Testosterone synthesis in the female songbird brain
Introduction
Studies on rapid regulation of neurosteroids have focused almost exclusively on the potent estrogen 17β-estradiol (E2). E2 is produced via the local aromatization of testosterone within many brain regions, and influences major functions and behaviors including social communication (Ball and Balthazart, 2010; Balthazart et al., 2009; Garcia-Segura, 2008; Hull and Dominguez, 2015; Yoder and Vicario, 2012). The local dynamics and acute actions of E2 on social communication are most evident in songbirds.
One region of songbird secondary auditory cortex (caudomedial nidopallium; NCM) is enriched with estrogen receptors and aromatase (Metzdorf et al., 1999; Yoder and Vicario, 2012). Both male and female zebra finches exhibit a rapid elevation in NCM E2 in social and auditory contexts (Remage-Healey et al., 2008, Remage-Healey et al., 2012). Increases in local E2 also directly enhance the auditory-evoked activity of NCM neurons (Remage-Healey et al., 2010; Remage-Healey and Joshi, 2012; Tremere et al., 2009; Tremere and Pinaud, 2011), while infusion of fadrozole, a potent and selective aromatase inhibitor, causes acute decreases in the auditory-evoked activity of NCM neurons and the behavioral preference for familiar songs (Remage-Healey et al., 2010). Therefore, local E2 acts as a neuromodulator to directly influence both local neuronal response properties and downstream sensorimotor integration (Remage-Healey et al., 2013; Vahaba and Remage-Healey, 2018). The rapid fluctuations in local E2 levels in the songbird brain predict that upstream precursors like testosterone also exhibit dynamic regulation.
Theoretically, song-induced elevations in NCM E2 can depend on three related mechanisms, including (a) a rapid increase of aromatase activity, (b) a rise of the precursor testosterone in the plasma and diffusion into the brain and/or (c) a direct local testosterone synthesis independent from the periphery. Rapid changes in aromatase activity have been well demonstrated in the avian brain (Balthazart et al., 2006; Cornil and Bournonville, 2017) including the telencephalon of zebra finches (Remage-Healey et al., 2009). Instead, this study focused specifically on the substrate fluctuation/synthesis hypotheses. This current work was conducted using female zebra finches for three reasons. First, females have lower peripheral testosterone levels than do males (Kabelik et al., 2011; Prior et al., 2014). Second, the key enzymes responsible for testosterone synthesis, 3β-hydroxysteroid-dehydrogenase (3β-HSD) and 17β-hydroxysteroid-dehydrogenase (17β-HSD), are expressed and active in the female NCM, exceeding that in males (London et al., 2003, London et al., 2006; Soma et al., 2004; Tomaszycki and Dzubur, 2013). Third, this high androgenic enzymatic activity is rapidly regulated during stress in females but not males (Soma et al., 2004). Therefore, testosterone synthesis in the female NCM could have a functional behavioral role in addition to providing a substrate for fast conversion to estrogens.
Section snippets
Animals
Females zebra finches (adults > 105 dph) were used for this study. Protocols were approved by the Institutional Animal Care and Use Committee at the University of Massachusetts. All animals were raised from our colony or came from a commercial supplier.
General procedures
Three main experiments were performed using in vivo microdialysis or blood measurements for steroids according to previously published protocols (Remage-Healey et al., 2008, Remage-Healey et al., 2012). All animals were housed in acoustically
Validations of the testosterone assay on NCM dialysates
Testosterone was measured in NCM dialysates per previously published protocols (Remage-Healey et al., 2008). Here we used sequential assays of both steroids in the same samples and drugs that can interfere with these measurements, five validation steps were thus necessary.
First, the fidelity of both the testosterone-only and the sequential assays were compared. Increasing concentrations of testosterone were added to control aCSF or to the mix that came from the previous E2 assay. This mix
Discussion
To our knowledge, this study provides the first evidence that testosterone is locally synthesized in the brain, in vivo. Our findings also clarify rapid neuroestrogen fluctuations in the forebrain. Neuroestrogens can fluctuate in several species, including within the hippocampus and hypothalamus (Kenealy et al., 2013; Sato and Woolley, 2016). Our findings replicated a song induced-elevation in local neuroestrogen levels that was previously observed in the NCM of male (Remage-Healey et al., 2008
Conclusions
Our findings demonstrate that testosterone is actively synthetized in the female brain and suggest that neuroandrogens might thus regulate brain function and behavior. These data focus attention on reproduction and cognition for neuroandrogen actions (Davison and Davis, 2003; Pluchino et al., 2015). It is also now important to evaluate the translational implications of androgen synthesis in the female brain, since steroidal drugs are used by women for sexual desire disorder and anti-cancer
Declaration of competing interest
None.
Acknowledgements
This work was supported by National Institutes of Health - Grant R01NS082179 (to LRH). CdB was supported by a Belgian American Education Foundation (BAEF) fellowship.
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Present address: GIGA Neuroscience, University of Liege, Liege, B-4000, Belgium.