Susana I. Sá

Estrogen modulates the sexually dimorphic synaptic connectivity of the ventromedial nucleus.

Neurons in the ventrolateral division of the hypothalamic ventromedial nucleus (VMNvl) display a remarkable estrogen‐dependent functional and structural plasticity, which is likely to be mediated, in part at least, by neuronal afferents. The present study was designed to determine whether the number of synapses per neuron and the size of individual synapses in the VMNvl vary across the estrus cycle and, also, whether they differ between the sexes. To accomplish this, the VMNvl of adult female rats at proestrus or diestrus day 1 and of age‐matched male rats was analyzed using electron microscopy. We found that a single VMNvl neuron receives around 7,000 synapses during diestrus and ∼10,000 during proestrus. This estrus cycle‐related variation is accounted for by increases in the number of all types of synapses. In males, the number of synapses received by each VMNvl neuron is similar to that of diestrus rats (∼7,500). However, in males the number of axodendritic and axospinous synapses is smaller than in proestrus rats, whereas the number of axosomatic synapses is higher than in diestrus rats. In addition, we found that the size of the postsynaptic densities of axospinous and axosomatic synapses is consistently larger in males than in females. Our results show that the synaptic organization of the VMNvl is sexually dimorphic, with females having more dendritic synapses and males more somatic synapses. They also show that the synaptic plasticity induced by estrogen in the VMNvl is characterized by changes in the number, but not the size, of the synapses. J. Comp. Neurol. 484:68–79, 2005.

Timed hypocaloric food restriction alters the synthesis and expression of vasopressin and vasoactive intestinal peptide in the suprachiasmatic nucleus

In mammals, the main circadian pacemaker is located in the suprachiasmatic nucleus (SCN) and its most potent synchronizer is the daily variation of the intensity of light. However, other nonphotic cues, such as timed food restriction, can induce changes in the circadian rhythms, leading also to the appearance of a food-entrained oscillator. The present study was designed to establish if the alterations of the circadian rhythms induced by timed hypocaloric food restriction are accompanied by structural changes in the SCN. Two groups of adult rats, both maintained on 12-h light/12-h dark cycles, were used; in one group, animals had permanent free access to food, whereas in the other they were subjected to a restricted hypocaloric early morning feeding during 7 months. Using stereological techniques and in situ hybridization, we have examined the structure of the SCN and the synthesis and expression of vasopressin (AVP) and vasoactive intestinal peptide (VIP). The volume of the SCN and the total number of neurons did not vary between the two groups. However, the total number of AVP- and VIP-immunoreactive neurons and the AVP and VIP mRNA levels were significantly decreased in timed hypocaloric food-restricted animals. The results indicate that timed hypocaloric food restriction has led to changes of AVP and VIP content of the neurons. They furthermore suggest the existence of a coupling between the food-entrainable oscillator and the light-entrainable pacemaker.

Role of neural afferents as mediators of estrogen effects on the hypothalamic ventromedial nucleus

The effects of estrogens on the ventrolateral division of the hypothalamic ventromedial nucleus (VMNvl) are essential for its role in the regulation of female sexual behavior. Enhanced synaptogenesis and induction of progesterone receptors (PRs) are hallmarks of the actions of estrogens on the VMNvl. To investigate the influence of neural afferents in mediating these effects, we estimated the number of spine and dendritic synapses per neuron and the total number of PR-immunoreactive neurons in ovariectomized rats treated with either estradiol benzoate or vehicle, after unilateral VMN deafferentation. The estimates were performed independently in the VMNvl of the deafferented and contralateral sides, and in the VMNvl of unoperated rats (controls). The administration of estradiol benzoate did not induce any increase in the number of synapses of the deafferented VMNvl. In the contralateral VMNvl, the synaptogenic effects of estrogen were apparent, but still reduced relative to the control VMNvl, where a 25% increase in the total number of synapses was observed after estrogenic stimulation. In the absence of estrogenic stimulation, i.e., in basal conditions, deafferentation reduced the number of dendritic and spine synapses, but particularly the latter. The reduction was also visible, but less marked, in the contralateral VMNvl. Contrary to synapses, the estrogen induction of PRs was unaffected by deafferentation, and the total number of PR-immunoreactive neurons was similar in the control, deafferented and contralateral VMNvl. The results show that estrogens enhance synaptogenesis in the VMNvl by acting through neural afferents and induce PR expression by acting directly upon VMN neurons.

Estrogen receptors α and β have different roles in the induction and trafficking of progesterone receptors in hypothalamic ventromedial neurons

Progesterone receptor (PR) activation in the ventrolateral division of the hypothalamic ventromedial nucleus (VMNvl) is essential for promoting female sexual behavior. Estrogen receptor (ER) α, in contrast to ERβ, has been implicated in the induction of PRs. The simultaneous activation of ERα and ERβ, although not increasing the number of PR‐immunoreactive neurons in the VMNvl, facilitates lordosis, which suggests that ERβ and/or the ERα–ERβ interaction might play a role in PR dynamics and/or PR expression by individual neurons. To address this question, we used western blot and immunohistochemical studies to determine the amounts and subcellular distributions of both PR isoforms in VMNvl neurons of ovariectomized rats injected with estradiol benzoate or with specific agonists of ERα and ERβ, alone or in association. The present data show that ERα activation does not change PR expression in individual neurons, but increases the number of PRs in the VMNvl, because it increases the number of neurons expressing PRs. Conversely, ERβ activation does not change the total number of PRs in the VMNvl, but increases the labeling intensity of the perikaryal cytoplasm, which suggests that it promotes the transport of PRs from neurites into cell bodies. In addition, the simultaneous activation of ERα and ERβ increases the expression of PRs by individual neurons and, consequently, increases the total number of PRs in the VMNvl. Our findings reveal that individual and simultaneous activation of ERα and ERβ have different effects on the levels and subcellular location of PRs in VMNvl neurons.

Role of plasma membrane estrogen receptors in mediating the estrogen induction of progesterone receptors in hypothalamic ventromedial neurons

Progesterone is well known for its role in the modulation of sexual behavior. In the ventromedial nucleus (VMN), a part of the mediobasal hypothalamus that regulates sexual behavior in female rodents, estrogens induce the expression of progesterone receptors (PRs). This effect is known to be dependent on the activation of nuclear estrogen receptors (ERs). However, recent studies have documented estrogen activation of genomic transcription triggered by protein–protein phosphorylation cascades initiated at membrane receptors. The aim of this study was to examine if membrane‐initiated estradiol (E2) stimulation is able to induce PR expression in the VMN or, at least, to modulate nuclear ER action. To achieve this goal, 2‐month‐old ovariectomized Wistar rats were injected bilaterally, in the vicinity of VMN, with free E2 and with E2 conjugated with bovine serum albumin (E2BSA), alone or in sequence, by using a two‐pulse injection paradigm. Stereological methods and western blot analysis were used to estimate the total number of PR‐immunoreactive neurons in the VMN and the PR protein content of the VMN, respectively. The results showed that the administration of E2BSA alone increases the number of PR‐immunoreactive neurons and the expression level of PR protein to values similar to those resulting from E2 administration. They also showed that the sequential administration of E2 and E2BSA potentiates the effects resulting from the injection of E2 or E2BSA alone. These data provide the first evidence that membrane‐initiated E2 stimulation is able to induce and to potentiate the genomic activation of PR expression in the VMN. J. Comp. Neurol. 522:298–307, 2014.

The endocannabinoid system expression in the female reproductive tract is modulated by estrogen

The endocannabinoid system (ECS) is involved in several physiological events that resulted in a growing interest in its modulation. Moreover, the uterine levels of anandamide (AEA), the major endocannabinoid, must be tightly regulated to create proper embryo implantation conditions. However, there are no evidences about the regulation of AEA in uterus by estrogen. Thus, the aim of this study is to elucidate whether estradiol benzoate (EB) and tamoxifen (TAM) administration to ovariectomized (OVX) rats can induce changes in the expression of cannabinoid receptors and AEA-metabolic enzymes in uterus by evaluating gene transcription and protein levels by qPCR, Western blot and immunohistochemistry. Moreover, the plasmatic and uterine levels of AEA and of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α), the major cyclooxygenase-2 (COX-2) products, were determined by UPLC-MS/MS. The immunohistochemistry showed that cannabinoid receptors, as well as AEA-metabolic enzymes are mainly located in the epithelial cells of both lumen and glands and, to a lesser extent, in the muscle cells. Moreover, EB administration to OVX rats significantly increased CB1, CB2, NAPE-PLD, FAAH and COX-2 expression and transcription. These effects were absent in TAM and TAM+EB treatments showing that this response is estrogen receptor dependent. Additionally, although uterine levels of AEA remained unchanged in EB or TAM treated animals, they showed a rise with EB treatment in plasma. The latter also produced a decrease in uterine PGE2 levels. In summary, these data collectively indicate that the expression of ECS components, as well as, the AEA and PGE2 levels in rat uterus is modulated by EB. Thus, estradiol may have a direct regulatory role in the modulation of ECS in female reproductive tissues.

Dynamics of progesterone and estrogen receptor alpha in the ventromedial hypothalamus

Cyclic fluctuations of estradiol and progesterone in females influence neuronal activity in the ventrolateral division of the ventromedial hypothalamic nucleus (VMNvl), through the activation of progesterone receptors (PRs) and estrogen receptors (ERs). The expression of ER and PR in the VMNvl is influenced by their cognate ligands and is a central upstream trigger in the pathway of VMNvl-dependent modulation of endocrine responses. By studying the role played by estradiol and progesterone in PR and ERa expression in the VMNvl along the estrous cycle and how the two receptors interact in the same neuron, we aim to evaluate the synergistic action of both ovarian hormones in the regulation of VMNvl activity. In animals at all phases of the estrous cycle, the number of VMN neurons expressing PR or ERa was estimated by stereological methods, and the percentage, and rostro-caudal distribution, of neurons simultaneously expressing both receptors was determined. The highest number of PR-immunoreactive neurons was seen at proestrus, and of ERa-immunoreactive neurons was seen at proestrus and metestrus. The ERa/PR co-localization is increased at caudal levels. Approximately half the neurons expressing PR co-express ERa, a proportion that stays constant along the estrous cycle. The percentage of ERa neurons co-expressing PR changes from 60% at proestrus to 40% at metestrus. Fluctuations in circulating ovarian hormone levels promote coordinated changes in PR and ERa expression and co-localization. This may be an important mechanism in the regulation of input relayed by the VMNvl, allowing a precise modulation of endocrine responses.

Regulation of NPY and α-MSH expression by estradiol in the arcuate nucleus of Wistar female rats: a stereological study.

Objectives: Feeding behavior in both animals and humans is modulated by estrogens, as shown by the increased adiposity observed in women and rats upon the drop of estradiol levels at menopause. Estradiol action on food intake is mediated through its cognate receptors within several hypothalamic nuclei, namely the arcuate nucleus (ARN). The ARN contains two neuronal populations expressing peptides that exert opposing effects on the central control of feeding: the orexigenic neuropeptide Y (NPY) and the anorexigenic α-melanocyte-stimulating hormone (α-MSH). Methods: To understand the role played by estradiol in the modulation of food intake, we have used an animal model of cyclic 17β-estradiol benzoate (EB) administration and stereological methods to estimate the total number of neurons immunoreactive for NPY and α-MSH in the ARN of ovariectomized rats. Results: Present results show that the experimentally induced EB cyclicity prompted a decrease in food consumption and in body weight. Data also show that ovariectomy induced an increase in NPY expression and a decrease in α-MSH expression in the ARN that were reverted by EB administration. Conversely, EB blocked the expression of NPY and increased the synthesis of α-MSH in ARN neurons, without affecting the overall sum of NPY and α-MSH neurons. Discussion: These results suggest that estradiol affects food intake and, consequently, body weight gain, through an overriding mechanism superimposed in the physiological balance between both peptides in the ARN of female rats.

The integrity of the nucleus of the lateral olfactory tract is essential for the normal functioning of the olfactory system

The nucleus of the lateral olfactory tract (nLOT) is a relatively small component of the cortical pallial amygdala, with peculiar neurogenic, neurochemical and connectivity patterns. Although it has been suggested that it might be involved in non-pheromonal olfactory-guided behaviors, particularly feeding, the functional implications of the nLOT have never been investigated. In view of this fact, we have tackled this subject by performing a series of behavioral tests and by quantifying biological and biochemical parameters in sexually naïve adult male rats that were submitted to bilateral excitotoxic lesions of the nLOT. nLOT-lesioned rats had severe olfactory deficits with inability to detect and discriminate between odors. Additionally, they did not display innate behavioral responses to biologically relevant chemosignals. Specifically, nLOT-lesioned rats did not show avoidance towards predator odors or aggressive behaviors towards intruders, and had severely impaired sexual behavior. In fact, nLOT lesions abolished preference for odors of receptive females, reduced chemoinvestigatory behavior and eliminated mounting behavior. nLOT-lesioned rats had normal circulating levels of testosterone, did not display anxiety- or depressive-like behaviors, and had unimpaired cognitive functions and fear acquisition and memory. Altogether, our results suggest that the nLOT integrity is required for the normal functioning of the olfactory system.

Induction and subcellular redistribution of progesterone receptor A and B by tamoxifen in the hypothalamic ventromedial neurons of young adult female Wistar rats.

The ventrolateral division of the hypothalamic ventromedial nucleus (VMNvl) is a brain center for estrogen-dependent triggering of female sexual behavior upon progesterone receptor (PR) activation. We examined the agonistic and antagonistic actions of tamoxifen in this nucleus by analyzing its effects on the total number of PR-immunoreactive neurons, PR mRNA and protein levels, and subcellular location of PRs in ovariectomized Wistar rats. The results show that tamoxifen has no agonistic action in the number of PR-immunoreactive neurons, but increases PR expression and labeling in the nucleus and cytoplasm of VMNvl neurons that constitutively express PRs. As an antagonist, tamoxifen partially inhibited the estradiol-dependent increase in the number of PR-immunoreactive neurons and in PR mRNA and protein levels, without interfering with the subcellular location of the protein. We suggest that tamoxifen influence on PR expression in the VMNvl critically depends on the presence or absence of estradiol.