Neurosteroids in Adult Hippocampus of Male and Female Rodents: Biosynthesis and Actions of Sex Steroids

The brain is not only the target of steroid hormones but also is able to locally synthesize steroids de novo. Evidence of the local production of steroids in the brain has been accumulating in various vertebrates, including teleost fish, amphibia, birds, rodents, non-human primates, and humans. In this review, we mainly focus on the local production of sex steroids in the hippocampal neurons of adult rodents (rats and mice), a center for learning and memory. From the data of the hippocampus of adult male rats, hippocampal principal neurons [pyramidal cells in CA1–CA3 and granule cells in dentate gyrus (DG)] have a complete system for biosynthesis of sex steroids. Liquid chromatography with tandem-mass-spectrometry (LC-MS/MS) enabled us to accurately determine the levels of hippocampal sex steroids including 17β-estradiol (17β-E2), testosterone (T), and dihydrotestosterone (DHT), which are much higher than those in blood. Next, we review the steroid synthesis in the hippocampus of female rats, since previous knowledge had been biased toward the data from males. Recently, we clarified that the levels of hippocampal steroids fluctuate in adult female rats across the estrous cycle. Accurate determination of hippocampal steroids at each stage of the estrous cycle is of importance for providing the account for the fluctuation of female hippocampal functions, including spine density, long-term potentiation (LTP) and long-term depression (LTD), and learning and memory. These functional fluctuations in female had been attributed to the level of circulation-derived steroids. LC-MS/MS analysis revealed that the dendritic spine density in CA1 of adult female hippocampus correlates with the levels of hippocampal progesterone and 17β-E2. Finally, we introduce the direct evidence of the role of hippocampus-synthesized steroids in hippocampal function including neurogenesis, LTP, and memory consolidation. Mild exercise (2 week of treadmill running) elevated synthesis of DHT in the hippocampus, but not in the testis, of male rats, resulting in enhancement of neurogenesis in DG. Concerning synaptic plasticity, hippocampus-synthesized E2 is required for LTP induction, whereas hippocampus-synthesized DHT is required for LTD induction. Furthermore, hippocampus-synthesized E2 is involved in memory consolidation tested by object recognition and object placement tasks, both of which are hippocampus-dependent.


Steroid Biosynthesis in the Hippocampus of Adult Male Rodents
Until about 15 years ago, it had not been elucidated whether adult hippocampal neurons have a complete system for synthesis of sex hormones (from cholesterol to androgens or estrogens) since P450(17α), which is required for synthesis of dehydroepiandrosterone (DHEA) from pregnenolone (PREG), had been thought to be absent in the brain of mammals. Any effort to demonstrate the existence of P450(17α) or its enzymatic activity had been unsuccessful (38)(39)(40)(41) despite the presence of DHEA in the rodent brain even after castration (1,2).
Although these results give information about the potential to synthesize steroids in the hippocampus, it remains unclear whether hippocampus-synthesized steroids are effective enough to modulate hippocampal functions. To answer this question, it is necessary to quantitatively determine the concentration of steroids in the hippocampus. From early 2000s, quantitative determination of steroids, such as PREG, DHEA, and T in brain with mass-spectrometry (MS) began to emerge (53)(54)(55)(56)(57)(58). The presence of pregnenolone sulfate (PREGS) in the brain of mammals had been a matter of debate (59).
To detect small amounts of steroids in the brain, purification of samples and selection of appropriate derivatization reagents are indispensable. Extracts from brain tissue contain various kinds of impurities (lipids and other steroids) which mask derivatization and ionization of the steroid of interest, resulting in decrease of detection efficiency of MS. Purification of the extracts by hybrid-SPE cartridges before LC-ESI-MS/MS enabled the detection of PREGS in the rat hippocampus (60). Concerning the detection of sex steroids, we removed impurities from hippocampal extracts and separated into fractions containing an individual steroid, with C18 column and normal phase HPLC before derivatization. Next, picolinoyl-derivatization was selected for steroids of interest (E2, T, DHT, and E1) to increase ionization efficiency (61,62). Concerning E2, further derivatization with pentafluorobenzyl was performed to elevate volatility. In combination with these improvements, LC-MS/MS enabled us to quantitatively determine the concentration of E2, T, DHT, and E1 in the hippo campus, with high accuracy and reproducibility (37). Caruso and collaborators also determined the levels of steroids including E2, T, and DHT in  c Data are expressed as mean and are represented as nanomolar. Concentration in nanomolar is calculated using the average volume of 0.14 mL for one whole hippocampus that has 0.14 ± 0.02 g wet weight (n = 86). We assume that tissue having 1 g of wet weight has an approximate volume of 1 mL, since the major part of tissue consists of water whose 1 mL weight is 1 g (47). rat hippocampus, with LC-MS/MS methods (63). These results support that the significant amount of sex steroids exists in the hippocampus.
Correlation between the level of hippocampal androgen (T and DHT) and that of plasma T (Figure 1) was observed (37,64). Hippocampal estrogen (E2), however, did not correlate with plasma T (Figure 1) (37,64). Using male and female rats, Melcangi's laboratory extensively analyzed the correlation of steroid levels among plasma, CSF and various brain regions (63). According to this work, the levels of E2, T, and DHT in the hippocampus or CSF positively correlated with those in plasma, but no significant correlation of E2 level was observed between in the hippocampus and CSF (63).

Synthesis and Fluctuation of Steroids in the Hippocampus of Adult Female Rodents
Equally important is to clarify whether local steroid production occurs in female hippocampus, because sex hormones have a great impact on functions of female hippocampus [reviewed in Ref. (65)]. The knowledge of the hippocampus-synthesized steroids, however, had been biased toward the data from males (11)(12)(13) because of the estrous cycle in female animals. In case of rats and mice, estrous cycle comprises of four stages [proestrus: Pro, estrus: Est, diestrus1 (also called metestrus): D1, and diestrus2 (also called diestrus): D2], and each stage switches in 1 day in this order (66). Hippo campal functions such as spatial memory (67)(68)(69)(70)(71), LTP (72,73), and spine/ synapse density (74)(75)(76)(77)(78), fluctuate across the estrous cycle. To investigate hippocampal steroid synthesis in female rodents, therefore, fourfold as much data as those of male must be acquired.
LC-MS/MS analysis revealed the accurate concentrations of progesterone (PROG), androstenedione (ADione), T, E1, and E2 in the hippocampus of adult female rats at each stage of estrous cycle ( Table 1) (79). The levels of plasma steroids exhibit typical estrous cycle dependent changes, in agreement with the previous study (80). The level of hippocampal E2 highly correlates with that of plasma E2, in agreement with other study (63). Concerning the correlation of PROG level between in hippocampus and in plasma, our data exhibit highly positive correlation, whereas others have no correlation (63). It may be due to the difference of samples used for calculation of correlations, rats of both sexes (male and diestrus female) in Caruso et al. (63) and only female (all four stages of the estrous cycle) in our study (79).
Penetration of plasma E2 into the hippocampus, however, is not able to account for the level of hippocampal E2 because hippocampal E2 is much higher than that in plasma. There are two possibilities for explanation of hippocampal E2 fluctuation. The first is the fluctuation of blood PROG which is well known to fluctuate across the estrous cycle (80). This peripherally produced PROG may penetrate hippocampus and be converted into E2, resulting in E2 fluctuation. The other is the fluctuation of activity of kinases including MAPK, Akt, and LIMK, across the estrous cycle (72,83,84). The activity of P450arom (E2 synthase) changes upon phosphorylation (85). If the activity of kinases fluctuates, then following the fluctuation of P450arom activity may generate hippocampal E2 fluctuation, even if the mRNA levels of steroidogenic enzymes do not change across the estrous cycle.

Regulation of local Production of Steroids in Hippocampus
A stimulation with NMDA for 30 min increases the levels of PREG and E2 in the hippocampal slices of adult male rats (42,43,47), suggesting that neural activity-dependent Ca 2+ influx drives local production of PREG and E2.
Reduction of P450arom activity by phosphorylation via kinases (PKA and PKC) is an important mechanism which regulates E2 synthesis. Balthazart et al. demonstrated that this phosphorylation occurred in the quail brain within 15 min (85)(86)(87). In the cultured hippocampal neurons of female rats, E2 application facilitated the phosphorylation of P450arom, suggesting negative feedback mechanism (88).
As slow/genomic modulators, cis-retinoic acid (89) and gonadotropin-releasing hormone (GnRH) (75) were examined using hippocampal slice culture from neonatal rats. Fortyeight-hour treatment with 1 µM of 9-cis-retinoic acid increased the expression levels of P450(17α) and P450arom in the cultured hippocampal slices from male rats, via retinoid X receptor signaling (89). On the other hand, 8 days of treatment with GnRH enhanced local E2 production (75,90). Hippocampal E2 synthesis was also increased by a stereotaxic injection of GnRH into the hippocampus of adult female rats (91).
Interestingly, behaviors, including social interaction (92, 93) and exercise (94), alter local production of steroids in the hippocampus. Social isolation (housing individually for 8 weeks) upregulated the mRNA levels of P450arom and StAR in the hippocampus of adult male rats, compared with pair housed rats (92), whereas environmental enrichment (housing in a group of nine in a large cage for 8 weeks) increased the mRNA levels of 5α-reductase type 1 and 3α-HSD (93).

Hippocampus-Synthesized DHT enhances neurogenesis in Dg
Adult hippocampal neurogenesis occurs in DG throughout life in mammals (95). Sex steroids (96)(97)(98)(99) and exercise (100,101) enhance adult hippocampal neurogenesis of rodents, but the involvement of sex steroids in the exercise-induced neurogenesis, had been poorly understood.
Recently, Okamoto et al. revealed that mild exercise (30 min/day for 2 weeks) increased synthesis of hippocampal DHT, resulting in the neurogenesis enhancement (94). Injection of flutamide, an AR antagonist, suppressed the exercise-induced increase in neurogenesis, suggesting the involvement of androgens. However, surprisingly, castration (depletion of androgen from blood circulation) did not suppress this effect, suggesting the involvement of hippocampus-synthesized androgens. Indeed, the increase in DHT and 5α-reductase (DHT synthase) mRNA, were observed in the hippocampus of castrated rats after exercise (94). This study provides the direct evidence of the role of hippocampus-synthesized steroids in hippocampal functions.

Modulation of lTP/lTD induced by Hippocampus-Synthesized Steroids
The physiological roles of hippocampus-synthesized sex steroids (E2 and DHT) in LTP/LTD were demonstrated in vitro studies using acute hippocampal slices and selective inhibitors of steroidogenic enzymes. A perfusion with letrozole, a selective inhibitor of P450arom, suppressed the magnitude of LTP in CA1-CA3 synapses of adult male rats (102), and in DG synapses of young (3-to 4-week old) male rats (103)(104), within 10-20 min. ICI182,780, a selective antagonist of ERα/β, mimicked this suppressive effect (102), suggesting that hippocampus-synthesized E2 is required for full induction of LTP via synaptic ER. Conversely, hippocampus-synthesized DHT is required for the induction of LTD, from the data showing that low frequency stimulation (1 Hz, 15 min)-induced LTD was suppressed in the presence of finasteride, an inhibitor of 5α-reductase (105). In addition to sex steroids, the effect of hippocampus-synthesized PREG is reported, in which the application of aminoglutethimide, an inhibitor of P450scc, decreased the field excitatory postsynaptic potentials in granule cells in 20 min (103). Although the molecular mechanism underlying these effects remains unclear, a possible explanation may be provided by analogy from the data, showing that exogenous application of E2 rapidly (within 30 min) enhanced LTP by driving kinase network (Erk MAPK, PKA, PKC, PI3K, and CaMKII) in a non-genomic manner (27,106).

Role of Hippocampus-Synthesized e2 in Hippocampus-Dependent Memory
Recently, the role of hippocampus-synthesized E2 in hippocampus-dependent memory consolidation was provided using OVX mice (107). Immediate after the training, bilateral infusion of letrozole into the dorsal hippocampus blocked the transient elevation of hippocampal E2 (within 30 min), and impaired object recognition and object placement memory consolidation (107). Under the same condition except for infusion of E2, this group previously demonstrated that E2 enhanced hippocampal memory consolidation via rapid activation of Erk MAPK and PI3K/Akt (108,109), suggesting that learning experienceinduced E2 elevation in the hippocampus rapidly activates kinase cascades.

COnClUSiOn
Hippocampus-synthesized steroids as well as circulationderived ones, are of importance for hippocampal functions.
A possible molecular mechanism for rapid effect of hippocampussynthesized steroids may be kinase networks which modulate hippocampal functions, including spinogenesis (106,110), LTP (27), learning, and memory (108,109).

AUTHOR COnTRiBUTiOnS
YH wrote the manuscript. SK brushed up the initial draft of the manuscript written by YH.