Center of Experimental Morphology

Departamento de Anatomia - Faculdade de Medicina da Universidade do Porto

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Estrogens and Synaptic Plasticity in the Mature Brain

Research Area: Health Sciences
Status: In progress  
Project leaders:  
In cooperation with
  • Faculdade de Medicina - Universidade do Porto
Funded by: FCT - Fundação para a Ciência e Tecnologia Funding: 0.00 AUD


The ventrolateral division of the hypothalamic ventromedial nucleus (VMNvl) is a region playing a prominent role in the control of feminine sexual behavior, namely the lordosis reflex. Around 80% and 40% of its neurons contain ER? and ER? mRNA, respectively. Activation of ER? in the VMNvl by elevated circulating concentrations of estradiol, secreted by ovaries in proestrus, is required for stimulation of female sexual receptivity. Otherwise, disruption of the receptor function resulted in suppression of lordosis reflex in ovariectomized mice in response to estradiol or estradiol plus progesterone treatment.

In contrast, ER? function is not crucial for reproductive behavior as shown in experiments with mice lacking ER? gene. In normally cycling rats, levels of both ERs fluctuate with changes in concentration of estradiol during estrous cycle. In proestrus, at increased concentrations of the hormone, the number of neurons, containing mRNA and protein of ER? or ER?, start to decrease. Otherwise, removal of circulating estradiol (ovariectomy) results in significant rise in ERs immunoreactivity. The mechanism of estradiol action on ER isoforms expression in VMNvl is revealed in regard to ER?. ER? activation was found to be necessary to decrease the number of ER? immunoreactive cells and attenuate ER?-dependent behavior. Antagonistic influence of ER? on ER? functioning accounts for a number of behavioral responses such as aggression, anxiety and stress.

Very little is known about how estradiol regulates ER? protein in the CNS. Likely, in some regions, ER? may mediate the action of hormone. Knockout of ER? function in mice suppressed changes of ER? immunoreactivity in response to estradiol. However, the mechanism of this effect of ER? remains unclear. In order to reveal the mechanism of estradiol influence on ER? protein expression, in this study was investigated how the hormone regulates the levels of nuclear ER isoforms in cultured VMNvl neurons of female rats. Nuclear ER isoforms are under sophisticated regulatory control, including several hormones regularly included in formulation of standard growth media. Therefore, to succeed with the goal of this study, we developed a method to culture neurons in hormone-free conditions of chemically-defined medium.

To examine how estrogens act, in vivo, to regulate VMNvl neuronal function, a series of studies were carried out in parallel using young adult female rats.

To examine how estrogens act, in vivo, to regulate VMNvl neuronal function, a series of studies were carried out in parallel using young adult female rats.

 In the first study, we have examined the role played by neural afferents in mediating the effects of estrogen on synapse formation and expression of progesterone receptors (PR) by VMNvl neurons. For this purpose, we have surgically isolated the right VMN in ovariectomized rats that were later treated with either vehicle or estradiol benzoate and have estimated the number of spine and dendritic synapses established per neuron and the number of PR-immunoreactive neurons in the deafferented VMNvl, contralateral VMNvl, and VMNvl of control (non-deafferented) rats.

In the second study, the objective was to identify which ER isoform, the ER? or the ER?, is activated to enable the induction of progesterone receptors (PR) by estrogens. The studies were carried out in ovariectomized rats that received subcutaneous injections of the specific agonist of the ER? propyl pyrazole triol (PPT), the specific agonist of the ER? diaryl-propionitrile (DPN) or a combination of both in a schedule and dose that it is known to induce changes in the connectivity of the VMNvl neurons. In the same experiment, ovariectomized rats were also treated with the ER modulator Tamoxifen (Tx), followed by estradiol benzoate or vehicle.


Developed method of culturing VMNvl neurons in hormone-free synthetic medium allowed us to get a further insight into the mechanism of regulation of nuclear ER isoforms. In detail:
  1. A method allowing long-term culturing of VMNvl neurons in hormone-free chemically-defined medium was developed.
  2. In the hormone-free medium, all neurons derived from the VMNvl region expressed both ER isoforms, in contrast to the previous studies in vivo. The ER? protein expression was twice-fold higher than that of ER?. Individual cells showed significant dispersion in the levels of ER? and ER? expression in terms of both the total content and concentration.
  3. Estradiol treatment significantly decreased the expression of ER? (202%) whereas the level of ER? slightly rose in some cells (on 20%). There was a tendency toward an increase in neuronal size (in average, 58%) in response to treatment with estradiol.
  4. Treatment of neurons with the ER? selective agonist – PPT – dramatically increased ER? expression (310%) in cell and nucleus (52%). Sizes of cell and nucleus rose on 330% and 16%, respectively.
  5. The selective agonist of ER? – DPN - reversed the effect of PPT if neurons were simultaneously treated with both compounds. ER? expression level and cell area slightly decreased in comparison to untreated neurons in the presence of 1nM PPT and 10 nM DPN.
  6. DPN alone inhibited ER? protein immunoreactivity in the neurons at a compatible level with 10 nM estradiol and slightly decreased average size of cell.
Therefore, we found that ER? mediates the decrease in ER? expression in response to estradiol, like it was reported for VMN of neonatal rat brain. In turn, our data show that ER? activates ER? gene expression. This finding is supported by the fact that ER? gene contains ERE sequence and PPT treatment augmented expression of ER? protein in T47D cancer cells. Thus, nuclear ERs are able to control each other expression in VMNvl neurons. Estradiol treatment resulted in increase in the size of cells and was able activate ER?-dependent gene transcription. However, the balance between the receptors is shifted toward down-regulation of ER? as its activation results in increase of inhibitory activity of ER?. It suggests that inhibition of ER? in vivo is likely a factor that regulates ER?-dependent transcription and behavior.

The study of the mediators of estrogen actions on the synaptic plasticity in the VMNvl showed that:
  1. The administration of estradiol benzoate does not promote the formation of new synapses in the deafferented VMNvl.
  2. In the contralateral VMNvl, there is also a decrease in the number of synapses, suggesting that the VMN receives afferents from both hemispheres.
  3. After deafferentation, the fall in the number of spine synapses was greater than that observed in dendritic synapses.
Data obtained suggests that the synaptogenic effects of estrogens in the VMNvl are mediated by trans-synaptic signaling mechanisms and that the afferents involved are likely to originate in brain regions that also express ERs. In contrast to synapses, VMN deafferentation did not prevent the estrogen induction of PRs, leading solely to a small reduction in the number of neurons expressing non-inducible PRs. Thus, the results indicate that the induction of PRs by estrogens results from the local and direct action of these hormones on VMNvl neurons.

In the second study it was found that the expression of PRs increased only after the administration of PPT and Tx followed by EB, and that this increase was smaller than that induced by EB alone. This fact suggests that the estrogen induction of PRs is mediated by membrane receptors alone or by the combined activation of nuclear and membrane receptors. This hypothesis is now being tested by direct injection of 17?-estradiol conjugated with bovine serum albumine (BSA) into the VMN of ovariectomized rats after subcutaneous administration, on the previous day, of PPT, DPN or vehicle.


Sá SI, Pereira PA, Paula-Barbosa MM, Madeira MD (2010) Role of neural afferents as mediators of estrogen effects on the hypothalamic ventromedial nucleus. Brain Res. 1366: 60-70.

Carvalho IP (2010) Two siblings concordant for male-to-female transsexualism: A case report. Int J Transgender 12(1): 15-20.


Carvalho IP (2010) Sexuality and transsexuality: A qualitative study. Eur J Sex Health 19(1): S141.


Carvalho IP (2010) Transsexualidade: Vivência do processo de transição no contexto dos serviços de saúde. Acta Med Port. 23(6): 1001-1010.

Carvalho IP. “Contra a socialização”: A identidade transsexual. In Actas do VII Simpósio Nacional de Investigação em Psicologia. Edts Nogueira C, Silva I, Lima L, Almeida AT, Cabecinhas R, Gomes R, Machado C, Maia A, Sampaio A e Taveira MC. Universidade do Minho, Braga, 2010; pp 393-403.


Francisco Allen Gomes, Sexologia para Psiquiatras.


Carvalho IP. Sexuality and Transsexuality: A Qualitative Study. 10th Congress of the European Federation of Sexology, Porto. Maio de 2010.

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