Distribution and subcellular localization of glucocorticoid receptor-immunoreactive neurons in the developing and adult male zebra finch brain

Stress has long lasting effects on physiology, development, behavior, reproductive success and the survival of an individual. These effects are mediated by glucocorticoids, such as corticosterone, via glucocorticoid receptors (GR), however the exact mechanisms underlying these effects are unknown. GR have been widely studied in mammals but little is known about GR in other vertebrate groups, especially songbirds. We investigated the distribution, quantity, and subcellular-localization of GR-immunoreactive (GRir) neurons in the brains of male zebra finches on P10 (post-hatch day 10, song nuclei formed), and in adulthood (post-hatch day 90 or older) using immunohistochemistry. GRir neurons were widely distributed in the brains of male zebra finches including two song nuclei HVC (acronym is a proper name) and RA (nucleus robustus arcopallii) and brain regions including HP (hippocampal formation), BSTl (lateral part of the bed nucleus of the stria terminalis), POM (nucleus preopticus medialis), PVN (nucleus paraventricularis magnocellularis), TeO (optic tectum), S (nucleus of the solitary tract), LoC (Locus coeruleus). Distribution did not vary at the two age points examined, however there were significant differences in staining intensity. Subcellular GR-immunoreactivity patterns were classified as cytoplasmic, nuclear, or both (cytoplasmic and nuclear) and there were significant differences in the overall number of GRir neurons and neurons with both nuclear and cytoplasmic staining in P10 and adult brains. However, there were no significant differences in the percentage of subcellular GR immunoreactivity patterns between P10 and adults. Our study of GRir neuronal distribution in the zebra finch brain may contribute towards understanding of the complex and adverse effects of stress on brain during two different stages of life history.     

17β-Estradiol levels in male zebra finch brain: Combining Palkovits punch and an ultrasensitive radioimmunoassay

Local aromatization of testosterone into 17β-estradiol (E₂) is often required for the physiological and behavioral actions of testosterone. In most vertebrates, aromatase is expressed in a few discrete brain regions. While many studies have measured brain aromatase mRNA or activity, very few studies have measured brain E₂ levels, particularly in discrete brain regions, because of technical challenges. Here, we used the Palkovits punch technique to isolate 13 discrete brain nuclei from adult male zebra finches. Steroids were extracted via solid phase extraction. E₂ was then measured with an ultrasensitive, specific and precise radioimmunoassay. Our protocol leads to high recovery of E₂ (84%) and effectively removes interfering brain lipids. E₂ levels were high in aromatase-rich regions such as caudal medial nidopallium and hippocampus. E₂ levels were intermediate in the medial preoptic area, ventromedial nucleus of the hypothalamus, lateral and medial magnocellular nuclei of anterior nidopallium, nucleus taeniae of the amygdala, and Area X. E₂ levels were largely non-detectable in the cerebellum, HVC, lateral nidopallium and optic lobes. Importantly, E₂ levels were significantly lower in plasma than in the caudal medial nidopallium. This protocol allows one to measure E₂ in discrete brain regions and potentially relate local E₂ concentrations to aromatase activity and behavior.     

Steroid receptors in the adult zebra finch syrinx: a sex difference in androgen receptor mRNA, minimal expression of estrogen receptor alpha and aromatase

The zebra finch syrinx (sound production organ) is a sexually dimorphic component of the song system. Only male zebra finches sing, and in parallel, the overall mass and size of fibers in the two largest syrinx muscles are greater in males than females. Despite these obvious sexual dimorphisms, little is known about the role of steroid hormones in the maintenance of the structure and/or function of the syrinx. In this report, we used in situ hybridization to assess the expression of androgen receptor (AR), estrogen receptor alpha (ERalpha), and aromatase (AROM) mRNAs in the syrinx of adult male and female zebra finches. Increased AR mRNA expression was noted in males compared to females in two regions, over the ventralis muscle and in a band of connective tissue neighboring cartilage (perichondria). In contrast, we did not detect specific ERalpha or AROM mRNA expression within the syrinx. However, substantial ERalpha mRNA was present in oviduct, and aromatase mRNA was expressed at high levels in ovary. In parallel, an assay for AROM detected activity in ovary, but not in syrinx tissue from males or females. Taken together, these data suggest that the adult syrinx is sensitive to androgens; that sex differences in function and morphology of the syrinx may in part be due to increased expression of AR in males compared to females. In contrast, estrogen receptor alpha and AROM appear to have limited roles.     

The specific estrogen receptor antagonist ICI 182,780 masculinizes development of the zebra finch syrinx

In zebra finches, the vocal organ (syrinx) is larger in males compared to females. The exact mechanism responsible for this sex difference is not known, but it may be related to steroid hormones. Previous studies have demonstrated that treatment with estradiol feminizes the mass as well as fiber size of the two largest syrinx muscles (ventralis and dorsalis) in males. Treating females with the aromatase inhibitor fadrozole, however, does not induce masculinization. As an alternative approach to further clarify this paradoxical effect of estrogens on syrinx development, we administered the specific estrogen receptor antagonist ICI 182,780 during the first 25 days post-hatching. Daily injections of this drug significantly increased ventralis and dorsalis muscle fiber size in both sexes. Data also demonstrate that in males, the ventralis muscle makes an earlier contribution to the sex difference in syrinx mass by becoming dimorphic prior to dorsalis. Taken together, these data suggest that estrogens can influence development of the syrinx by feminizing morphology of this tissue. However, the lack of reported sex differences during development in steroid receptors, plasma steroid levels, and aromatase enzyme, indicate that hormones are not solely responsible for sex differences in this organ. Thus, similar to the neural forebrain regions that control song, complete sexual differentiation of the zebra finch syrinx likely involves additional factors.     

Dehydroepiandrosterone metabolism by 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase in adult zebra finch brain: sex difference and rapid effect of stress

Dehydroepiandrosterone (DHEA) is a precursor to sex steroids such as androstenedione (AE), testosterone (T), and estrogens. DHEA has potent effects on brain and behavior, although the mechanisms remain unclear. One possible mechanism of action is that DHEA is converted within the brain to sex steroids. 3beta-Hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase (3beta-HSD) catalyzes the conversion of DHEA to AE. AE can then be converted to T and estrogen within the brain. We test the hypothesis that 3beta-HSD is expressed in the adult brain in a region- and sex-specific manner using the zebra finch (Taeniopygia guttata), a songbird with robust sex differences in song behavior and telencephalic song nuclei. In zebra finch brain, DHEA is converted by 3beta-HSD to AE and subsequently to estrogens and 5alpha- and 5beta-reduced androgens. 3beta-HSD activity is highest in the diencephalon and telencephalon. In animals killed within 2-3 min of disturbance, baseline 3beta-HSD activity in portions of the telencephalon is higher in females than males. Acute restraint stress (10 min) decreases 3beta-HSD activity in females but not in males, and in stressed animals, telencephalic 3beta-HSD activity is greater in males than in females. Thus, the baseline sex difference is rapidly reversed by stress. To our knowledge, this is the first demonstration of 1) brain region differences in DHEA metabolism by 3beta-HSD, 2) rapid modulation of 3beta-HSD activity, and 3) sex differences in brain 3beta-HSD and regulation by stress. Songbirds are good animal models for studying the regulation and functions of DHEA and neurosteroids in the nervous system.     

The expression of the sex steroid-synthesizing enzymes CYP11A1, 3beta-HSD, CYP17, and CYP19 in gonads and adrenals of adult and developing zebra finches

Songbirds have emerged as important animal models for understanding how sex steroids influence brain and behavior, particularly how they direct the sexually dimorphic development of the neural circuits controlling song and then activate adult song behavior. Presumably, sex steroids synthesized in the gonads are responsible for these actions on brain. However, experiments do not always reveal a direct relationship between gonadal function, circulating sex steroids, and activation and/or organization of song. Thus, it is critical that we understand more about the sites and mechanisms of sex steroid synthesis in this group of birds. Toward this end, we have established the use in zebra finches of chicken cDNA probes to the principal androgen synthetic enzymes, CYP11A1, 3beta-HSD, and CYP17. On Northern blots, these probes recognized bands of the appropriate size and in tissues similar to those seen in chickens. With these probes, and a probe to CYP19 specific to the zebra finch, we used in situ hybridization to examine the cellular expression of these enzymes in gonads and adrenals of adult and developing zebra finches (1 to 20 days posthatching). In adults, we identified significant expression of CYP11A1 and CYP17 in large ovarian follicles, particularly the thecal cell layer and over the testicular interstitial area. 3beta-HSD was expressed by both theca and granulosa and in testicular interstitial and seminiferous tubular cells. In adrenals, CYP11A1 and 3beta-HSD are abundant with lesser amounts of CYP17. Developmentally, we identified high expression of CYP11A1 and 3beta-HSD in the adrenals, CYP17 in both testes and ovaries, and CYP19 in ovaries only. These results suggest that the ovaries but not the testes may secrete estrogen developmentally and the adrenals may contribute precursors for gonadal steroidogensis.     

Estrogen synthesis in the brain—Role in synaptic plasticity and memory

Estrogen and androgen are synthesized from cholesterol locally in hippocampal neurons of adult animals. These neurosteroids are synthesized by cytochrome P450s and hydroxysteroid dehydrogenases (HSDs) and 5alpha-reductase. The expression levels of enzymes are as low as 1/200–1/50,000 of those in endocrine organs, however these numbers are high enough for local synthesis. Localization of P450(17alpha), P450arom, 17beta-HSD and 5alpha-reductase is observed in principal glutamatergic neurons in CA1, CA3 and the dendate gyrus. Several nanomolar levels of estrogen and androgen are observed in the hippocampus.Estrogen modulates memory-related synaptic plasticity not only slowly but also rapidly in the hippocampus. Rapid action of 17beta-estradiol via membrane receptors is demonstrated for spinogenesis and long-term depression (LTD). The enhancement of LTD by 1–10 nM estradiol occurs within 1 h. The density of spine is increased in CA1 pyramidal neurons within 2 h after application of estradiol. The density of spine-like structure is, however, decreased by estradiol in CA3 pyramidal neurons. ERalpha, but not ERbeta, induces the same enhancement/suppression effects on both spinogenesis and LTD.     

Sexually dimorphic expression of trkB, a Z-linked gene, in early posthatch zebra finch brain

Sexual differentiation of the zebra finch (Taeniopygia guttata) neural song circuit is thought to be initiated by sex differences in sex chromosome gene expression in brain cells. One theory is that Z-linked genes, present in the male's ZZ genome at double the dose of females' (ZW), are expressed at higher levels and trigger masculine patterns of development. We report here that trkB (tyrosine kinase receptor B) is Z-linked in zebra finches. trkB is the receptor for neurotrophic factors BDNF and neurotrophin 4, and mediates their influence on neuronal survival, migration, and specification. trkB mRNA is expressed at a higher level in the male telencephalon or whole brain than in corresponding regions of the female in adulthood, and at posthatch day (P) 6, when the song circuit is undergoing sexual differentiation. Moreover, this expression is higher in the song nucleus high vocal center (HVC) than in the surrounding telencephalon at P6, and in males relative to females. In addition, trkB protein is expressed more highly in male than female whole brain at P6. These results establish trkB as a candidate factor that contributes to masculine differentiation of HVC because of its Z-linkage, which leads to sex differences in expression. BDNF is known to be stimulated by estrogen and to be expressed at higher levels in males than females at later ages in HVC. Thus, the trkB-BDNF system may be a focal point for convergent masculinizing influences of Z-linked factors and hormones.     

Localized neuronal activation in the zebra finch brain is related to the strength of song learning

Songbirds (Oscines) learn their songs from a tutor. It is not known where in the brain the memories of these learned sounds are stored. Recent evidence suggests that song perception in songbirds involves neuronal activation in brain regions that have not traditionally been implicated in the control of song production or song learning, notably the caudal part of the neostriatum (NCM) and of the hyperstriatum ventrale. Zebra finch males (Taeniopygia guttata castanotis) were reared without their father and exposed to a tape-recorded song during the sensitive period for song learning. When, as adults, they were reexposed to the tutor song, the males showed increased expression of the protein products of the immediate early genes egr-1 (ZENK) and c-fos in the NCM and caudal hyperstriatum ventrale, but not in the conventional "song-control nuclei." The strength of the immediate early gene response (which is a reflection of neuronal activation) in the NCM correlated significantly and positively with the number of song elements that the birds had copied from the tutor song. These results show localized neural activation in response to tutor song exposure that correlates with the strength of song learning.     

Analysis of steroids in songbird plasma and brain by coupling solid phase extraction to radioimmunoassay

It is a common practice to extract steroids from plasma, serum, or tissue samples prior to steroid measurement by radioimmunoassay (RIA) or enzyme immunoassay (EIA). Steroid extraction is critical because it can remove substances that interfere with the RIA or EIA. Steroid extraction is commonly achieved using organic solvents, such as diethyl ether or dichloromethane. However, organic solvent extractions can suffer from low recovery, imprecise recovery, or incomplete removal of assay interference. Here, we describe validations of a simple protocol to extract steroids (e.g., dehydroepiandrosterone, corticosterone, and estradiol) from avian plasma, serum, and brain tissue using solid phase extraction (SPE) with commercially available C18 columns. We compare various methods for (1) eluting steroids from columns, (2) drying eluates, and (3) resuspending dried eluates prior to RIA. The SPE method yields high and consistent recoveries. The SPE method also effectively separates steroids from interfering substances, even when extracting steroids from lipid-rich plasma and brain tissue. These data indicate that SPE is superior to organic solvent extraction on several measures. SPE should be broadly useful for extracting steroids from plasma or tissue samples.     

Differential regulation of brain aromatase by androgen in adult and fetal ferrets

The contribution of androgens to the regulation of aromatase activity (AA), measured by quantifying the in vitro formation of [3H]estrone from 19-[3H]hydroxyandrostenedione precursor, was studied in equivalent microdissected brain regions of adult and fetal ferrets. In adulthood, AA was similar in the bed nucleus of the stria terminalis, medial (M) and lateral (L) preoptic area (POA), medial (MA) and lateral amygdala (LA), ventromedial hypothalamus (VMH), and parietal cortex of gonadectomized males and females given no concurrent steroid treatment. Daily sc injections of the androgen dihydrotestosterone propionate significantly stimulated AA in MPOA, MA, and VMH of adult males and in MA of females; a similar trend was seen in MPOA and VMH of females. By contrast, no evidence of androgenic regulation of AA was obtained in these three brain regions microdissected from fetuses killed on embryonic day 35 (E35; 41-day gestation). Transplacental administration of the antiandrogen flutamide beginning on day E24 failed to affect AA in MPOA, LPOA, MA, LA, or parietal cortex, although this treatment significantly reduced AA in bed nucleus of the stria terminalis of fetal males. The results suggest that the responsiveness of aromatizing enzymes to androgenic induction is similar in several subcortical brain regions of adult ferrets of both sexes. In breeding males such an action of androgen may augment the neural production of estrogen, which has previously been implicated in the control of sexual behavior and the feedback regulation of LH secretion. By contrast, androgen apparently contributes minimally to the regulation of AA in brain regions of fetal ferrets, particularly in the MPOA, in which a sexually dimorphic nucleus differentiates in males around E37 in response to estrogen produced locally.     

Sex steroid levels in developing and adult male and female zebra finches (Poephila guttata)

Serum samples from male and female zebra finches ranging in age from 1 day before hatch to 54 days posthatch were assayed for 17 beta-estradiol (E), androgen, testosterone (T), or 5 alpha-dihydrotestosterone (DHT). Additional samples were assayed from intact and gonadectomized adults, gonadectomized adults with intraperitoneal implants of testosterone propionate (TP) or estradiol benzoate (EB), gonadectomized nestlings, and nestlings injected subcutaneously with EB. DHT levels of developing birds did not vary as a function of either sex or age. During development, average androgen and T levels were highest during the nestling period, prior to sexual maturation, and were higher in females than in males. Endogenous androgen levels of most subjects that were sampled repeatedly rose and then declined between 24 and 49 days. TP implants produced higher T levels in adult females than in adult males. Levels of E were higher in both sexes during the hatching period (Days -1 through 0) than during the nestling period (Days 2 through 14). A greater number of males than females had relatively high E levels on Days 12 and 14 and during the second week overall. There was no sex difference in levels of E in adults, and gonadectomized adults had markedly higher E levels than intact adults. Gonadectomized nestlings had the same androgen and E levels as intact nestlings of the same age; EB injected nestlings had elevated E levels. The present results indicate specific endocrine changes that mirror events crucial to sexual differentiation of endocrine and behavioral components of reproduction, and have important implications for models of sexual differentiation in zebra finches.     

Two separate areas of the brain differentially guide the development of a song control nucleus in the zebra finch.

A brain nucleus that is important for the generation of song in the adult male zebra finch (Poephila guttata), the robust nucleus of the archistriatum (RA), receives dual inputs from two other telencephalic song nuclei: the hyperstriatum ventrale pars caudale (HVc) and the lateral magnocellular nucleus of the anterior neostriatum (L-MAN). We lesioned each of these afferent inputs to the RA early in development, either by themselves or both at the same time in the same side of the brain, to determine what influences each of these nuclei exerts on the normal development of the RA into adulthood. We found that lesioning the HVc in a 20-day-old male zebra finch prevents the later increase in RA volume and soma size that would normally occur around 35 days post-hatching. MAN lesions at this same early age, on the other hand, had a large effect on reducing the volume and cell number of RA neurons, without affecting soma size. Lesioning both inputs early in development induced considerable RA neuronal cell death and atrophy of the nucleus. This study shows that the development of the RA is affected differently by each of its two input nuclei.     

Immunolocalization of 3beta-HSD and 17beta-HSD in the testis of the spotted ray Torpedo marmorata

Using polyclonal antibodies, we examined the localization of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) as markers of the site of steroidogenetic activity during the spermatogenesis of Torpedo marmorata. These enzymes play a central role in the biosynthesis of steroid hormones, including androgen and oestrogen production. We demonstrated that in the spotted ray testis, Sertoli and Leydig cells, as well as spermatogonia, show a positive reaction to anti 3beta-HSD and 17beta-HSD antibodies. In particular, we demonstrated that Sertoli cells show a positive reaction to anti 3beta-HSD and 17beta-HSD antibodies in cysts containing spermatogonia and spermatozoa, while Leydig cells present a positive reaction only when they are located between cysts containing meiotic cells. This study strongly suggests that, as hypothesised in our previous study [Prisco, M., Liguoro, A., D'Onghia, B., Ricchiari, L., Andreuccetti, P., Angelini, F., 2002. Fine structure of Leydig and Sertoli cells in the testis of immature and mature spotted ray Torpedo marmorata. Mol. Reprod. Dev. 63, 192-201.], Sertoli and Leydig cells are differently involved in the hormonal control of spermatogenesis: Sertoli cells before the beginning of meiosis and after spermiation, Leydig cells only during meiosis phase. Moreover, the present paper deals with the possibility that also spermatogonia are engaged in the production of androgen hormones, as they are characterized by the presence of 3beta-HSD and 17beta-HSD enzymes, and show the ultrastructural features of steroid hormone-producing cells.     

Brain aromatase (Cyp19A2) and estrogen receptors, in larvae and adult pejerrey fish Odontesthes bonariensis: Neuroanatomical and functional relations

Although estrogens exert many functions on vertebrate brains, there is little information on the relationship between brain aromatase and estrogen receptors. Here, we report the cloning and characterization of two estrogen receptors, α and β, in pejerrey. Both receptors’ mRNAs largely overlap and were predominantly expressed in the brain, pituitary, liver, and gonads. Also brain aromatase and estrogen receptors were up-regulated in the brain of estradiol-treated males.In situ hybridization was performed to study in more detail, the distribution of the two receptors in comparison with brain aromatase mRNA in the brain of adult pejerrey. The estrogen receptors’ mRNAs exhibited distinct but partially overlapping patterns of expression in the preoptic area and the mediobasal hypothalamus, as well as in the pituitary gland. Moreover, the estrogen receptor α, but not β, were found to be expressed in cells lining the preoptic recess, similarly as observed for brain aromatase.Finally, it was shown that the onset expression of brain aromatase and both estrogen receptors in the head of larvae preceded the morphological differentiation of the gonads. Because pejerrey sex differentiation is strongly influenced by temperature, brain aromatase expression was measured during the temperature-sensitive window and was found to be significantly higher at male-promoting temperature.Taken together these results suggest close neuroanatomical and functional relationships between brain aromatase and estrogen receptors, probably involved in the sexual differentiation of the brain and raising interesting questions on the origin (central or peripheral) of the brain aromatase substrate.