR led to activation of MAPK and Src kinases. Importantly, progesterone or Src activation induced release of kiss from E2-primed mHypoA51 neurons. Consistent with previous studies, the present results provide compelling evidence that the interaction of E2 and progesterone stimulates kiss expression and release. Further, these results demonstrate a mechanism though which peripheral E2 may prime kiss neurons to respond to neuroP, mediating estrogen positive feedback.

Rodent Models of Non-classical Progesterone Action Regulating Ovulation.

It is becoming clear that steroid hormones act not only by binding to nuclear receptors that associate with specific response elements in the nucleus but also by binding to receptors on the cell membrane. In this newly discovered manner, steroid hormones can initiate intracellular signaling cascades which elicit rapid effects such as release of internal calcium stores and activation of kinases. We have learned much about the translocation and signaling of steroid hormone receptors from investigations into estrogen receptor α, which can be trafficked to, and signal from, the cell membrane. It is now clear that progesterone (P4) can also elicit effects that cannot be exclusively explained by transcriptional changes. Similar to E2 and its receptors, P4 can initiate signaling at the cell membrane, both through progesterone receptor and via a host of newly discovered membrane receptors (e.g., membrane progesterone receptors, progesterone receptor membrane components). This review discusses the parallels between neurotransmitter-like E2 action and the more recently investigated non-classical P4 signaling, in the context of reproductive behaviors in the rodent.

Actions of Steroids: New Neurotransmitters.

Over the past two decades, the classical understanding of steroid action has been updated to include rapid, membrane-initiated, neurotransmitter-like functions. While steroids were known to function on very short time spans to induce physiological and behavioral changes, the mechanisms by which these changes occur are now becoming more clear. In avian systems, rapid estradiol effects can be mediated via local alterations in aromatase activity, which precisely regulates the temporal and spatial availability of estrogens. Acute regulation of brain-derived estrogens has been shown to rapidly affect sensorimotor function and sexual motivation in birds. In rodents, estrogens and progesterone are critical for reproduction, including preovulatory events and female sexual receptivity. Membrane progesterone receptor as well as classical progesterone receptor trafficked to the membrane mediate reproductive-related hypothalamic physiology, via second messenger systems with dopamine-induced cell signals. In addition to these relatively rapid actions, estrogen membrane-initiated signaling elicits changes in morphology. In the arcuate nucleus of the hypothalamus, these changes are needed for lordosis behavior. Recent evidence also demonstrates that membrane glucocorticoid receptor is present in numerous cell types and species, including mammals. Further, membrane glucocorticoid receptor influences glucocorticoid receptor translocation to the nucleus effecting transcriptional activity. The studies presented here underscore the evidence that steroids behave like neurotransmitters to regulate CNS functions. In the future, we hope to fully characterize steroid receptor-specific functions in the brain.

Ablation of KNDy Neurons Results in Hypogonadotropic Hypogonadism and Amplifies the Steroid-Induced LH Surge in Female Rats.

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 17ß-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 17ß-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.

Estradiol Membrane-Initiated Signaling and Female Reproduction.

The discoveries of rapid, membrane-initiated steroid actions and central nervous system steroidogenesis have changed our understanding of the neuroendocrinology of reproduction. Classical nuclear actions of estradiol and progesterone steroids affecting transcription are essential. However, with the discoveries of membrane-associated steroid receptors, it is becoming clear that estradiol and progesterone have neurotransmitter-like actions activating intracellular events. Ultimately, membrane-initiated actions can influence transcription. Estradiol membrane-initiated signaling (EMS) modulates female sexual receptivity and estrogen feedback regulating the luteinizing hormone (LH) surge. In the arcuate nucleus, EMS activates a lordosis-regulating circuit that extends to the medial preoptic nucleus and subsequently to the ventromedial nucleus (VMH)--the output from the limbic and hypothalamic regions. Here, we discuss how EMS leads to an active inhibition of lordosis behavior. To stimulate ovulation, EMS facilitates astrocyte synthesis of progesterone (neuroP) in the hypothalamus. Regulation of GnRH release driving the LH surge is dependent on estradiol-sensitive kisspeptin (Kiss1) expression in the rostral periventricular nucleus of the third ventricle (RP3V). NeuroP activation of the LH surge depends on Kiss1, but the specifics of signaling have not been well elucidated. RP3V Kiss1 neurons appear to integrate estradiol and progesterone information which feeds back onto GnRH neurons to stimulate the LH surge. In a second population of Kiss1 neurons, estradiol suppresses the surge but maintains tonic LH release, another critical component of the estrous cycle. Together, evidence suggests that regulation of reproduction involves membrane action of steroids, some of which are synthesized in the brain.

Neurokinin 3 Receptor-Expressing Neurons in the Median Preoptic Nucleus Modulate Heat-Dissipation Effectors in the Female Rat.

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-17ß (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.

Classical and membrane-initiated estrogen signaling in an in vitro model of anterior hypothalamic kisspeptin neurons.

The neuropeptide kisspeptin is essential for sexual maturation and reproductive function. In particular, kisspeptin-expressing neurons in the anterior rostral periventricular area of the third ventricle are generally recognized as mediators of estrogen positive feedback for the surge release of LH, which stimulates ovulation. Estradiol induces kisspeptin expression in the neurons of the rostral periventricular area of the third ventricle but suppresses kisspeptin expression in neurons of the arcuate nucleus that regulate estrogen-negative feedback. To focus on the intracellular signaling and response to estradiol underlying positive feedback, we used mHypoA51 cells, an immortalized line of kisspeptin neurons derived from adult female mouse hypothalamus. mHypoA51 neurons express estrogen receptor (ER)-α, classical progesterone receptor (PR), and kisspeptin, all key elements of estrogen-positive feedback. As with kisspeptin neurons in vivo, 17ß-estradiol (E2) induced kisspeptin and PR in mHypoA51s. The ERα agonist, 1,3,5-Tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, produced similar increases in expression, indicating that these events were mediated by ERα. However, E2-induced PR up-regulation required an intracellular ER, whereas kisspeptin expression was stimulated through a membrane ER activated by E2 coupled to BSA. These data suggest that anterior hypothalamic kisspeptin neurons integrate both membrane-initiated and classical nuclear estrogen signaling to up-regulate kisspeptin and PR, which are essential for the LH surge.

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.

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.

Spatial behavior and seizure-induced changes in c-fos mRNA expression in young and old rats.

The subcellular processes of gene induction and expression in the hippocampus are likely to underlie some of the known age-related impairments in spatial learning and memory. It is well established that immediate-early genes are rapidly and transiently induced in response to neuronal activity and this expression is required for stabilization of durable memories. To examine whether age-related memory impairment might be caused, in part, by differences in the level of cellular activation or subcellular processing, c-fos expression in CA1 pyramidal and dentate gyrus granule cells in the dorsal hippocampus of young and old rats was determined using fluorescence in situ hybridization and reverse transcription polymerase chain reaction. No significant age differences were found in the numbers of pyramidal or granule cells that show c-fos expression; however, c-fos mRNA transcripts were altered in these 2 cell types in aged animals. These findings suggest that though the networks of cells that participate in behavior or seizure-induced activity are largely maintained in aged rats, their RNA transcript levels are altered. This might, in part, contribute to cognitive deficits frequently observed with advancing age.

Role for kisspeptin/neurokinin B/dynorphin (KNDy) neurons in cutaneous vasodilatation and the estrogen modulation of body temperature.

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 17ß-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.

Arcuate kisspeptin/neurokinin B/dynorphin (KNDy) neurons mediate the estrogen suppression of gonadotropin secretion and body weight.

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 17ß-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 17ß-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.