Can distinctly different rapid estrogen actions share a common mechanistic step?

Contribution to Special Issue on Fast effects of steroids. This paper reviews early evidence for the existence of rapid, non-genomic effects of estrogens on neurons, and, further, proposes that these rapid effects are often synergistic with later, genomic effects. Finally, suggestions about potential molecular mechanisms underlying the rapid effects of estrogens are offered. A mechanistic step we propose to be common among rapid estrogenic actions includes membrane ER's binding to histamine, and NMDA receptors and subsequent dimerization, and clustering (respectively) in a manner that enhances histamine and NMDA actions.

Rapid increases in immature synapses parallel estrogen-induced hippocampal learning enhancements.

Dramatic increases in hippocampal spine synapse density are known to occur within minutes of estrogen exposure. Until now, it has been assumed that enhanced spinogenesis increased excitatory input received by the CA1 pyramidal neurons, but how this facilitated learning and memory was unclear. Delivery of 17ß-estradiol or an estrogen receptor (ER)-α (but not ER-ß) agonist into the dorsal hippocampus rapidly improved general discrimination learning in female mice. The same treatments increased CA1 dendritic spines in hippocampal sections over a time course consistent with the learning acquisition phase. Surprisingly, estrogen-activated spinogenesis was associated with a decrease in CA1 hippocampal excitatory input, rapidly and transiently reducing CA1 AMPA activity via a mechanism likely reflecting AMPA receptor internalization and creation of silent or immature synapses. We propose that estrogens promote hippocampally mediated learning via a mechanism resembling some of the broad features of normal development, an initial overproduction of functionally immature connections being subsequently "pruned" by experience.

Development of Electrophysiological Properties of Nucleus Gigantocellularis Neurons Correlated with Increased CNS Arousal.

Many types of data have suggested that neurons in the nucleus gigantocellularis (NGC) in the medullary reticular formation are critically important for CNS arousal and behavioral responsiveness. To extend this topic to a developmental framework, whole-cell patch-recorded characteristics of NGC neurons in brainstem slices and measures of arousal-dependent locomotion of postnatal day 3 (P3) to P6 mouse pups were measured and compared. These neuronal characteristics developed in an orderly, statistically significant monotonic manner over the course of P3-P6: (1) proportion of neurons capable of firing action potential (AP) trains, (2) AP amplitude, (3) AP threshold, (4) amplitude of inward and outward currents, (5) amplitude of negative peak currents, and (6) steady state currents (in I-V plot). These measurements reflect the maturation of sodium and certain potassium channels. Similarly, all measures of locomotion, latency to first movement, total locomotion duration, net locomotion distance, and total quiescence time also developed monotonically over P3-P6. Most importantly, electrophysiological and behavioral measures were significantly correlated. Interestingly, the behavioral measures were not correlated with frequency of excitatory postsynaptic currents or the proportion of neurons showing these currents, responses to a battery of neurotransmitter agents, or rapid activating potassium currents (including IA). Considering the results here in the context of a large body of literature on NGC, we hypothesize that the developmental increase in NGC neuronal excitability participates in causing the increased behavioral responsivity during the postnatal period from P3 to P6.

Estradiol regulates responsiveness of the dorsal premammillary nucleus of the hypothalamus and affects fear- and anxiety-like behaviors in female rats.

Research suggests a causal link between estrogens and mood. Here, we began by examining the effects of estradiol (E2 ) on rat innate and conditioned defensive behaviors in response to cat odor. Second, we utilized whole-cell patch clamp electrophysiological techniques to assess noradrenergic effects on neurons within the dorsal premammillary nucleus of the hypothalamus (PMd), a nucleus implicated in fear reactivity, and their regulation by E2 . Our results show that E2 increased general arousal and modified innate defensive reactivity to cat odor. When ovariectomized females treated with E2 as opposed to oil were exposed to cat odor, they showed elevations in risk assessment and reductions in freezing, indicating a shift from passive to active coping. In addition, animals previously exposed to cat odor showed clear cue + context conditioning 24 h later. However, although E2 persisted in its effects on general arousal in the conditioning task, its effects on fear disappeared. In the patch clamp experiments noradrenergic compounds that typically induce fear clearly excited PMd neurons, producing depolarizations and action potentials. E2 treatment shifted some excitatory effects of noradrenergic agonists to inhibitory, possibly by differentially affecting α- and ß-adrenoreceptors. In summary, our results implicate E2 in general arousal and fear reactivity, and suggest these may be governed by changes in noradrenergic responsivity in the PMd. These effects of E2 may have ethological relevance, serving to promote mate seeking even in contexts of ambiguous threat and shed light on the involvement of estrogen in mood and its associated disorders.

Histaminergic responses by hypothalamic neurons that regulate lordosis and their modulation by estradiol.

How do fluctuations in the level of generalized arousal of the brain affect the performance of specific motivated behaviors, such as sexual behaviors that depend on sexual arousal? A great deal of previous work has provided us with two important starting points in answering this question: (i) that histamine (HA) serves generalized CNS arousal and (ii) that heightened electrical activity of neurons in the ventromedial nucleus of the hypothalamus (VMN) is necessary and sufficient for facilitating the primary female sex behavior in laboratory animals, lordosis behavior. Here we used patch clamp recording technology to analyze HA effects on VMN neuronal activity. The results show that HA acting through H1 receptors (H1R) depolarizes these neurons. Further, acute administration of estradiol, an estrogen necessary for lordosis behavior to occur, heightens this effect. Hyperpolarization, which tends to decrease excitability and enhance inhibition, was not affected by acute estradiol or mediated by H1R but was mediated by other HA receptor subtypes, H2 and H3. Sampling of mRNA from individual VMN neurons showed colocalization of expression of H1 receptor mRNA with estrogen receptor (ER)-alpha mRNA but also revealed ER colocalization with the other HA receptor subtypes and colocalization of different subtypes with each other. The latter finding provides the molecular basis for complex "push-pull" regulation of VMN neuronal excitability by HA. Thus, in the simplest causal route, HA, acting on VMN neurons through H1R provides a mechanism by which elevated states of generalized CNS arousal can foster a specific estrogen-dependent, aroused behavior, sexual behavior.

Estradiol modulation of phenylephrine-induced excitatory responses in ventromedial hypothalamic neurons of female rats.

Estrogens act within the ventromedial nucleus of the hypothalamus (VMN) to facilitate lordosis behavior. Estradiol treatment in vivo induces alpha(1b)-adrenoreceptor mRNA and increases the density of alpha(1B)-adrenoreceptor binding in the hypothalamus. Activation of hypothalamic alpha(1)-adrenoceptors also facilitates estrogen-dependent lordosis. To investigate the cellular mechanisms of adrenergic effects on VMN neurons, whole-cell patch-clamp recordings were carried out on hypothalamic slices from control and estradiol-treated female rats. In control slices, bath application of the alpha(1)-agonist phenylephrine (PHE; 10 microM) depolarized 10 of 25 neurons (40%), hyperpolarized three neurons (12%), and had no effect on 12 neurons (48%). The depolarization was associated with decreased membrane conductance, and this current had a reversal potential close to the K(+) equilibrium potential. The alpha(1b)-receptor antagonist chloroethylclonidine (10 microM) blocked the depolarization produced by PHE in all cells. From estradiol-treated rats, significantly more neurons in slices depolarized (71%) and fewer neurons showed no response (17%) to PHE. PHE-induced depolarizations were significantly attenuated with 4-aminopyridine (5 mM) but unaffected by tetraethylammonium chloride (20 mM) or blockers of Na(+) and Ca(2+) channels. These data indicate that alpha(1)-adrenoceptors depolarize VMN neurons by reducing membrane conductance for K(+). Estradiol amplifies alpha(1b)-adrenergic signaling by increasing the proportion of VMN neurons that respond to stimulation of alpha(1b)-adrenergic receptors, which is expected in turn to promote lordosis.

Kv2.1 expression in giant reticular neurons of the postnatal mouse brain.

Previous experiments charted the development of behavioral arousal in postnatal mice. From Postnatal Day 3 (P3) to Postnatal Day 6 (P6) mice (a) become significantly more active, "arousable"; and (b) in large reticular neurons, nucleus gigantocellularis (NGC), patch clamp recordings reveal a significantly increased ability to fire high frequency trains of action potentials as are associated with elevated cortical arousal. These action potential trains depend on delayed rectifiers such as Kv2.1. Here we report tracking the development of expression of a delayed rectifier, Kv2.1 in NGC neurons crucial for initiating CNS arousal. In tissue sections, light microscope immunohistochemistry revealed that expression of Kv2.1 in NGC neurons is greater at day P6 than at P3. Electron microscope immunohistochemistry revealed Kv2.1 labeling on the plasmalemmal surface of soma and dendrites, greater on P6 than P3. In brainstem reticular neuron cell culture, Kv2.1 immunocytochemistry increased monotonically from Days-In-Vitro 3-10, paralleling the ability of such neurons to fire action potential trains. The increase of Kv2.1 expression from P3 to P6, perhaps in conjunction with other delayed rectifier currents, could permit the ability to fire action potential trains in NGC neurons. Further work with genetically identified NGC neurons is indicated.

Potassium channels and the development of arousal-relevant action potential trains in primary hindbrain neurons.

Neurons in nucleus gigantocellularis (NGC) have been shown by many lines of evidence to be important for regulating generalized CNS arousal. Our previous study on mouse pups suggested that the development of NGC neurons' capability to fire action potential (AP) trains may both lead to the development of behavioral arousal and may itself depend on an increase in delayed rectifier currents. Here with whole-cell patch clamp we studied delayed rectifier currents in two stages. First, primary cultured neurons isolated from E12.5 embryonic hindbrain (HB), a dissection which contains all of NGC, were used to take advantage of studying neurons in vitro over using neurons in situ or in brain slices. HB neurons were tested with Guangxitoxin-1E and Resveratrol, two inhibitors of Kv2 channels which mediate the main bulk of delayed rectifier currents. Both inhibitors depressed delayed rectifier currents, but differentially: Resveratrol, but not Guangxitoxin-1E, reduced or abolished action potentials in AP trains. Since Resveratrol affects the Kv2.2 subtype, the development of the delayed rectifier mediated through Kv2.2 channels may lead to the development of HB neurons' capability to generate AP trains. Stage Two in this work found that electrophysiological properties of the primary HB neurons recorded are essentially the same as those of NGC neurons. Thus, from the two stages combined, we propose that currents mediated through Kv2.2 are crucial for generating AP trains which, in turn, lead to the development of mouse pup behavioral arousal.

Arousal of cerebral cortex electroencephalogram consequent to high-frequency stimulation of ventral medullary reticular formation.

We have theorized that large neurons in the ventral and medial reticular formation of the medulla are critical for both autonomic and cortical arousal. To test this theory, we anesthetized rats with urethane, lowered concentric bipolar stimulating electrodes into the medullary reticular formation, and implanted electroencephalogram (EEG) and ECG recording electrodes. We stimulated in the medulla with pulse frequencies ranging from 50 to 300 Hz while recording cortical EEG and ECG. These female rats were ovariectomized, and one subgroup was administered estradiol. Electrical stimulation at either 200 or 300 Hz among the large medullary reticular neurons in nucleus paragigantocellularis (PGi) caused a significant reduction in the portion of the EEG power spectrum represented by delta-waves (0.1-4 Hz) and -waves (4.1-8 Hz). Correspondingly, there were increases in gamma-wave power (22-50 Hz), especially when using 300 Hz. Stimulation at

Equation representing the dark-entrained transition from inaction to action in male and female mice.

The activation of behaviour in a daily rhythm governed by the light cycle is a universal phenomenon among humans, laboratory mammals and other vertebrates. For mice, the active period is during the dark. We have quantified the increase in activity when the lights shut off (Light to Dark, L to D) using a generalized CNS arousal assay with 20 ms resolution, rather than traditional running wheels. Data analysis yielded the rare demonstration of an equation which precisely tracks this behavioural transition and, surprisingly, its reverse during D to L. This behavioural dynamic survives in constant darkness (experiment 2) and is hormone-sensitive (experiment 3). Finally (experiment 4), mice on a light schedule analogous to one which proved troublesome for U.S. Navy sailors, had dysregulated activity bursts which did not conform to the transitions between D and L. These experiments show the lawfulness of a behavioural phase transition and the consequence of deviating from that dynamic pattern. And, in a new way, they bring mathematics to the realm of behavioural neuroscience.

Concepts and mechanisms of generalized central nervous system arousal.

A concept of generalized arousal of the CNS is presented and given an operational definition that leads to quantitative physical measures. Because this primitive arousal function underlies all motivated behavioral responses, cognitive functions, and emotional expression, disorders of generalized arousal can be associated with a large number of problems in medicine and public health, including vegetative states, attentional disorders, depression, occupational hazards, and problems with sleep and anesthesia. Some of its known mechanisms are briefly reviewed, at the levels of neuroanatomy, neurophysiology, and functional genomics. Generalized arousal contributes to the excitement and the activation of behaviors during specific arousal states. Data are summarized for four genomic/neurochemical systems through which changes in generalized arousal could affect sexual arousal, two of which heighten, and the other two of which reduce arousal.

Oestrogen receptor expression and neuronal nitric oxide synthase in the clitoris and preputial gland structures of mice.

OBJECTIVE: To study the presence of oestrogen receptors (ER) and neuronal nitric oxide synthase (nNOS) in the mouse clitoris. MATERIALS AND METHODS: A series of sections of the pelvic area, including the preputial glands and clitoris, of 10 mice were assessed by immunocytochemical studies specific for ER-alpha and -beta, and nNOS; selected sections were also stained with Masson's trichrome. RESULTS: ER alpha was detected in the epithelium of the gland of the clitoris, and in the glandular tissue, preputial and apocrine gland. ER alpha was detected in the nuclei of stromal cells around the cavernous tissue and near the epithelium of the clitoris. Cytoplasm ER alpha was detected in a few cells in an area ventral to the clitoral gland. There was also nuclear staining in the connective tissue cells surrounding the clitoris. Very light ER beta immunostaining was detected in the clitoris and in the tissue related to it. There were some cells with nuclear staining in the vessels of the cavernous tissue of the clitoris. nNOS immunostaining was detected in the clitoris, the preputial gland and the connective tissue. CONCLUSION: ER alpha and beta isoforms, and nNOS, are present in the clitoris and preputial glands of female mice in different cellular locations and with differing levels of receptivity. Functional studies would further elucidate the role of receptor functions and their relationship to the neuronal expression of NO.

Oestrogen receptors and their relation to neural receptive tissue of the labia minora.

OBJECTIVE: To assess the cellular distributions of oestrogen receptors alpha and beta (ER alpha and ER beta) and neuronal nitric oxide synthase (nNOS) in the labia minora, as knowledge about ER type and function may clarify the role of oestrogens in vaginal scar formation and improve outcomes in female genital surgery. SUBJECTS AND METHODS: Labial samples were taken from 10 girls (aged 2-9 years) who underwent surgery for labial fusion. The waste tissue strips obtained were used for immunohistochemical identification of ER alpha and ER beta, and nNOS in the labia minora. RESULTS: There was ER alpha nuclear staining in the stroma of the labia minora close to the clitoris, and basal and suprabasal in the epidermal cells membrane restricted to superficial sections of the labia minora. ER beta was found in the stroma of the labia minora closer to the clitoris and in superficial sections, in the basal epidermal cells membrane and apocrine glandular epithelial cells membrane. There was also ER beta cell membrane staining in the basal and suprabasal epithelial cells and fibroblasts in the lamina propria. CONCLUSIONS: Established ER presence allows the consideration of the introitus of the vagina as a target for oestrogen therapy in various clinical and surgical situations. Continuing elucidation of the immunohistochemistry of this external genital tissue might assist in the development of molecular tools to treat genital abnormalities. Details of this immunohistochemistry may also advance the understanding of the effects of sexual differentiation on the brain and other organ systems.

Significance of topical estrogens to labial fusion and vaginal introital integrity.

PURPOSE: One theory for labial fusion is low prepubertal estrogen levels. Topical estrogens remains the mainstay of therapy. Some patients require surgical lysis of the adhesion. Estrogen's action in regard to collagen may influence recurrent adhesions and adhesions that form after manual disruption or surgical separation. This study assesses the efficacy of topical estrogen to separate the labia, recurrence, and estrogen related side effects. Estrogens may have a role in vaginal healing in genital surgery. MATERIALS AND METHODS: Retrospective chart review of 109 girls from 3 months to 10 years old (mean 44 months) who had labial fusion. Data was collected on the length of topical estrogen treatment, rate of successful separation, rate of recurrence, percentage requiring surgery, and post-operative outcomes. RESULTS: Mean length of topical estrogen treatment was 3.7 months (range 0 to 36 months), with separation in 79% (85/107) of patients. Forty-one percent (44/107) had recurrence of labial fusion one to five times (range 2 to 72 months). Surgery was required in 21% (22/107). Ten percent of patients (2/21) had recurrence of labial fusion post-operatively (age 6,10 years, at 1, 18 month post-operatively). Neither of these two patients was responsive to topical estrogen cream post-operatively and required a second surgical separation. Side effects of estrogen were minimal breast development in 6 children and vaginal bleeding in one child. Discontinuance resulted in resolution of side effects. CONCLUSION: Topical estrogens were effective treatment for labial fusion. After surgical separation of adhesions there was a 10% recurrence rate. Estrogen therapy initiated after surgical failure was unsuccessful.

Rapid estrogen actions on ion channels: A survey in search for mechanisms.

A survey of nearly two hundred reports shows that rapid estrogenic actions can be detected across a range of kinds of estrogens, a range of doses, on a wide range of tissue, cell and ion channel types. Striking is the fact that preparations of estrogenic agents that do not permeate the cell membrane almost always mimic the actions of the estrogenic agents that do permeate the membrane. All kinds of estrogens, ranging from natural ones, through receptor modulators, endocrine disruptors, phytoestrogens, agonists, and antagonists to novel G-1 and STX, have been reported to be effective. For actions on specific types of ion channels, the possibility of opposing actions, in different cases, is the rule, not the exception. With this variety there is no single, specific action mechanism for estrogens per se, although in some cases estrogens can act directly or via some signaling pathways to affect ion channels. We infer that estrogens can bind a large number of substrates/receptors at the membrane surface. As against the variety of subsequent routes of action, this initial step of the estrogen's binding action is the key.

Analyses of rapid estrogen actions on rat ventromedial hypothalamic neurons.

Rapid estrogen actions are widely diverse across many cell types. We conducted a series of electrophysiological studies on single rat hypothalamic neurons and found that estradiol (E2) could rapidly and independently potentiate neuronal excitation/depolarizations induced by histamine (HA) and N-Methyl-d-Aspartate (NMDA). Now, the present whole-cell patch study was designed to determine whether E2 potentiates HA and NMDA depolarizations - mediated by distinctly different types of receptors - by the same or by different mechanisms. For this, the actions of HA, NMDA, as well as E2, were investigated first using various ion channel blockers and then by analyzing and comparing their channel activating characteristics. Results indicate that: first, both HA and NMDA depolarize neurons by inhibiting K(+) currents. Second, E2 potentiates both HA and NMDA depolarizations by enhancing the inhibition of K(+) currents, an inhibition caused by the two transmitters. Third, E2 employs the very same mechanism, the enhancement of K(+) current inhibition, thus to rapidly potentiate HA and NMDA depolarizations. These data are of behavioral importance, since the rapid E2 potentiation of depolarization synergizes with nuclear genomic actions of E2 to facilitate lordosis behavior, the primary female-typical reproductive behavior.

Metabolic pathways that mediate inhibition of hypothalamic neurons by glucose.

Neurons in the ventromedial hypothalamus mediate some counterregulatory responses to hypoglycemia and 2-deoxyglucose, but the mechanisms that mediate these responses to glucose are unclear. In the present study, ventromedial hypothalamus neurons were identified on the basis of their inhibition by the transition from 5 to 20 mmol/l glucose. Tolbutamide, which activates glucose-stimulated neurons, failed to inhibit or activate glucose-inhibited neurons. Inhibitors of glucose transport and glycolysis, in particular by the glucokinase inhibitor glucosamine, blocked the effect of glucose on glucose-inhibited neurons. Furthermore, the glucose-inhibited neurons were activated by 2-deoxyglucose, which also activates counterregulatory responses. Conversely, glucose-inhibited neurons were inhibited by glycolytic metabolites, including lactate, but not by pyruvate. These data indicate that hypoglycemia induces electrical activity in glucose-inhibited neurons by attenuating glycolysis in those neurons. Thus, counterregulatory failure could be due to relatively enhanced glycolysis in glucose-stimulated neurons during hypoglycemia and attenuation of glycolysis in glucose-inhibited neurons might reverse counterregulatory failure.

Generalized arousal of mammalian central nervous system.

A fundamental capacity of the mammalian CNS is becoming amenable to study with the techniques of functional genomics. Emphasized in this review are ascending connections from the medullary reticular formation and descending connections from the paraventricular nucleus of the hypothalamus. In particular, sex hormone effects on neurons allow us to relate generalized arousal to a specific form of arousal which is required for reproductive behaviors.

Integration of steroid hormone initiated membrane action to genomic function in the brain.

Estrogen is a ligand for the estrogen receptor (ER), which on binding 17beta-estradiol, functions as a ligand-activated transcription factor and regulates the transcription of target genes. This is the slow genomic mode of action. However, rapid non-genomic actions of estrogen also exist at the cell membrane. Using a novel two-pulse paradigm in which the first pulse rapidly initiates non-genomic actions using a membrane-limited estrogen conjugate (E-BSA), while the second pulse promotes genomic transcription from a consensus estrogen response element (ERE), we have demonstrated that rapid actions of estrogen potentiate the slower transcriptional response from an ERE-reporter in neuroblastoma cells. Since rapid actions of estrogen activate kinases, we used selective inhibitors in the two-pulse paradigm to determine the intracellular signaling cascades important in such potentiation. Inhibition of protein kinase A (PKA), PKC, mitogen activated protein kinase (MAPK) or phosphatidylinositol 3-OH kinase (PI-3K) in the first pulse decreases potentiation of transcription. Also, our data with both dominant negative and constitutive mutants of Galpha subunits show that Galpha(q) initiates the rapid signaling cascade at the membrane in SK-N-BE(2)C neuroblastoma cells. We discuss two models of multiple kinase activation at the membrane Pulses of estrogen induce lordosis behavior in female rats. Infusion of E-BSA into the ventromedial hypothalamus followed by 17beta-estradiol in the second pulse could induce lordosis behavior, demonstrating the applicability of this paradigm in vivo. A model where non-genomic actions of estrogen couple to genomic actions unites both aspects of hormone action.

Analgesia after lesions of nucleus reticularis magnocellularis: differential effect on supraspinal versus spinal pain reflexes.

The present studies examine the effect of bilateral electrolytic lesions of descending fibers arising from nucleus reticularis magnocellularis (NMC) on responding to noxious peripheral thermal or mechanical stimulation and on spinal cord monoamine levels. The lesion effects were quantified by examining two supraspinally organized pain responses, the hot plate latency and vocalization threshold and two spinally organized nociceptive reflexes, tail flick latency and hind limb flexion threshold. Following interruption of descending NMC fibers, a profound analgesia was observed on supraspinally organized pain responses. Assay of spinal cord serotonin (5-HT) indicated that the NMC lesions also destroyed appreciable numbers of descending 5-HT fibers of passage originating in nucleus raphe magnus (RM). A modest hypersensitivity to pain occurred after control lesions in RM suggesting that the analgesia observed after NMC lesions would have been even more pronounced if RM fibers of passage had not been concomitantly destroyed. To assess whether the analgesia observed after NMC lesions was due to non-specific destruction of a given volume of reticular tissue, control lesions were placed in nucleus subcoeruleus (NSC). NSC lesions resulted in a hypersensitivity to pain and significant depletion of spinal cord noradrenaline (NA). These data suggest both that the analgesia observed after NMC lesions was not due to non-specific destruction of the reticular formation, and that descending NSC NA fibers tonically suppress pain. The above analgesic effects were observed exclusively on supraspinally organized pain responses, not spinally organized nociceptive reflexes. This supported previous studies demonstrating differential descending control of nociception in the spinal versus intact preparations. In summary, the present data suggest that descending NMC fibers tonically suppress ascending pain transmission.