Steroidal and gonadal effects on neural cell proliferation in vitro in an adult songbird

Abstract

Neurogenesis in the adult songbird brain occurs along the ventricular zone (VZ), a specialized cell layer surrounding the lateral ventricles. To examine the acute effects of sex steroids on VZ cell proliferation, male and female adult zebra finch brain slices containing the VZ were exposed to 5-bromo-2′-deoxyuridine-5′-monophosphate (BrdU) in vitro. Slices from one hemisphere served as the control, while contralateral slices were treated with steroids, steroidogenic enzyme inhibitors or gonadal tissue itself. There were no significant effects on VZ cell proliferation in either sexes by acute exposure to 17β-estradiol (E2), dihydrotestosterone (DHT), a cocktail of four sex steroids, and inhibitors of sex steroid synthesis (aminoglutethimide, ketoconazole, and fadrozole), or by activation of a mitochondrial cholesterol transporter. By contrast, dehydroepiandrosterone (DHEA) suppressed VZ cell proliferation in males, but not females, replicating previous observations involving treatments with corticosterone and RU-486. This suggests that DHEA suppresses proliferation in males via a glucocorticoid receptor-related mechanism. These results suggest that neurosteroidogenesis per se has little effect on acute VZ cell proliferation. Co-incubation with an ovary of female, but not male, slices significantly increased VZ cell proliferation; testicular tissue had no impact on proliferation in males or females. This suggests a role for a non-steroidal ovarian factor on adult female VZ cell proliferation. We also have evidence that previously reported sex-differences in BrdU-labeling along the adult VZ (males > females) result from a more rapid loss of cells in females. Sex differences in steroid action and cell death along the VZ may contribute to the maintenance of the sexually dimorphic song system.

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.