The song control nuclei of the zebra finch brain contain more neurones of larger diameter in the male than in the female. This sexual dimorphism is thought to result from differential growth of neurones in the two sexes. Using neurohistological techniques and radioactive tracers, we have studied the development of several forebrain nuclei involved in the control of song and find that the dimorphism arises from neuronal atrophy and death in the female brain as well as from an increase in cell-body size and afferent terminals from other forebrain nuclei in the male. Although the timing of these events varies from nucleus to nucleus, the sequence is essentially similar in all of them except area X. Here we describe the events in one of these nuclei, the robust nucleus of archistriatum (RA), as an example.

Songbirds use a complex network of discrete brain areas and connecting fiber tracts to sing their song, but our knowledge of this circuitry may be incomplete. The forebrain area, "caudal mesopallium" (CM), has received much attention recently for its song-related activities. HVC, a prominent song system nucleus, projects to a restricted area of the CM known as the avalanche nucleus (Av). However, the other connections of Av remain unknown. Here we used tract-tracing methods to examine the connections of Av to other song system nuclei. Injections of biotinylated dextran amine (BDA) into Av labeled both afferent terminals and neurons in HVC and the interfacial nucleus of the nidopallium (NIf), suggesting that there is complex feedforward and feedback communication between these nuclei (HVC<-->Av<-->NIf). Labeled neurons were also found in the uvaeform nucleus (Uva), which was substantiated by BDA injections into Uva that labeled terminals in Av. Double fluorescent tracing experiments confirm that both HVC and Uva project to Av. The present study adds complex new connections that expand the traditional song system circuitry into the caudal mesopallium. These new pathways are likely to have broad implications for deciphering how this intricate system works.

The song nuclei of the male zebra rinch (Poephila guttata) contain larger neurons than those of the female. This gender difference arises after hatching as a result of cell atrophy in the female and cell growth in the male. Implantation of estrogen in female chicks induces masculine differentiation of neurons in their song nuclei. The effects of estrogen on neuron size decline steeply after posthatching day 35 when neuronal atrophy begins. Estrogen loses its masculinizing effects completely after day 45 when the adult level of neuronal atrophy is reached. Thus, the end and the intensity of hormone action appear to be correlated with the timing of neuronal atrophy. and day 45. We chose for study two of the forebrain song nuclei, the ventral nucleus of hyperstriatum caudal section (HVc) and the robust nucleus of archistriatum (RA). The cross-sectional area of neuronal somata serves as a sensitive measure of their growth and atrophy. Birds were perfused with saline and 10% formalin. Brains were cut into parasagittal sections of 30 ttm in thickness and stained with creyslviolet. For the measurement of cross-sectional area, the perikaryal outlines of 50 randomly chosen neurons per bird were drawn with a Zeiss camera lucida and the area within the outlines was calculated by a computer-aided planimetric method.Administration of gonadal steroids early in life can induce the development of brain anatomical and behavioral attributes unique to one or the other gender. The inductive action of hormone is generally limited to a short period before or after birth (1). However, the factors controlling the length of the critical period and variation in the intensity of hormone action during the period are not known in most cases. This lack of knowledge is partly due to reliance on descriptive behavioral criteria for hormone action. Sexually dimorphic neurons and neuronal populations are convenient for the establishment of quantitative relationships between the timing and effect of hormone administration (2, 3). The brain nuclei that control song in the zebra finch (Poephila guttata) show marked sexual dimorphism (4). The gender differences do not exist at birth but arise later from a decrease in cell size and cell death in the female nuclei as well as from an increase in cell size in the male (5). An increase in cell number in one of the male nuclei may also contribute to the differences (6). Administration of estrogen to a female chick induces the development of masculine cell size in her song control system (7,8). This report describes how the time of estrogen implantation affects neuron size in two of the female song nuclei. MATERIALS AND METHODSZebra finch eggs were collected from our own breeding stock and incubated in an incubator so that the exact dates of their hatching could be determined. The chicks were raised by Bengalese finches as foster parents. Estrogen (50 ,ug of 17,8-estradiol) was administered as a slow-release (Silastic) subcutenous implant. Removal of the implant after various lengths of time showed ...

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.

The distribution of brain-derived neurotrophic factor (BDNF) in the song system of male zebra finches changes with posthatching age. At day 20, the hyperstriatum ventrale, pars caudale is the only song nucleus in which neurons showed BDNF immunoreactivity. At day 45, the staining in hyperstriatum ventrale, pars caudale was denser than at day 20 and the robust nucleus of the archistriatum, another song nucleus, showed BDNF labeling. By day 65, two additional song nuclei, area X and the lateral magnocellular nucleus of the anterior neostriatum, have become immunoreactive. In the adult, however, the amount of BDNF labeling in all of these brain nuclei is sharply reduced. These sequential events, the anatomical connections between these song nuclei, and the labeling of relevant axons and terminals suggest anterograde transport of BDNF. Furthermore, the timing of BDNF expression coincident with the development of singing behavior suggests that this neurotrophin may be directly involved with the differentiation of the song system.

The vocal control system of zebra finches shows auditory gating in which neuronal responses to the individual bird's own song vary with behavioral states such as sleep and wakefulness. However, we know neither the source of gating signals nor the anatomical connections that could link the modulatory centers of the brain with the song system. Two of the song-control nuclei in the forebrain, the HVC (used as the proper name) and the interfacial nucleus of the nidopallium, both show auditory gating, and they receive input from the uvaeform nucleus (Uva) in the thalamus. We used a combination of anterograde and retrograde tracing methods to show that the dorsal part of the reticular formation and the medial habenula (MHb) project to the Uva. We also show by choline acetyl transferase immunohistochemistry that the MHb is cholinergic and sends cholinergic fibers to the Uva. Our findings suggest that the Uva might serve as a hub to coordinate neuromodulatory input into the song system. auditory gating ͉ cholinergic ͉ song system ͉ thalamus T he song system is a discrete, interconnected series of brain nuclei in songbirds that controls singing, which is a learned vocal behavior ( Fig. 1) (1). In addition to displaying song motor activity, neurons of most song nuclei selectively respond to playback of the individual bird's own song (BOS) (2). Responses to the BOS are not created de novo in each song nucleus (3) but are relayed from the HVC to a vocal motor pathway that includes the robust nucleus of the arcopallium (RA) and the tracheosyringeal part of the hypoglossal nucleus. Auditory information also is transmitted from the HVC to an anterior forebrain pathway that includes area X within the songbird medial striatum; the nucleus dorsolateralis anterior thalami, pars medialis; the lateral magnocellular nucleus of the anterior nidopallium; and the RA. The source of auditory input to the HVC is thought to be the interfacial nucleus of the nidopallium (NIf), in which selectivity for the BOS is not as exclusive as in the HVC (4). Both the degree and selectivity of BOS-evoked responses in the HVC vary with natural and induced behavioral states such as sleeping, wakefulness, and anesthesia. This phenomenon is called ''auditory gating'' (5) and provides evidence that links the modulation of auditory input with the vocal control system. The first site of gating in the chain of song nuclei is thought to be the NIf (6). However, little is known about the source of signals that control gating in the NIf. Although several candidate sources for neural modulation have been postulated (7,8), the connections between these areas and the song system remain tenuous. The present study provides anatomical evidence that the uvaeform nucleus (Uva), which projects to both the NIf and HVC, receives cholinergic fibers from neurons in the medial habenula (MHb) and also afferents from the nucleus reticularis superior, pars dorsalis (RSd). These thalamic areas are neuromodulatory centers in other vertebrates, suggesting that the Uva in songbirds ma...

The robust nucleus of the archistriatum (RA) is one of the forebrain nuclei that control song production in birds. In the zebra fmch (Poephila guttata), this nucleus contains more and larger neurons in the male than in the female. A single injection of tritiated thymidine into the egg on the 6th or 7th day of incubation resulted in labeling of many RA neurons with tritium. The size of tritium-labeled neurons and the tissue volume containing them did not differ between the sexes at 15 days after hatching. In the adult brain, tritiumlabeled neurons and the tissue volume containing them were much larger in the male than in the female. Also, tritiumlabeled RA neurons were large in females which received an implant of estrogen immediately after hatching. The gender differences in the neuron size and nuclear volume of the zebra finch RA are, therefore, due not to the replacement of old neurons by new ones during development but to the growth and atrophy of neurons born before hatching. Similarly, the masculinizing effects of estrogen on the female RA are due not to neuronal replacement but to the prevention of atrophy and promotion of growth in preexisting neurons.In many bird species, the male sings and the female does not. This sexual dimorphism in behavior finds its morphological correlates in the brain nuclei that control song (1). All song nuclei are larger in the male than in the female. The volume differences are due to differences in both cell size and number (2-5). In the zebra finch (Poephila guttata), the size and number of neurons in the robust nucleus of the archistriatum (RA), one of the forebrain song nuclei, are not different between the sexes until about 20 days after hatching. Gender differences in RA neuron size and number emerge rapidly between posthatching days 30 and 40 (3, 4). The simplest explanation for these observations is to assume that RA neurons grow in the male and undergo atrophy and death in the female (3). Another explanation is to assume neuronal replacement during development. New neurons continue to be born in the forebrain ofthe male zebra finch during the first month after hatching (6, 7). The original embryonic RA neurons may later be replaced by larger neurons in the male and smaller ones in the female. Female zebra finches that have received an implant of estrogen during the first 40 days of life develop larger neuron size and nuclear volume in their RA than normal females (2,5,8,9). Estrogen implants either prevent neuronal atrophy and death or facilitate neuronal replacement in the RA. In both normal and estrogen-treated birds, the discrimination between neuronal replacement and growth or atrophy would be aided by the specific labeling of the neurons destined to occupy the embryonic RA. For this purpose, we labeled dividing neuroblasts with tritiated thymidine and observed their fate in both sexes from an early age to adulthood. MATERIALS AND METHODSA colony of zebra finches in our animal quarters provided fertilized eggs of known dates of laying. Cells that were des...

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