Reinhold Metzdorf

Distribution of aromatase, estrogen receptor, and androgen receptor mRNA in the forebrain of songbirds and nonsongbirds

Androgens and estrogens are crucial for the differentiation and function of the vocal control system of songbirds. A major source of estrogens in songbirds is the cerebral aromatization of circulating testosterone by aromatase (ARO). In the vocal control system, songbirds have a unique estrogen receptor (ER)‐containing area, the nucleus hyperstriatalis ventrale pars caudale (HVC) of the caudal neostriatum. Work in the zebra finch has demonstrated ARO expression adjacent to but not in the HVC. Compared with other songbirds, such as the canary, the HVC of adult zebra finches contains only few ERs. To determine whether the disjunctive distribution of ERs and ARO in the forebrain is a songbird‐specific feature, the authors investigated ARO and ER mRNA expression in songbirds (canary, house sparrow, and zebra finch) and in nonsongbirds (budgerigar, ring dove, swift, grey partridge, and barn owl) of five avian orders. In addition, the coexpression of androgen receptor (AR) and ARO mRNAs was studied. Preoptic hypothalamic areas showed similar expression of ARO in all species. In the caudal neostriatum, ARO, AR, and ER transcripts were found only in songbirds. ARO and ER mRNA expression in the caudal forebrain was spatially separated, i.e., the HVC contained ER mRNA but very little or no ARO mRNA, and the caudomedial neostriatum contained high levels of ARO mRNA but few if any ERs. ARO and AR mRNAs, however, were coexpressed in the caudomedial neostriatum. The coexpression of ARO mRNA with AR mRNA but not with ER mRNA was found in further brain areas, such as the nucleus posterior lateralis hypothalami. The area‐specific coexpression of AR, ER, and ARO suggests various possibilities for the steroid‐dependent regulation of ARO and for the role of ARO in controlling AR‐ and ER‐dependent mechanisms. J. Comp. Neurol. 407:115–129, 1999.

Distribution and Dynamics in the Expression of Androgen and Estrogen Receptors in Vocal Control Systems of Songbirds

Developmental and seasonal changes in the production of androgens and estrogens seem to control sex-specific differentiation and seasonal changes in sexual behaviors such as singing of songbirds. These steroids affect the brain by binding to intracellular located receptors. Here we analyze whether the expression of androgen receptors (AR) and estrogen receptors (ER) is a limiting factor for differentiation of the vocal pattern and the vocal control system of zebra finches and canaries. AR and ER are localised in the brain using in situ hybridizations with cRNA probes of the AR and ER of the zebra finch. AR are widely expressed in the vocal control system and allow androgen-dependent alterations of the development and function of most vocal control areas. The expression of AR in some vocal control areas such as NIF, DLM, and AVT differs between individuals. This individual variability suggests genetic differences or transient steroid-independent expression of AR. ER are found only in the HVC and thus restrict estrogen-dependent developmental and functional changes of the singing to the HVC area. AR- and ER-mRNA expression per cell in the HVC of adult canaries undergoes seasonal changes so that ER are higher expressed from fall to the early breeding season. During ontogeny, ER start to occur in the zebra finch HVC at posthatching day 15 and in the canary HVC at posthatching day 30. As the HVC is already sexual dimorphic in size at these times, HVC-based estrogen-ER-dependent mechanisms seem not to be important for the initial sexual dimorphic development of the HVC.

The Sexually Dimorphic Expression of Androgen Receptors in the Song Nucleus Hyperstriatalis Ventrale Pars Caudale of the Zebra Finch Develops Independently of Gonadal Steroids

The development of sex differences in brain structure and brain chemistry (“brain sex”) of vertebrates is frequently thought to depend entirely on gonadal steroids such as androgens and estrogens, which act on the brain at the genomic level by binding to intracellular transcription factors, the androgen receptors (ARs) and estrogen receptors (ERs). These hormone actions are thought to shift the brain from a monomorphic to a dimorphic phenotype. One prominent such example is the nucleus hyperstriatalis ventrale pars caudale (HVc) of the zebra finch (Poephila guttata), a set of cells in the caudal forebrain involved in the control of singing. In contrast with previous studies using nonspecific cell staining techniques, the size and neuron number of the HVc measured by the distribution of AR mRNA is already sexually dimorphic on posthatching day (P)9. No ARs or ERs are expressed in the HVc before day 9. Slice cultures of the caudal forebrain of P5 animals show that the sexually dimorphic expression of AR mRNA in HVc is independent of the direct action of steroids on this nucleus or any of its immediate presynaptic or postsynaptic partners. Therefore, gonadal steroids do not appear to be directly involved in the initial sex difference in the expression pattern of AR mRNA, size, and neuron number of the HVc. Furthermore, we demonstrate that the initial steroid-independent size and its subsequent steroid-independent growth by extension linearly with the extension of the forebrain explains 60–70% of the masculine development of the HVc. Thus, we suggest that epigenetic factors such as the gonadal steroids modify but cannot overwrite the sex difference in HVc volume determined autonomously in the brain.

Spatial ability is impaired and hippocampal mineralocorticoid receptor mRNA expression reduced in zebra finches (Taeniopygia guttata) selected for acute high corticosterone response to stress.

In mammals, stress hormones have profound influences on spatial learning and memory. Here, we investigated whether glucocorticoids influence cognitive abilities in birds by testing a line of zebra finches selectively bred to respond to an acute stressor with high plasma corticosterone (CORT) levels. Cognitive performance was assessed by spatial and visual one-trial associative memory tasks. Task performance in the high CORT birds was compared with that of the random-bred birds from a control breeding line. The birds selected for high CORT in response to an acute stressor performed less well than the controls in the spatial task, but there were no significant differences between the lines in performance during the visual task. The birds from the two lines did not differ in their plasma CORT levels immediately after the performance of the memory tasks; nevertheless, there were significant differences in peak plasma CORT between the lines. The high CORT birds also had significantly lower mineralocorticoid receptor mRNA expression in the hippocampus than the control birds. There was no measurable difference between the lines in glucocorticoid receptor mRNA density in either the hippocampus or the paraventricular nucleus. Together, these findings provide evidence to suggest that stress hormones have important regulatory roles in avian spatial cognition.

Melatonin affects the temporal organization of the song of the zebra finch.

In birds and mammals, including humans, melatonin‐binding sites are abundant in brain areas that have no known clock function. Although the role of such binding sites is still unclear, it is assumed that these sites link neural functions to circadian or circannual demands of neuroendocrine homeostasis and reproduction. To investigate a possible direct role of melatonin in motor control, we studied the song and neural song system of the zebra finch. Neurons of two sensory‐motor areas of the descending song control circuit that are crucial for the organization of the song pattern, the HVC and RA, express the melatonin‐1B receptor (Mel1B), while the hypoglossal motor neurons of the song circuit express melatonin‐1C receptors (Mel1C). Application of melatonin to brain slices decreases the firing‐rate of RA‐neurons. Systemic administration of a Mel1B antagonist at the beginning of the night shortens the song and motif length and affects the song syllable lengths produced the next day. The temporal pattern of the song, however, does not undergo daily changes. Thus, melatonin is likely to affect a non‐circadian motor pattern by local modulation of song control neurons and in consequence alters a sexual signal, the song of the zebra finch.

The ontogeny of the canary HVC revealed by the expression of androgen and estrogen receptors

THE vocal control nucleus HVC (nucleus hyperstriatalis ventrale, pars caudale) of the canary (Serinus canaria) is a model in which to study the relationship between anatomical plasticity and vocal developmental learning. Much of the structural plasticity of the HVC is sensitive to the action of androgenic and oestrogenic gonadal hormones that affect the brain by binding to androgen receptors (AR) and oestrogen receptors (ER). Here we report developmental changes of AR and ER expression in the HVC using in situ hybridization with avian specific cRNA probes. AR and ER are first expressed in the HVC at post-hatching day 10 (P10) and P30, respectively and are, therefore, present in the HVC throughout the singing-learning period of the canary. Because AR occur only in the caudal neostriatum of the HVC we mapped the size of the HVC with this marker. The size and neurone number of the AR-defined HVC reaches adult values at P30 and differentiates, therefore, independently of the singing activity and probably independently of oestrogens. Continuing neurogenesis in the HVC requires neuronal death and replacement from P30 on.

Bi-Directional Sexual Dimorphisms of the Song Control Nucleus HVC in a Songbird with Unison Song

Sexually dimorphic anatomy of brain areas is thought to be causally linked to sex differences in behaviour and cognitive functions. The sex with the regional size advantage (male or female) differs between brain areas and species. Among adult songbirds, males have larger brain areas such as the HVC (proper name) and RA (robust nucleus of the arcopallium) that control the production of learned songs. Forest weavers (Ploceus bicolor) mated pairs sing a unison duet in which male and female mates learn to produce identical songs. We show with histological techniques that the volume and neuron numbers of HVC and RA were ≥1.5 times larger in males than in females despite their identical songs. In contrast, using in-situ hybridizations, females have much higher (30–70%) expression levels of mRNA of a number of synapse-related proteins in HVC and/or RA than their male counterparts. Male-typical and female-typical sexual differentiation appears to act on different aspects of the phenotypes within the same brain areas, leading females and males to produce the same behaviour using different cellular mechanisms.

Differential expression pattern and steroid hormone sensitivity of SNAP-25 and synaptoporin mRNA in the telencephalic song control nucleus HVC of the zebra finch

Gonadal steroid hormones play an important role in the process of sexual differentiation of brain areas and behavior such as singing and song learning in songbirds. These hormones affect behavior controlling circuits on both the gross morphological and ultrastructural levels. Here we study whether the expression of genes coding for synaptic proteins is sensitive to gonadal steroid hormones and whether such altered expression coincides with changes in brain area size. We treated adult male zebra finches with the aromatase inhibitor fadrozole, to reduce estrogen synthesis and analyzed the mRNA expression of the synaptic proteins synaptoporin (SPO) and synaptosomal‐associated protein 25 kDa (SNAP‐25) in song control areas and surrounding tissues of adult male zebra finches. SPO and SNAP‐25 are differently expressed throughout the song system. Generally, the telencephalic song nuclei expressed SNAP‐25 at high intensity whereas SPO expression was area‐specific. Elevated levels of SNAP‐25 mRNA were present in the nucleus hyperstriatalis ventrale pars caudale (HVC) and in the robust nucleus of the archistriatum (RA). SPO mRNA was found in moderate levels in the HVC, in low levels in the lateral nucleus magnocellularis (lMAN) and Area X, and was absent in the RA. The treatment significantly increased the mRNA level of SPO in the HVC, whereas SNAP‐25 expression level was not affected. These expression patterns are not explained by the decrease of HVC volume after treatment. The decreased HVC size is not area‐specific but correlates with an overall reduction in size and an overall increase in cell density of the forebrain. J. Comp. Neurol. 475:83–94, 2004.