Estrus cyclicity of spinogenesis: underlying mechanisms

For more than a decade, it has been known that estrogens influence synaptic plasticity in the hippocampus. Gould et al. (1990) were the first to show that ovariectomy of female rats resulted in a decrease of dendritic spine density in CA1 pyramidal neurons in the hippocampus. Accordingly, systemic estradiol treatment of these ovariectomized animals caused a rescue of impaired spinogenesis in this particular hippocampal region. Consistently, spine synapse density varied in response to fluctuating estrogen levels during the estrus cycle in female rats (Woolley et al. 1990). Along these lines, a decrease in spine density was observed over the 24-h period between the late proestrus and the late estrus phases of the cycle, which is characterized by a distinct decline in plasma estrogen level. Subsequently, during diestrus dendritic spine synapse density cycles back from low values to high values. Further studies have demonstrated that a variety of synaptic markers are also upregulated after systemic estrogen application (for review see: McEwen 2002), confirming the supportive role of estrogens on synapse formation. Along these lines, estradiol increases the magnitude of LTP at CA3-CA1 synapses in the hippocampus (Cordoba Montoya and Carrer 1997; Yun et al. 2007). This has been related to the memory-enhancing effects of this hormone (Walf and Frye 2006). Warren et al. (1995) were the first to demonstrate enhanced synaptic activity in proestrus rats and Cordoba Montoya and Carrer (1997) found that estrogen facilitates the induction of LTP in the hippocampus of awake rats. Acute application of estradiol to native hippocampal slices in vitro increases NMDA and AMPA receptor transmission and LTP (Foy et al. 1999; Good et al. 1999). In more recent studies, Smith and McMahon (2005, 2006) demonstrated that the estrogen-induced increase in the magnitude of LTP occurs only when the ratio of NMDA transmission to AMPA transmission is increased.

The results of these initial studies on estrogen-induced synaptic plasticity and the fact that gonads are the main source of estrogen, strongly suggest an endocrine regulation of spinogenesis in the hippocampus by gonadal estradiol. The latter reaches its target, the hippocampus, via the plasma. We used hippocampal dispersed cultures and hippocampal slice cultures in an attempt to adopt this paradigm to hippocampal culture models for further experimental approaches in vitro. Slice cultures allow preservation of neuronal connectivity and can be maintained for 4 weeks (Frotscher et al. 1995). In these cultures neither high pharmacological nor physiological serum concentrations of estradiol increased the number of spine synapses and spines. These results questioned the endocrine effect of gonadal estrogens on synapse formation (Kretz et al. 2004). Here, we review our previous findings and add novel results in addressing the discrepancy between our in vitro findings and estrus cyclicity of synaptogenesis in animals (Wehrenberg et al. 2001; Rune et al. 2002; Prange-Kiel et al. 2003; Kretz et al. 2004; Rune et al. 2006; Prange-Kiel et al. 2008).

Neurosteroids are defined as steroids that accumulate in the brain even in the absence of steroidogenic glands and are synthesized in the brain from endogenous precursors by enzymes that are present in situ (Baulieu and Robel 1990). The precursor of all steroid hormones is cholesterol, which is transported into the mitochondria, where steroidogenesis starts by conversion of cholesterol to pregnenolone. This cholesterol transfer across the mitochondrial membranes is considered to be the rate-limiting step of steroid biosynthesis and is mediated by the Steroid Acute Regulatory Protein (StAR). Using in situ hybridization and immunohistochemistry, we showed the expression of StAR mRNA as well as StAR protein in the hippocampus (Wehrenberg et al. 2001). Pyramidal neurons, granule cells of the dentate gyrus, and interneurons showed specific staining with both methods. A comparable expression pattern was demonstrated for StAR in the hippocampus by Furukawa et al. (1998). It is taken for granted that StAR-positive cells always contain side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase (Furukawa et al. 1998). These enzymes synthesize the substrates for aromatization of androgen precursors, whose aromatization eventually leads to the synthesis of estrogens. Furthermore, aromatase, the enzyme that converts testosterone to estradiol, and StAR show an overlapping hippocampal expression on mRNA and protein level (Wehrenberg et al. 2001). This coexpression strongly suggests that hippocampal neurons are able to synthesize estrogens de novo.

In order to prove the hypothesis of a de novo estradiol synthesis in hippocampal neurons, we established a pure neuronal hippocampal dispersion culture, obtained from hippocampi of adult (10-week-old) rats (Prange-Kiel et al. 2003). These neurons were cultivated under serum- and steroid-free conditions. An RIA revealed the presence of estradiol in the medium, indicating the ability of hippocampal cell cultures to synthesize and release estradiol. Subsequent experiments with hippocampal slice cultures and hippocampal dispersion cultures obtained from postnatal rats confirmed these findings (Kretz et al. 2004). 17β-Estradiol must be synthesized de novo from cholesterol, the precursor of all steroid hormones, since the medium of the dispersion cultures contained neither serum nor steroids.

The use of an aromatase inhibitor, letrozole, confirmed the aromatase dependency of estrogen synthesis. Supplementation of the medium with letrozole resulted in a significant decrease in the amount of synthesized estrogen. The effect was most prominent in dispersion cultures obtained from adult hippocampi, where the estradiol synthesis was almost completely downregulated (Prange-Kiel et al. 2003). In cultures originating from postnatal (p5) animals, the inhibiting effect of letrozole on estrogen synthesis was demonstrated to be dose-dependent (Kretz et al. 2004).

Transfection of the cells with siRNA against StAR was performed to confirm the results after letrozole treatment. We chose siRNA against StAR, instead of siRNA against aromatase, to ensure that the effects seen after aromatase inhibition are due to the lack of estrogen rather than to elevated levels of precursors in estrogen synthesis (see below). The transfection did not influence StAR expression, as immunostaining of StAR in siRNA-StAR control cells did not differ from non-transfected cells. Measurement of estradiol in media collected after 7 days revealed a downregulation of estradiol synthesis by approximately 20% in siRNA against StAR transfected cells compared to siRNA controls (Fester et al. 2006; Fester et al. 2009). These experiments prove that hippocampal neurons synthesize estradiol de novo from cholesterol.

In order to stimulate aromatase activity by offering elevated levels of precursors of estrogen synthesis, which serve as substrates of aromatase, we treated dispersed cultures with cholesterol and testosterone. To demonstrate the effects of cholesterol, we used water-soluble, methyl-β-cyclodextrin-encapsuled cholesterol, which is able to pass through the plasma membrane (Daniel et al. 2004; Daniel et al. 2007). This is a common approach to load cells with cholesterol without using lipoproteins. Testosterone was used as a control as it is aromatized to estradiol by aromatase and, as a consequence, increases spine density in the rat hippocampus (Leranth et al. 2004). Treatment of our cultures with testosterone or cholesterol resulted in a highly significant increase in estradiol release in the supernatant, showing that elevated substrate levels do indeed increase estradiol synthesis (Fester et al. 2009).

We used the same dose of cholesterol and testosterone in order to prove the hypothesis that the dose of cholesterol required for affecting synaptic plasticity has to be far higher than the doses of steroid metabolites (Pfrieger 2003). At first glance, this hypothesis seems to be plausible, considering the twofold function of cholesterol: it serves as building material for different synaptic components and alternatively, as a precursor for steroid biosynthesis. The degree of upregulation was notably higher in response to cholesterol than after testosterone treatment, which very likely results from cholesterol depletion under serum-free conditions, such as our dispersed cultures. Cholesterol depletion has severe consequences in neuronal culture preparations (de Chaves et al. 1997; Michikawa and Yanagisawa 1999; Thiele et al. 2000; Chamberlain et al. 2001; Lang et al. 2001; Fan et al. 2002). We did not observe any effect on cell viability, namely because neurons cultivated under serum- and glia-free conditions produce enough cholesterol to survive and to differentiate axons and dendrites (Mauch et al. 2001). Supplementation of the cultures with cholesterol, however, compensates for cholesterol depletion under serum-free conditions. Thus, it is not unexpected that the increase in estradiol synthesis in dispersed cultures is very dramatic in response to cholesterol and less strong in response to testosterone (Fester et al. 2009).

The fact that hippocampal neurons are capable of producing estrogens raises the question of the functional significance of this endogenous steroid. As an internal control we studied the expression of ERα and β. In dispersion cultures, ERα was upregulated, whereas ERβ expression was decreased in response to estradiol. Vice versa, ERα was downregulated and ERβ upregulated in response to letrozole. Since ERs are ligand-inducible transcription factors, these data show that the cultures are responsive to estrogen-treatment (Prange-Kiel et al. 2003).

In view of the estrus cycle dependency of spinogenesis (Woolley et al. 1990), gender differences in hippocampal synaptogenesis should be a logical consequence. In vitro we did not find differences between hippocampal cultures that originated from males and females (Fig. 1). The expression pattern of aromatase mRNA is similar in the hippocampi of males and females, as shown by in situ hybridization (Wehrenberg et al. 2001). ERs density differed neither in vivo nor in vitro (Rune et al. 2002) and in hippocampal cultures obtained from males and females, the estradiol levels in the medium were in the same order of magnitude. Furthermore, spine synapse density did not differ in hippocampal slice cultures originating from male and female animals. Finally, estradiol treatment of hippocampal slices does not result in an increase in spine synapse density, irrespective of the gender of the animals providing the tissue (Kretz et al. 2004).

Obviously, there is a discrepancy between our findings on the unique role of endogenous estradiol in the regulation of spinogenesis and the fact that spine synapse density in females varies with the estrus cycle. We therefore hypothesized that hippocampal and ovarian estradiol synthesis are synchronized by a common factor. In view of abundant GnRH-R mRNA expression in the hippocampus (Bard and Pelletier 1987; Reubi et al. 1987; Jennes et al. 1988) and the direct effect of GnRH on estradiol synthesis in gonadal cells (for review: Janssens et al. 2000), we tested GnRH as a potential regulator of hippocampal estradiol synthesis. GnRH is the key regulator of reproduction, as its pulsative release from the hypothalamus controls the secretion of follicle stimulating hormone and luteinizing hormone in the pituitary, which, in turn, regulate steroid hormone synthesis in the gonads. In recent years, however, numerous in vitro and in vivo studies in mammals have revealed that GnRH also influences estradiol synthesis directly in the ovary (for review: Janssens et al. 2000). Treatment of dispersed hippocampal neurones with GnRH affected the release of estradiol into the cell culture medium in a specific dose-dependent manner (Fig. 2a). Intermediate doses of 10⁻⁸ and 10⁻⁷ M GnRH resulted in a significant increase in estradiol synthesis as compared to controls. At the highest dose of GnRH (5 × 10⁻⁷ M), estradiol synthesis was inhibited as compared to the intermediate doses and did not differ from the untreated control. Notably, an inverted U-shaped dose–response curve of estradiol synthesis has also been described for cultured granulosa cells that were treated with the GnRH agonist buserelin (Parinaud et al. 1988). This type of dose–response curve, caused by receptor desensitization, is typical of G-protein-coupled receptors such as GnRH-R. Receptor desensitization, mainly due to receptor internalization, accounts for this phenomenon (for review: McArdle et al. 2002).

Our next step was to test whether the effect of GnRH on estradiol synthesis is aromatase-dependent. We applied therefore, GnRH at a dose that would increase estradiol synthesis in combination with the aromatase inhibitor letrozole. The co-treatment with letrozole blocked the positive effect of GnRH on estradiol synthesis, demonstrating that the effect of GnRH is mediated by aromatase (Fig. 2b. The effect of GnRH on estradiol synthesis was abolished by co-treatment of cell cultures with the GnRH antagonist antide (Fig. 2b). This further validated the specificity of the GnRH effect.

In subsequent experiments we demonstrated that GnRH affects the expression of the postsynaptic protein spinophilin and the formation of spine synapses by regulating estrogen synthesis. The application of GnRH to hippocampal cultures increased the expression of spinophilin (Fig. 3) and the formation of spine synapses in a dose-dependency that mirrors that of estradiol synthesis. Moreover, when GnRH-induced hippocampal estradiol synthesis was blocked by the aromatase inhibitor letrozole, the spinophilin-stimulating effect of GnRH was abolished (Fig. 4). This finding shows that the GnRH effect on spine formation is mediated by its influence on estradiol synthesis.

Previous studies have shown that estradiol decreases GnRH-R expression in ovarian cells and in a gonadotrope-derived cell line (Nathwani et al. 2000; McArdle and Poch 1992). This raises the question of whether the expression of GnRH-R in the hippocampus is influenced by endogenous estradiol. The treatment of hippocampal neurons with letrozole resulted in an increase in GnRH-R immunoreactivty in the cells, compared to untreated cells. These findings argue for an inhibition of hippocampal GnRH-R expression by endogenously produced estradiol and may indicate a negative-feedback mechanism that prevents excessive estradiol production, thus, balancing the system.

Spine synapse counting in the brains of females at estrus and proestrus revealed that estrus cyclicity of spine density occurs specifically in the hippocampus, but not in the frontal cortex. Quantitative RT-PCR proved that GnRH-R expression is fivefold higher in the hippocampus than in the cortex (Fig. 5), which points to the specific responsiveness of the hippocampus to GnRH. A recent study by Quintanar et al. (2007) further supports this idea, as it shows that only 7% of the cortical neurons are immunoreactive for GnRH-R, whereas the GnRH binding capacity in the hippocampus is considerably high (Bard and Pelletier 1987; Reubi et al. 1987; Jennes et al. 1988).

Our data strongly suggest that estrus cyclic synaptogenesis in the female hippocampus results from the cyclic regulation of hippocampal estradiol synthesis in response to the pulsative release of GnRH. Thus, GnRH may synchronize both gonadal and hippocampal estradiol synthesis, and as a consequence, estradiol serum levels and hippocampal spine density change in parallel.