The gonadotropin-releasing hormone (GnRH) neurons of the hypothalamus are the principal regulators of reproductive function and are strongly modulated by estrogen (E₂). Several studies indicate that E₂ is able to influence GnRH neurons, both with "classical" long-term transcriptional effects, and with rapid non-transcriptional effects. One most interesting action of E₂ is that of modulating intracellular calcium concentration [Ca2+]I: this has been shown to happen in many different cell types, including embryonic models of GnRH neurons. The aim of this project was to evaluate if these rapid effects of E₂ on [Ca2+]I also happen at the level of adult GnRH neurons. In order to study the acute effects of E₂ on calcium dynamics, a novel transgenic mouse line was generated, that allows real-time measurement of [Ca2+]I selectively in GnRH neurons in an acute brain slice preparation. Using this mouse line, our group has previously shown that these cells show spontaneous activity in the form of Ca2+ transients. A first set of experiments was designed to define the effects of E₂ on spontaneous activity. E₂ was found to modulate [Ca2+]I in a activity-dependent manner: it increased the frequency of [Ca2+]I transients in about 50% of GnRH neurons with low spontaneous activity, whereas it decreased the frequency of the transients in more than 80% spontaneously active GnRH neurons. Different experiments were then performed in order to determine the molecular pathways that generates these opposite effects. The inhibitory effect was reproduced by the membrane-impermeable compound E2-6-BSA, indicating that it happens through a membrane receptor. The E₂ isomer l7α-estradiol was also able to reproduce the inhibitory effect of E₂, suggesting the involvement of some non-classical receptor. This is also confirmed by the presence of this effect in estrogen-receptor β (ER-β) knock-out mice, which exclude the involvement of this receptor. The stimulatory effect was found to be generated through a novel, indirect mechanism. It cannot be reproduced by E2-6-BSA nor by l7α-estradiol, and it is still present in the ER-β knock-out mice. The stimulation, though, can be reproduced in about 50% of cells with an ER-α selective agonist. As this receptor is not present in GnRH neurons, an indirect mechanism must be generating the stimulatory effect. Blockage of action potential mediated synaptic transmission with tetrodotoxin (TTX) did not block E₂ effects, but blockage of non-action potential mediated GABAergic transmission using the GABA[A] selective blocker gabazine completely abolished them. Our hypothesis is therefore that E₂ stimulates the generation of [Ca2+]I transients through estrogen-receptor a (ER-α) located in the terminals of GABAergic afferents. This modulation, in turn, is able to determine release of Ca2+ from IP₃-sensitive intracellular stores. To confirm this, we applied exogenous GABA to the neurons and found that it was able to initiate [Ca2+]I transients. Furthermore, removal of tonic GABAergic tone with gabazine was able to block spontaneous activity. To further analyse the effects of E₂, Ca2+ imaging experiments were performed together with cell-attached patch clamp electrophysiological recordings in order to correlate the electrical activity with the calcium activity. Simultaneous recordings revealed a strong correlation between [Ca2+]I transients and bursts of action currents in adult GnRH neurons. E₂ was able to increase the electrical activity of GnRH neurons with low spontaneous activity, and inhibit that of highly active ones. Application of GABA to GnRH neurons resulted in increased firing, accompanied by an increase in [Ca2+]I. These observations provide evidence for a complex mechanism of E₂ action on adult GnRH neurons, that may be important for the generation of the pulsatile release of this hormone.
|Publication status||Published - 2009|
- non genomic effects