Research ReportSteroidal and gonadal effects on neural cell proliferation in vitro in an adult songbird
Research highlights
►Steroidal and gonadal effects on VZ cell proliferation in adult zebra finch brain. ►Sex steroids do not affect VZ cell proliferation in adult zebra finch brain. ►DHEA reduces VZ cell proliferation only in adult male zebra finches. ►Ovaries increase VZ cell proliferation in adult female zebra finches.
Introduction
Neurogenesis persists from birth to adulthood along the ventricular zone (VZ) surrounding the lateral ventricles (Eriksson et al., 1998, Gould et al., 1999, Alvarez-Bullya et al., 1994). In adult songbirds, neurogenesis occurs to a particularly high degree in specialized “hot spots” along the lateral ventricles that contain pluripotent stem cells and radial glia along which newly born neurons migrate (Goldman & Nottebohm, 1983, Garcia-Verdugo et al., 2002, DeWulf & Bottjer, 2005). Some of these newly born cells differentiate into functional neurons within nuclei of the telencephalic efferent motor pathway for learned vocalization (Alvarez-Buylla & Garcia-Verdugo, 2002, Barnea, 2009). This feature of the songbird brain makes these birds an excellent model to investigate factors regulating neural proliferation.
A variety of factors have been identified that influence adult neural proliferation including peptidergic signals such as cholecystokinin (CCK) and neuropeptide Y (NPY) (Stanic et al., 2008), brain derived neurotrophic factor (BDNF), nitric oxide (NO) (Torroglosa et al., 2007), and pro- and anti-inflammatory secreted molecules (Galvao et al., 2008, Battista et al., 2006). There is also evidence that adrenal and gonadal steroids regulate cell incorporation into developing and adult neural circuits with some debate about direct effects on proliferation, differentiation and migration (Goldman, 1998, Baulieu, 2001, Belelli et al., 2006, Schumacher et al., 2007, Ghoumari et al., 2003). Estradiol (E2), dehydroepiandrosterone (DHEA), progesterone (PROG), allopregnenolone and its sulfate derivatives have all been shown to stimulate neural proliferation in the mammalian brain (Karishma & Herbert, 2002, Keller et al., 2004, Mayo et al., 2005, Galea, 2008). Despite recent advances characterizing the steroidal effects on neuronal cell proliferation, we know relatively little about how steroids derived from the brain (neurosteroids) or synthesized in gonads, impact adult neural proliferation. To address these issues, we exposed slices of the adult zebra finch brain containing the lateral ventricles to BrdU to label cells undergoing DNA synthesis. BrdU has been used previously to “birthdate” mitotically active progenitor cells along the VZ (Nowakowski et al., 1989, Eisch & Mandyam, 2007). In our experiments described below, cells that incorporated BrdU were identified immunocytochemically after a 2-h exposure to BrdU. Hereafter we equate BrdU-labeling and proliferation, acknowledging that cell cycle rates might also differ across treatment groups and was untested in this experimental design.
A variety of steroids can be synthesized de novo in the vertebrate brain including active sex steroids (Baulieu, 2001, Belelli et al., 2006, Schumacher et al., 2007, Charalampopoulos et al., 2008). Steroid synthesis in the avian brain has been a focus of particular interest (Tsutsui and Schlinger, 2001). In the zebra finch (Taeniopygia guttata), the full suite of steroidogenic factors (enzymes and cholesterol transporters; Fig. 1) is expressed at multiple neural sites (London et al., 2006, London & Schlinger, 2003, London & Schlinger, 2007) with the estrogen-synthesizing enzyme aromatase expressed to an especially high degree compared to other birds and mammals (Schlinger & Arnold, 1993, Shen et al., 1995, Saldanha et al., 2000). Developmentally, steroidogenic enzymes are expressed along the VZ suggesting that products of their catalytic reactions regulate proliferation. In adults, however, there is conflicting evidence for a steroid role in neuronal mitosis (proliferation) or post-mitotic (recruitment) phases of neuronal plasticity (Brown et al., 1993, Alvarez-Buylla & Kim, 1997, Hidalgo et al., 1995, Lee et al., 2007). Whereas the rate of cell division measured in vivo in the VZ of adult canaries is sex steroid-independent (Brown et al., 1993), adult zebra finches may show a different pattern (Lee et al., 2007).
Previously, we found that acute exposure to the adrenal steroid corticosterone (CORT) and its agonist RU-486 significantly reduced VZ cell proliferation in adult males, with no effects in females (Katz et al., 2008). Pregnenolone, progesterone and trilostane, an inhibitor of the 3β-HSD enzyme, had no effects on VZ proliferation in either sex suggesting that the effect of CORT acting through the glucocorticoid receptor was specific to CORT and restricted to males. Here, we examined the effects of additional sex steroids on adult VZ proliferation including the androgen precursor dehydroepiandrosterone (DHEA), the potent androgen dihydrotestosterone (DHT) and estradiol (E2). We tested the possibility that steroids synthesized in brain de novo impacted VZ proliferation by inhibiting steroidogenic enzymes or by inducing mitochondrial cholesterol transport.
Section snippets
Impact of sex steroids/steroid synthesis enzyme inhibitors/cholesterol transporter on adult brain VZ cell proliferation
As expected, BrdU-labeled cells were detected only along the surface of the walls of lateral ventricles (Fig. 2). All labeling was confined to the VZ border; no labeled cells were ever observed away from the VZ. We observed no significant effects on the numbers of BrdU-labeled cells after treatment of female brain slices with 1 μM E2 (t = 2.02, p = 0.09) or male brain slices with 100 nM E2 (t = 0.77, p = 0.47; data not shown). In addition, we detected no significant effects on proliferation with 1 μM DHT
Discussion
In this study we examined the acute effects of sex steroids and gonadal tissues on adult VZ cell proliferation using BrdU to label mitotically active precursor cells. Our results suggest that sex steroids from either the periphery or produced within the brain itself do not increase number of BrdU-labeled cells along the walls of the lateral ventricles of the adult zebra finch brain. In contrast, our results indicate that DHEA reduces the number of BrdU-labeled VZ cells in males but not females,
Animals
Adult zebra finches (> 100 days of age; n = 123) were used for these studies and obtained from our breeding colony located in the UCLA Life Sciences vivarium. Birds were housed under 14/10 h light: dark cycle with food and water available ad libitum. All protocols were approved by the UC Chancellor's Committee on Animal Care and Use following NIH guidelines.
Reagents
dl-Aminoglutethimide (Amino) and 17β-Estradiol (E2) were purchased from Sigma. dehydroepiandrosterone (DHEA), ketoconazole (Keto) (Sigma),
Acknowledgments
Funding was provided by NIMH-061944 and HD07228-26.
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