Expression of the GABA(A) receptor gamma4-subunit gene in discrete nuclei within the zebra finch song system.

The acquisition, production and maintenance of song by oscine birds is a form of audition-dependent learning that, in many ways, resembles the process by which humans learn to speak. In songbirds, the generation of structured song is determined by the activity of two interconnected neuronal pathways (the anterior forebrain pathway and the vocal motor pathway), each of which contains a number of discrete nuclei that together form the song system. It is becoming increasingly evident that inhibitory GABAergic mechanisms are indispensable in counterbalancing the excitatory actions of glutamate and, thus, likely shape the neuronal firing patterns of neurons within this network. Furthermore, there is compelling evidence for the involvement of GABA(A) receptors, although the molecular composition of these has, to date, remained elusive. Here we describe the isolation of a complementary DNA for the zebra finch GABA(A) receptor gamma4 subunit, and map the expression pattern of the corresponding gene within the zebra finch (Taeniopygia guttata) brain. Our findings show, remarkably, that the gamma4-subunit transcript is highly enriched in the major nuclei of the song system, including the lateral magnocellular nucleus of the anterior nidopallium (LMAN), the medial magnocellular nucleus of the anterior nidopallium (MMAN), Area X, the robust nucleus of the arcopallium (RA) and the HVC (used as the proper name), as well as Field L, which innervates the area surrounding HVC. In summary, we have demonstrated the presence of the mRNA for the gamma4 subunit of the GABA(A) receptor, the major inhibitory receptor in brain, in most of the nuclei of the two neural circuits that mediate song production in the zebra finch. This not only marks the beginning of the characterization of the GABA(A) receptor subtype(s) that mediates the actions of GABA in the song system but it also provides a robust molecular marker with which to distinguish song system-specific brain structures.

Seasonal changes in astrocytes parallel neuronal plasticity in the song control area HVc of the canary.

In the song control area HVc of the canary, intercellular dye-coupling among astrocytes was studied by intracellular injection of neurobiotin into identified single astrocytes. Injection of individual astrocytes into acute slices resulted in dye spread to neighboring astrocytes, covering a sphere of up to 1 mm in diameter. The astrocytic nature of the dye-coupled cells was verified by double labeling of neurobiotin-filled cells with antisera for the astrocytic filament proteins GFAP or vimentin. The similarity in the number of dye-coupled cells and the total number of astrocytes labeled by immunocytochemical markers indicate that dye-coupling is specific for astrocytes and labels almost the entire local astrocytic population. Within the major nucleus for vocal control (HVc), approximately 25% more astroglial cells were present than in the surrounding forebrain tissue. There is no apparent hindrance of dye spread at the border of the HVc. The density of dye-coupled astrocytes and the expression of cytoskeletal filament proteins differed markedly between the reproductive period in spring and the quiescent period in autumn. While vimentin is the major astroglial filament in autumn, GFAP is strongly expressed in spring. The density of dye-coupled astrocytes reveals a marked increase in the reproductive period, followed by a reduction in autumn. The data indicate that the astrocytic population in the avian forebrain undergoes significant changes coincident with the known functional changes in the vocal control nuclei during periods of song production.

Testosterone elevates expression of tenascin-R and oligomannosidic carbohydrates in developing male zebra finches.

The song system of zebra finches is a model for studying the influence of steroids on neural connectivity and behavior during development. To investigate the molecular mechanisms underlying the song-related and gonadal hormone-regulated development of neural activity, we have chosen to investigate the expression of recognition molecules in the brain nuclei associated with motor control of song production. Here we show that testosterone accelerates expression of the predominantly oligodendroglia-, but also neuron-associated extracellular matrix glycoprotein tenascin-R and the oligomannosidic carbohydrate L3 during the third and seventh posthatching week in the higher vocal center (HVC) and robust nucleus of the archistriatum (RA), but not in other brain regions. The results suggest that recognition molecules and associated carbohydrate structures can be regulated by testosterone and that an increased expression of these molecules correlates with testosterone-induced modifications of song behavior.

Inhibitory properties coexist with plasticity in the song system of the canary.

The expression of neuronal growth regulating factors (Ni 35/250, GAP-43) were studied immunohistochemically in the plastic telencephalic song system and in the cerebellum of the canary. In the myelinated song nuclei (HVc and RA) growth promoting and inhibiting molecules could be detected, whereas in the white matter tracts of the cerebellum only the neurite growth inhibitor could be detected. The morphodynamic effects of these molecules were assessed by in vitro assays on cryosections. Neurites were formed by 20-30% of the E7 chick cortical neuroblasts cultured in myelinated areas of the vocal control system, neurite outgrowth from the white matter of the cerebellum was completely inhibited.

Exogenous testosterone alters expression pattern of cell recognition molecules in brains of juvenile zebra finches.

We assessed spatio-temporal expression of tenascin and janusin by immunocytochemistry in testosterone-treated zebra finches and in untreated controls. These cell recognition molecules and components of the extracellular matrix mediate neurone-glia interactions, which are crucial for differentiation of ordered neural structures. We describe boundaries for the spatio-temporal expression of cell recognition molecules that highly correlate with the song motor centres as defined by combined neuroanatomical and ethological studies on song vocalization. The expression of janusin is accelerated by exogenous testosterone.

Exogenous testosterone differentially affects myelination and neurone soma sizes in the brain of canaries.

We assessed the temporal and functional relationship of myelin maturation and of neuronal development in the telencephalic song motor centres of the canary at different developmental stages. Brain differentiation was modulated by exogenous testosterone, its effects on song quality were also monitored. Myelin maturation was studied by using computer aided analysis of silver impregnated brain sections, whereas the differentiation of neurones was determined by measuring neuronal soma sizes. At an early developmental stage, testosterone triggers growth of neurones but does not affect myelination. At a later developmental stage, both neurone soma sizes and myelination are enhanced by testosterone.

Antioestrogen inhibits myelination in brains of juvenile Zebra finches.

The effects of the antioestrogen Keoxifene on the ontogenetic process of myelination, on the differentiation of neurones of telencephalic song motor centres, on cerebellar structures and on behaviour were studied in male Zebra finches. Brain differentiation was studied by using computer aided analysis of silver impregnated brain sections and by measuring soma sizes of neurones after Nissl staining. An antioestrogen induced inhibition of myelination could be detected in the song motor centre robust nucleus of the archistriatum (RA) and in the cerebellum, whereas the region magnocellular nucleus of the anterior neostriatum (MAN) showed no difference between treated birds and controls.

Estrogen receptors in the avian brain: survey reveals general distribution and forebrain areas unique to songbirds.

Estrogens play an important role in the control and differentiation of species-typical behavior and in endocrine homeostasis of birds, but the distribution and evolution of cells that contain estrogen receptors in the avian brain are poorly understood. This study therefore surveys 26 species in the avian orders Anseriformes (1 species), Galliformes (2), Columbiformes (3), Psittaciformes (1), Apodiformes (2), and Passeriformes (3 suboscines, 14 oscines). Indirect immunocytochemistry with the estrogen receptor (ER) antibody H222Spy revealed a general pattern of ER-antibody-immunoreactive cells (ER-IRC) in all 26 species, with ER-IRC in consistent, well-defined locations in the limbic forebrain, the midbrain striatum, the hippocampus, the hindbrain, and especially in the preoptic area and the tuberal hypothalamus. For some species, the microdistribution of ER-IRC in some of these general areas differed, such as in the hippocampus and the anterior hypothalamus of suboscine species and in the preoptic area of the Japanese quail. Brains of oscine songbirds of both sexes, unlike brains of nonsongbirds, had ER-IRC in three specific structures of the nonlimbic forebrain: in the area surrounding the nucleus robustus archistriatalis; in the rostral forebrain; and, for all individuals, in the caudale neostriatum, including the nucleus hyperstriatalis ventrale, pars caudale (HVc). Among songbird families or subfamilies, adult males of the Estrildinae had much lower numbers of ER-IRC in HVc than did adult males of the Fringillidae, Paridae, Sturnidae, and Ploceinae. Differences occurred, too, among closely related species: the songbird canary (Serinus canaria) had an ER-IRC area in the rostral forebrain that was lacking in all other songbird species, including other cardueline finches. The cells with ER that are found only in the songbird forebrain but not in reptiles, nonpasserine birds, and nonoscine passerine birds very likely coevolved with steroid-dependent differentiation of vocal control areas. The songbird-specific expression of ER in the forebrain could be an example in which taxon-specific behavior is due to taxon specific neurochemical properties of the brain.

Prolactin and gonadal steroids during the reproductive cycle of the Bengalese finch (Lonchura striata var. domestica, Estrildidae), a nonseasonal breeder with biparental care.

Plasma levels of prolactin (Prl), testosterone (T), and progesterone were determined throughout the breeding cycle in pairwise caged Bengalese finches (Lonchura striata var. domestica, Estrildidae), a monogamous altricial passerine with nonseasonal breeding, in which parental behavior including incubation is equally shared between males and females. In both sexes, Prl titers increased 4- to 10-fold during incubation period compared to values during nestbuilding. Likewise, Prl levels after fledging of the young were significantly lower compared to titers during incubation. In males, T increased significantly (15-fold) during nestbuilding compared to all other stages of the breeding cycle. In females, T remained basal throughout the entire breeding cycle. Thus, T levels were elevated only during stages with low Prl titers and vice versa. Progesterone titers were elevated during egg-laying in females and fluctuated without any obvious pattern in males. These results suggest that (1) high levels of Prl are involved in the control of parental behavior of male and female Bengalese finches and (2) Prl secretion is controlled by proximate factors of the early reproductive cycle since reproduction is independent of seasonal factors in this species.

Application of testosterone accelerates oligodendrocyte maturation in brains of zebra finches.

The effects of exogenously applied hormones on glial cell maturation of telencephalic song motor centers, midbrain and cerebellar structures was studied in juvenile male zebra finches. Testosterone was administered and the development of oligodendrocytes was studied using immunochemistry and computer aided image analysis on silver impregnated brain sections. A testosterone induced acceleration of oligodendrocyte maturation could be detected in several brain areas by using the monoclonal antibody O 10 recognizing an oligodendrocyte-specific cell surface antigen and by the silver impregnation for myelin. The increase in myelin density was higher in the testosterone treated animals than in the control animals in the forebrain and in the cerebellum, whereas two regions in the midbrain showed no difference between treated and controls.

Immunocytochemical localization of estrogen-binding neurons in the songbird brain.

This study used monoclonal antibodies against estrogen receptor for the identification and localization of estrogen-binding cells in the avian brain. The distribution of estrogen-binding neurons in the songbird brain conformed to the general vertebrate pattern with highest labelling in hypothalamus and preoptic area. For the first time, estrogen-binding neurons were found in the song control system: these neurons might provide a substrate for the direct action of estrogen on the song system.