Relationship Between Circulating Metabolic Hormones and Their Central Receptors During Ovariectomy-Induced Weight Gain in Rats.

Although increasing research focuses on the phenomenon of body weight gain in women after menopause, the complexity of body weight regulation and the array of models used to investigate it has proven to be challenging. Here, we used ovariectomized (OVX) rats, which rapidly gain weight, to determine if receptors for ghrelin, insulin, or leptin in the dorsal vagal complex (DVC), arcuate nucleus (ARC), or paraventricular nucleus (PVN) change during post-ovariectomy weight gain. Female Sprague-Dawley rats with ad libitum access to standard laboratory chow were bilaterally OVX or sham OVX. Subgroups were weighed and then terminated on day 5, 33, or 54 post-operatively; blood and brains were collected. ELISA kits were used to measure receptors for ghrelin, insulin, and leptin in the DVC, ARC, and PVN, as well as plasma ghrelin, insulin, and leptin. As expected, body weight increased rapidly after ovariectomy. However, ghrelin receptors did not change in any of the areas for either group, nor did circulating ghrelin. Thus, the receptor:hormone ratio indicated comparable ghrelin signaling in these CNS areas for both groups. Insulin receptors in the DVC and PVN decreased in the OVX group over time, increased in the PVN of the Sham group, and were unchanged in the ARC. These changes were accompanied by elevated circulating insulin in the OVX group. Thus, the receptor:hormone ratio indicated reduced insulin signaling in the DVC and PVN of OVX rats. Leptin receptors were unchanged in the DVC and ARC, but increased over time in the PVN of the Sham group. These changes were accompanied by elevated circulating leptin in both groups that was more pronounced in the OVX group. Thus, the receptor:hormone ratio indicated reduced leptin signaling in the DVC and PVN of both groups, but only in the OVX group for the ARC. Together, these data suggest that weight gain that occurs after removal of ovarian hormones by ovariectomy is associated with selective changes in metabolic hormone signaling in the CNS. While these changes may reflect behavioral or physiological alterations, it remains to be determined whether they cause post-ovariectomy weight gain or result from it.

Selected omissions and capstone presentations: a new approach to student-centered integrative physiology education.

Here, we describe a pedagogical approach that combines didactics with active learning to facilitate integration across physiological systems in a team-taught, graduate-level physiology course. We covered the major physiological systems, with each system preceded by an overview of its evolution/ontogeny to provide a broader perspective. Lectures provided a framework for integration by giving examples of how each system interacted with systems that preceded and followed. In lieu of a final exam, the course culminated in capstone presentations by small groups to promote student-centered learning of integrative physiology. At the beginning of the semester, students were assigned to groups; each group chose from predetermined topics. This allowed them to accumulate information throughout the semester and required them to attend to lecture content to assess how the material applied to their topic, thereby facilitating learning and retention. Faculty were deliberate in choosing material that was presented in each system, and material that was strategically omitted, establishing "gaps" that students filled in their capstone presentations. The final week was dedicated to student preparation for their presentations, which promoted peer-to-peer teaching and problem solving by the group, assisted by faculty as necessary. Capstone presentations demonstrated students' mastery of basic physiological principles and their ability to integrate among physiological systems, and they rated capstone presentations highly in helping with integration and critical thinking. Thus students showed a better understanding of systems physiology and the importance of integration across systems in normal function and in responding to homeostatic challenges.

Estradiol and body weight during temporally targeted food restriction: Central pathways and peripheral metabolic factors.

We used temporally-targeted food restriction (TTFR), in which ovariectomized rats had chow only for 2 h/day, to test the hypothesis that estradiol benzoate (EB) suppresses feeding and decreases body weight during brief (4 day) TTFR, as it does during ad libitum feeding. All rats lost weight during TTFR, but the loss was greater with EB treatment. However, OIL and EB-treated rats ate comparable amounts of chow during TTFR. We next investigated central nervous system pathways and peripheral hormonal and metabolic changes that accompany the effects of TTFR to determine the mechanism for this effect. Immunolabeling for fos in the nucleus of the solitary tract, the terminal site of vagal afferents from the gastrointestinal tract, was increased when rats on TTFR had access to chow for 1 h on the test day, indicating neuronal activation associated with consumption of the meal. However, fos immunolabeling was not affected by EB treatment, nor were numbers of the α subtype of estrogen receptors. TTFR had the expected effects on carbohydrate and lipid metabolites and metabolic hormones, with only slight differences in plasma glucose, triglycerides, and free fatty acids attributable to EB treatment. Interestingly, plasma corticosterone levels were greater in EB-treated rats on TTFR, and increased further after eating. Given that corticosterone affects metabolism, these findings suggest that elevated corticosterone may explain the persistence of EB-induced differences in body weight during TTFR despite the lack of effect on food intake.

Breastfeeding and women's interest in specific food tastes.

To determine whether breastfeeding alters women's interest in eating foods of different taste categories, we surveyed women at their 6-week post-partum check-up, asking them to rate their interest in eating various foods. Regardless of whether women responded in English or Spanish, they indicated greatest interest in eating sweet-tasting foods and least interest in eating sour-tasting foods, independent of whether they were breastfeeding. In general, the interest in eating foods of all taste qualities foods was increased in women who were breastfeeding; however, interest in eating salty and sour foods was not altered by breastfeeding in Spanish respondants. It is noteworthy that interest in eating foods of specific taste categories correlated with ratings of hunger in women who were not breastfeeding, but not in women who were breastfeeding. Thus, although breastfeeding women had a greater interest in eating foods of all taste categories, their interest does not appear to be driven solely by hunger. Finally, independent of breastfeeding, the interest in eating specific foods within taste categories differed between English and Spanish respondants, with Spanish respondants reporting greater interest in eating both nuts and bananas compared to English respondants. Together, these findings represent an initial approach to assess the impact of breastfeeding on interest in eating different types of food, and of how reproductive status and cultural differences may interact to affect food preferences and thereby to alter food choices.

Estrogen effects on oxytocinergic pathways that regulate food intake.

Multiple stimulatory and inhibitory neural circuits control eating, and these circuits are influenced by an array of hormonal, neuropeptide, and neurotransmitter signals. For example, estrogen and oxytocin (OT) both are known to decrease food intake, but the mechanisms by which these signal molecules influence eating are not fully understood. These studies investigated the interaction between estrogen and OT in the control of food intake. RT-qPCR studies revealed that 17ß-estradiol benzoate (EB)-treated rats showed a two-fold increase in OT mRNA in the paraventricular nucleus of the hypothalamus (PVN) compared to Oil-treated controls. Increased OT mRNA expression may increase OT protein levels, and immunohistochemistry studies showed that EB-treated rats had more intense OT labeling in the nucleus of the solitary tract (NTS), a region known to integrate signals for food intake. Food intake measurements showed that EB treatment reduced food intake, as expected. EB-treated rats lost weight over the course of the experiment, as expected, and EB-treated rats that received the highest dose of OT lost more weight than EB-treated rats that did not receive OT. Finally, OT antagonist administered to EB-treated rats reversed the effect of EB on food intake, suggesting that estrogen effects to decrease food intake may involve the oxytocinergic pathway.

Temporal and Site-Specific Changes in Central Neuroimmune Factors During Rapid Weight Gain After Ovariectomy in Rats.

Systemic inflammation is present in obesity and emerging evidence, primarily from studies using male rodents fed high-fat diets, suggests neuroimmune signaling also is involved. We investigated early changes in neuroimmune signaling during the weight gain that follows ovariectomy in rats. Ovariectomized (OVX) rats were given standard rat chow and terminated 5 days (baseline), 4 or 8 weeks after ovariectomy. Levels of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) in plasma and periuterine adipose were not affected by ovariectomy. In contrast, compared to baseline levels, IL-6 expression in the arcuate nucleus (ARC) and dorsal vagal complex (DVC) decreased by 4 weeks after OVX, but was not affected in the paraventricular nucleus (PVN). MCP-1 expression decreased by 4 weeks in the ARC and by 8 weeks in the PVN, but was not affected in the DVC. Increased glial fibrillary acidic protein (GFAP) expression in the PVN indicated astrocyte activation; decreased toll-like receptor 4 (TLR4) expression in the ARC, but not other regions, suggested early effects on innate immune factors. Importantly, in reproductively intact rats, IL-6 and MCP-1 levels in plasma, periuterine adipose, and brain regions were not affected after 8 weeks. Unlike OVX rats, GFAP expression in the DVC of intact rats was decreased at 8 weeks, and TLR4 expression in the ARC was increased at 8 weeks. Taken together, these dynamic and selective changes in neuroimmune factors co-incident with post-ovariectomy weight gain provide insight into the role of neuroimmune signaling in obesity, particularly in females.

Increased locomotor activity in estrogen-treated ovariectomized rats is associated with nucleus accumbens dopamine and is not reduced by dietary sodium deprivation.

Estrogens are well known to increase locomotor activity in laboratory rodents; however, the underlying mechanism remains unclear. We used voluntary wheel running by female rats as an index of locomotor behavior to investigate this issue. We first determined whether the estrogen-induced increase in locomotion was susceptible to inhibition by a physiological challenge, and next whether it was associated with dopaminergic activation in the central reward area, nucleus accumbens. Ovariectomized rats were given estradiol or the oil vehicle and housed in cages with or without running wheels. All rats were given regular rodent chow for 1 week, a sodium-deficient diet for the next week, and then were returned to a regular diet for another week. At the end of the last week, all rats were killed, brains were extracted and dopamine levels in the nucleus accumbens were measured. As expected, estradiol treatment increased distance run. Surprisingly, dietary sodium deprivation further increased running, but this appeared to be related to experience with wheel running, rather than to sodium deprivation, per se. Dopamine was greater in the nucleus accumbens of estradiol-treated rats that ran compared to all other groups. Thus, the estrogen-induced increase in locomotion is a robust phenomenon that is not inhibited by a body sodium challenge and is associated with elevated levels of dopamine in reward pathways. These findings raise the possibility that the estrogen-induced increase in locomotor activity, which occurs during a hormonal milieu conducive to reproduction, may reflect mate-seeking behavior and, thereby, maximize reproductive success.

Aging affects isoproterenol-induced water drinking, astrocyte density, and central neuronal activation in female Brown Norway rats.

Age-dependent impairments in the central control of compensatory responses to body fluid challenges have received scant experimental attention, especially in females. In the present study, we found that water drinking in response to ß-adrenergic activation with isoproterenol (30 µg/kg, s.c.) was reduced by more than half in aged (25 mo) vs. young (5 mo) ovariectomized female Brown Norway rats. To determine whether this age-related decrease in water intake was accompanied by changes in central nervous system areas associated with fluid balance, we assessed astrocyte density and neuronal activation in the SFO, OVLT, SON, AP and NTS of these rats using immunohistochemical labeling for GFAP and c-fos, respectively. GFAP labeling intensity was increased in the SFO, AP, and NTS of aged females independent of treatment, and was increased in the OVLT of isoproterenol-treated rats independent of age. Fos immunolabeling in response to isoproterenol was reduced in both the SFO and the OVLT of aged females compared to young females, but was increased in the SON of female rats of both ages. Finally, fos labeling in the AP and caudal NTS of aged rats was elevated after vehicle control treatment and did not increase in response to isoproterenol as it did in young females. Thus, age-related declines in water drinking are accompanied by site-specific, age-related changes in astrocyte density and neuronal activation. We suggest that astrocyte density may alter the detection and/or processing of signals related to isoproterenol treatment, and thereby alter neuronal activation in areas associated with fluid balance.

Behavioral responses and fluid regulation in male rats after combined dietary sodium deficiency and water deprivation.

Most investigators use a single treatment such as water deprivation or dietary sodium deficiency to evaluate thirst or sodium appetite, which underlie behavioral responses to body fluid challenges. The goal of the present experiments was to assess the effects of combined treatments in driving behaviors. Therefore, we evaluated the effect of combined overnight water deprivation and dietary sodium deficiency on water intake and salt intake by adult male rats in 2-bottle (0.5M NaCl and water) tests. Overnight water deprivation alone increased water intake, and 10days of dietary sodium deficiency increased 0.5M NaCl intake, with a secondary increase in water intake. During combined water deprivation and dietary sodium deficiency, water intake was enhanced and 0.5M NaCl was reduced, but not eliminated, suggesting that physiologically relevant behavioral responses persist. Nonetheless, the pattern of fluid intake was altered by the combined treatments. We also assessed the effect of these behaviors on induced deficits in body sodium and fluid volume during combined treatments and found that, regardless of treatment, fluid ingestion partially repleted the induced deficits. Finally, we examined urine volume and sodium excretion during dietary sodium deficiency with or without overnight water deprivation and found that, whether or not rats were water deprived, and regardless of water consumption, sodium excretion was minimal. Thus, the combination of water deprivation and dietary sodium deficiency appears to arouse drives that stimulate compensatory behavioral responses. These behaviors, in conjunction with physiological adaptations to the treatments, underlie body sodium and volume repletion in the face of combined water deprivation and dietary sodium deficiency.

Early oxytocin inhibition of salt intake after furosemide treatment in rats?

Body fluid homeostasis requires a complex suite of physiological and behavioral processes. Understanding of the role of the central nervous system (CNS) in integrating these processes has been advanced by research employing immunohistochemical techniques to assess responses to a variety of body fluid challenges. Such techniques have revealed sex/estrogen differences in CNS activation in response to hypotension and hypernatremia. In contrast, it has been difficult to conclusively identify specific CNS areas and neurotransmitter systems that are activated by hyponatremia using these techniques. In part, this difficulty is due to the temporal disconnect between the physiological effects of treatments commonly used to deplete body sodium and the behavioral response to such depletion. In some methods, sodium ingestion is delayed in association with increased oxytocin (OT), suggesting an inhibitory role for OT in sodium intake. Urinary sodium loss increases within an hour after treatment with furosemide, a natriuretic-diuretic, but sodium intake is delayed for 18-24h. Accordingly, we hypothesized that acute furosemide-induced sodium loss activates centrally-projecting OT neurons which provide an initial inhibition of sodium intake, and tested this hypothesis in ovariectomized Sprague-Dawley rats with or without estrogen using immunohistochemical methods. Neuronal activation in the hypothalamic paraventricular nuclei (PVN) after administration of furosemide corresponded to the timing of the physiological effects. The activation was not different in estrogen-treated rats, nor did estrogen alter the initial suppression of sodium intake. However, virtually no fos immunoreactive (fos-IR) neurons in the parvocellular PVN were also immunolabeled for OT. Thus, acute sodium loss after furosemide produces neural activation and an early inhibition of sodium intake that does not appear to involve activation of centrally-projecting OT neurons and is not influenced by estrogen.

Estrogen and voluntary exercise interact to attenuate stress-induced corticosterone release but not anxiety-like behaviors in female rats.

The beneficial effects of physical exercise to reduce anxiety and depression and to alleviate stress are increasingly supported in research studies. The role of ovarian hormones in interactions between exercise and anxiety/stress has important implications for women's health, given that women are at increased risk of developing anxiety-related disorders, particularly during and after the menopausal transition. In these experiments, we tested the hypothesis that estrogen enhances the positive impact of exercise on stress responses by investigating the combined effects of exercise and estrogen on anxiety-like behaviors and stress hormone levels in female rats after an acute stressor. Ovariectomized female rats with or without estrogen were given access to running wheels for one or three days of voluntary running immediately after or two days prior to being subjected to restraint stress. We found that voluntary running was not effective at reducing anxiety-like behaviors, whether or not rats were subjected to restraint stress. In contrast, stress-induced elevations of stress hormone levels were attenuated by exercise experience in estrogen-treated rats, but were increased in rats without estrogen. These results suggest that voluntary exercise may be more effective at reducing stress hormone levels if estrogen is present. Additionally, exercise experience, or the distance run, may be important in reducing stress.

Divergent effects of ERα and ERß on fluid intake by female rats are not dependent on concomitant changes in AT1R expression or body weight.

Estradiol (E2) decreases both water and saline intakes by female rats. The ERα and ERß subtypes are expressed in areas of the brain that control fluid intake; however, the role that these receptors play in E2's antidipsogenic and antinatriorexigenic effects have not been examined. Accordingly, we tested the hypothesis that activation of ERα and ERß decreases water and saline intakes by female rats. We found a divergence in E2's inhibitory effect on intake: activation of ERα decreased water intake, whereas activation of ERß decreased saline intake. E2 decreases expression of the angiotensin II type 1 receptor (AT1R), a receptor with known relevance to water and salt intakes, in multiple areas of the brain where ERα and ERß are differentially expressed. Therefore, we tested for agonist-induced changes in AT1R mRNA expression by RT-PCR and protein expression by analyzing receptor binding to test the hypothesis that the divergent effects of these ER subtypes are mediated by region-specific changes in AT1R expression. Although we found no changes in AT1R mRNA or binding in areas of the brain known to control fluid intake associated with agonist treatment, the experimental results replicate and extend previous findings that body weight changes mediate alterations in AT1R expression in distinct brain regions. Together, the results reveal selective effects of ER subtypes on ingestive behaviors, advancing our understanding of E2's inhibitory role in the controls of fluid intake by female rats.

Neuroanatomical association of hypothalamic HSD2-containing neurons with ERα, catecholamines, or oxytocin: implications for feeding?

This study used immunohistochemical methods to investigate the possibility that hypothalamic neurons that contain 11-ß-hydroxysteroid dehydrogenase type 2 (HSD2) are involved in the control of feeding by rats via neuroanatomical associations with the α subtype of estrogen receptor (ERα), catecholamines, and/or oxytocin (OT). An aggregate of HSD2-containing neurons is located laterally in the hypothalamus, and the numbers of these neurons were greatly increased by estradiol treatment in ovariectomized (OVX) rats compared to numbers in male rats and in OVX rats that were not given estradiol. However, HSD2-containing neurons were anatomically segregated from ERα-containing neurons in the Ventromedial Hypothalamus and the Arcuate Nucleus. There was an absence of OT-immunolabeled fibers in the area of HSD2-labeled neurons. Taken together, these findings provide no support for direct associations between hypothalamic HSD2 and ERα or OT neurons in the control of feeding. In contrast, there was catecholamine-fiber labeling in the area of HSD2-labeled neurons, and these fibers occasionally were in close apposition to HSD2-labeled neurons. Therefore, we cannot rule out interactions between HSD2 and catecholamines in the control of feeding; however, given the relative sparseness of the appositions, any such interaction would appear to be modest. Thus, these studies do not conclusively identify a neuroanatomical substrate by which HSD2-containing neurons in the hypothalamus may alter feeding, and leave the functional role of hypothalamic HSD2-containing neurons subject to further investigation.

Estradiol and osmolality: Behavioral responses and central pathways.

Regulation of appropriate osmolality of body fluid is critical for survival, yet there are sex differences in compensatory responses to osmotic challenges. Few studies have focused on the role of sex hormones such as estradiol in behavioral responses to increases or decreases in systemic osmolality, and even fewer studies have investigated whether central actions of estrogens contribute to these responses. This overview integrates findings from a series of ongoing and completed experiments conducted in my laboratory to assess estradiol effects on water and NaCl intake in response to osmotic challenges, and on activity in central pathways that mediate such responses.

Dendritic arbor of neurons in the hypothalamic ventromedial nucleus in female prairie voles (Microtus ochrogaster).

Female mating behavior in rats is associated with hormone-induced changes in the dendritic arbor of neurons in the ventromedial nucleus of the hypothalamus (VMH), particularly the ventrolateral portion. Regulation of mating behavior in female prairie voles differs substantially from that in rats; therefore, we examined the dendritic morphology of VMH neurons in this species. Sexually naïve adult female prairie voles were housed with a male to activate the females' reproductive endocrine system. Following 48 h of cohabitation, females were tested for evidence of reproductive activation by assessing the level of male sexual interest, after which their brains were processed using Golgi impregnation, which allowed ventrolateral VMH neurons to be visualized and analyzed. Dendritic arborization in the female prairie vole VMH neurons was strikingly similar to that of female rats. The key difference was that in the prairie voles the long primary dendrites extended considerably further than those observed in rats. Although most female voles paired with males exhibited sexual activation, some females did not. These two groups displayed specific differences in their VMH dendrites. In particular, the long primary dendrites were longer in the reproductively active females compared with those in the non-activated females. Overall, dendrite lengths were positively correlated with plasma estradiol levels in females exposed to males, but not in unpaired females. Although causal relationships between the neuroendocrine events, dendrite length, and the outward, behavioral manifestation of reproductive activation cannot be determined from this study, these results suggest an association between ventrolateral VMH dendrite morphology and female mating behavior in prairie voles, akin to what has been observed in female rats.

Regional differences in serotonin content in the nucleus of the solitary tract of male rats after hypovolemia produced by polyethylene glycol.

Serotonin (5-HT) has been implicated in centrally mediated compensatory responses to volume loss in rats. Accordingly, we hypothesized that slowly developing, non-hypotensive hypovolemia increases serotonin in the hindbrain nucleus of the solitary tract (NTS). We produced volume loss in adult male rats by administering hyperoncotic polyethylene glycol (PEG) and then assessed 5-HT levels in the NTS using measurements of tissue 5-HT content or 5-HT immunohistochemistry. The results show selective increases of 5-HT in the caudal NTS after PEG treatment, but no change in the primary 5-HT metabolite, 5-HIAA. Moreover, the intensity of 5-HT immunolabeled fibers in the caudal NTS was increased after PEG treatment. These findings suggest that, after PEG-induced hypovolemia, 5-HT accumulates in neural elements in the caudal NTS. We propose that this accumulation is attributable to an initial release of 5-HT that then acts at presynaptic autoreceptors to inhibit subsequent 5-HT release.

Estradiol selectively reduces central neural activation induced by hypertonic NaCl infusion in ovariectomized rats.

We recently reported that the latency to begin drinking water during slow, intravenous infusion of a concentrated NaCl solution was shorter in estradiol-treated ovariectomized rats compared to oil vehicle-treated rats, despite comparably elevated plasma osmolality. To test the hypothesis that the decreased latency to begin drinking is attributable to enhanced detection of increased plasma osmolality by osmoreceptors located in the CNS, the present study used immunocytochemical methods to label fos, a marker of neural activation. Increased plasma osmolality did not activate the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT), or the nucleus of the solitary tract (NTS) in either oil vehicle-treated rats or estradiol-treated rats. In contrast, hyperosmolality increased fos labeling in the area postrema (AP), the paraventricular nucleus of the hypothalamus (PVN) and the rostral ventrolateral medulla (RVLM) in both groups; however, the increase was blunted in estradiol-treated rats. These results suggest that estradiol has selective effects on the sensitivity of a population of osmo-/Na(+)-receptors located in the AP, which, in turn, alters activity in other central areas associated with responses to increased osmolality. In conjunction with previous reports that hyperosmolality increases blood pressure and that elevated blood pressure inhibits drinking, the current findings of reduced activation in AP, PVN, and RVLM-areas involved in sympathetic nerve activity-raise the possibility that estradiol blunts HS-induced blood pressure changes. Thus, estradiol may eliminate or reduce the initial inhibition of water intake that occurs during increased osmolality, and facilitate a more rapid behavioral response, as we observed in our recent study.

Time course of behavioral, physiological, and morphological changes after estradiol treatment of ovariectomized rats.

Previous studies showed that treatment with 17-ß-estradiol-3-benzoate (EB) reduces isoproterenol (ISOP) stimulated water intake by ovariectomized rats. This effect was observed 48h after the second of two EB injections, suggesting that the attenuation is attributable to classic EB actions to alter gene expression. However, in addition to classic, slowly-occurring, genomic effects, estrogens have more rapidly-occurring effects that may be nongenomic or 'nonclassical' genomic effects. Thus, it is possible that the EB attenuation of water intake stimulated by ISOP is genomic, nongenomic, or both. Accordingly, we measured ISOP-induced water intake by OVX rats at different times after EB injections, using time points likely to indicate classic genomic effects (48h or 24h) or nonclassical genomic or nongenomic effects (90min). We also examined EB effects on body weight, uterine weight, and plasma volume and Na(+) concentration in the same animals using the same time points and EB dose. EB treatment decreased water intake stimulated by ISOP in both the 24-h and 48-h groups; however, water intake in the 90-min group was not affected by EB. Uterine weight was unchanged 90min after EB, but was increased 24h after the first injection of EB. In contrast, body weight decreased after EB, but not until 48h after the second EB injection. Finally, EB did not alter plasma Na(+) concentration or hematocrit, though plasma protein concentration increased transiently 24h after EB treatment. Taken together, these findings suggest that the behavioral, morphological, and physiological effects of EB likely are attributable to slowly-occurring, classic genomic actions of estrogens. Moreover, the time course of the observed effects varied, suggesting tissue-specific differences in estrogen receptor density or subtype, or in co-activators or co-repressors that, ultimately, determine the timing and direction of EB effects.