Sarah W. Bottjer

Circuits, hormones, and learning: Vocal behavior in songbirds

Species-typical vocal patterns subserve species identification and communication for individual organisms. Only a few groups of organisms learn the sounds used for vocal communication, including songbirds, humans, and cetaceans. Vocal learning in songbirds has come to serve as a model system for the study of brain-behavior relationships and neural mechanisms of learning and memory. Songbirds learn specific vocal patterns during a sensitive period of development via a complex assortment of neurobehavioral mechanisms. In many species of songbirds, the production of vocal behavior by adult males is used to defend territories and attract females, and both males and females must perceive vocal patterns and respond to them. In both juveniles and adults, specific types of auditory experience are necessary for initial song learning as well as the maintenance of stable song patterns. External sources of experience such as acoustic cues must be integrated with internal regulatory factors such as hormones, neurotransmitters, and cytokines for vocal patterns to be learned and produced. Thus, vocal behavior in songbirds is a culturally acquired trait that is regulated by multiple intrinsic as well as extrinsic factors. Here, we focus on functional relationships between circuitry and behavior in male songbirds. In that context, we consider in particular the influence of sex hormones on vocal behavior and its underlying circuitry, as well as the regulatory and functional mechanisms suggested by morphologic changes in the neural substrate for song control. We describe new data on the architecture of the song system that suggests strong similarities between the songbird vocal control system and neural circuits for memory, cognition, and use-dependent plasticity in the mammalian brain.

Neurotrophins Suppress Apoptosis Induced by Deafferentation of an Avian Motor-Cortical Region

Studies of the developing nervous system led to the general view that growth factors promote neuronal survival in a “retrograde” manner. For example, release of NGF from postsynaptic peripheral targets followed by uptake and retrograde transport by presynaptic neurons provided a widely accepted conceptual framework for the action of neurotrophins. In contrast, although presynaptic or “anterograde” influences on the survival of developing neurons have been recognized for some time, the mechanisms by which afferent input regulates the survival of postsynaptic cells have received considerably less attention. In the forebrain network for learned vocal behavior in zebra finches, lesions of a cortical region for song control, the lateral magnocellular nucleus of the anterior neostriatum (lMAN), remove presynaptic input to a motor-cortical song region, the robust nucleus of the archistriatum (RA), and cause massive RA neuron death in young birds that are entering the sensitive period for song learning. Here we report that lesions of lMAN followed by infusions of neurotrophins directly into RA completely suppress neuronal apoptosis in RA. Moreover, we show that lMAN neurons are able to transport neurotrophins in the anterograde direction to RA, that neurotrophin-like immunoreactivity is present in cells in lMAN and RA, and that neurotrophin receptor-like immunoreactivity is present in RA. Expression of neurotrophins in lMAN and RA suggests that lMAN presynaptic input could regulate RA neuron survival by synthesizing, transporting, and releasing neurotrophins anterogradely or by regulating the auto/paracrine release of neurotrophins within RA, or perhaps by both. These data provide the first in vivodemonstration that neurotrophins can prevent the death of deafferented cortical neurons, and they raise the possibility that nonretrograde signaling by neurotrophins may be a common means of promoting neuronal survival in the vertebrate telencephalon. Anterograde and auto/paracrine neurotrophin signaling, along with the more established view that neurotrophins regulate neuron survival via retrograde mechanisms, suggests multidirectional neurotrophin signaling in the vertebrate telencephalon.

Changes in neuronal number, density and size account for increases in volume of song-control nuclei during song development in zebra finches

The caudal nucleus of the ventral hyperstriatum (HVc) and the robust nucleus of the archistriatum (RA) are two anatomically discrete brain regions that are known to be involved with song production in adult passerine birds. Both the HVc and RA increase greatly in volume during a restricted period of song development in male zebra finches, while brain regions not involved with song control show little or no increase in size. We report here that the increased volume of the HVc is attributable to an increase in the number of neurons during this period of song learning, whereas the growth of the RA is due to an increase in the somal size of neurons and a decrease in neuronal density. The pattern of results described is consistent with the idea that the HVc matures prior to the RA, and that the development of the RA may depend on the ingrowth of axons from the HVc and other song-control regions.

An immunohistochemical and pathway tracing study of the striatopallidal organization of area X in the male zebra finch.

Area X is a nucleus within songbird basal ganglia that is part of the anterior forebrain song learning circuit. It receives cortical song‐related input and projects to the dorsolateral medial nucleus of thalamus (DLM). We carried out single‐ and double‐labeled immunohistochemical and pathway tracing studies in male zebra finch to characterize the cellular organization and circuitry of area X. We found that 5.4% of area X neuronal perikarya are relatively large, possess aspiny dendrites, and are rich in the pallidal neuron/striatal interneuron marker Lys8‐Asn9‐neurotensin8–13 (LANT6). Many of these perikarya were found to project to the DLM, and their traits suggest that they are pallidal. Area X also contained several neuron types characteristic of the striatum, including interneurons co‐containing LANT6 and the striatal interneuron marker parvalbumin (2% of area X neurons), interneurons containing parvalbumin but not LANT6 (4.8%), cholinergic interneurons (1.4%), and neurons containing the striatal spiny projection neuron marker dopamine‐ and adenosine 3′,5′‐monophosphate‐regulated phosphoprotein (DARPP‐32) (30%). Area X was rich in substance P (SP)‐containing terminals, and many ended on area X neurons projecting to the DLM with the woolly fiber morphology characteristic of striatopallidal terminals. Although SP+ perikarya were not detected in area X, prior studies suggest it is likely that SP‐synthesizing neurons are present and the source of the SP+ input to area X neurons projecting to the DLM. Area X was poor in enkephalinergic fibers and perikarya. The present data support the premise that area X contains both striatal and pallidal neurons, with the striatal neurons likely to include SP+ neurons that project to the pallidal neurons. J. Comp. Neurol. 469:239–261, 2004.

Neurogenesis in adult canary telencephalon is independent of gonadal hormone levels

Neurons generated in adulthood are found throughout the canary telencephalon. We are interested in the factors that control the rate of proliferation of stem cells that give rise to these new neurons. The rate of incorporation of newly generated neurons into vocal-control regions varies seasonally. This difference could reflect a higher rate of neurogenesis, a lower rate of cell death, or an altered migration. We examined the incidence of thymidine-labeled cells in the telencephalic ventricular zone of adult canaries as a function of variations in gonadal hormone levels. Adult female canaries maintained on a short-day photoperiod were anesthetized and gonadectomized. Four separate groups of birds received systemic exposure to either testosterone, estradiol, a combination of an anti-androgen and an inhibitor of estrogen synthesis, or nothing. All birds were also implanted with an osmotic minipump that released 3H-thymidine for 3 d and were killed 4 or 7 d following the onset of treatment. Analysis of autoradiograms revealed no differences between groups in the incidence of labeling within the ventricular zone either at the level of the anterior commissure or directly adjacent to the vocal-control nucleus HVC (higher vocal center). These results suggest that sex steroids do not regulate the rate of cell division in the ventricular zone. Seasonal differences in the incorporation of labeled cells into HVC may therefore be due to regulation of neurogenesis by photoperiodic factors other than gonadal steroids or to some other cellular mechanism, such as differential migration or survival of neurons.

Axonal connections of the high vocal center and surrounding cortical regions in juvenile and adult male zebra finches.

Neuronal connections of the High Vocal Center (HVC), a cortical nucleus of songbirds necessary for learned vocal behavior, and the region adjacent to HVC called paraHVC (pHVC), were studied in adult and juvenile male zebra finches. Extremely small injections of fluorescent dextran amines or biocytin were made within subregions of HVC and pHVC to define the precise nature and development of these pathways. In adults, all HVC injections produced an even, nontopographic distribution of retrograde label throughout the medial magnocellular nucleus of the anterior neostriatum (mMAN), the interfacial nucleus (NIf), and the uvaeform nucleus of the thalamus (Uva) and an even distribution of anterograde label within area X of the striatum and the robust nucleus of the archistriatum (RA). These same patterns of projections were present in juvenile birds 20–23 days of age, including the projection from HVC to RA, which has previously been reported to develop only after 25–30 days of age. Results also establish a novel efferent projection from HVC to pHVC in both juvenile and adult birds. Injections into pHVC indicate that this region receives afferent input from song control areas HVC, mMAN, medial regions of the parvicellular shell of lateral MAN, NIf, and Uva and projects to Area X, caudomedial regions of striatum, and regions of the caudomedial neostriatum (NCM). Thus, neuronal connections of pHVC are highly integrated with circuitry important for vocal behavior and are distinct from those of HVC. Such differences establish HVC and pHVC as separate brain areas and suggest that each may serve a different function in vocal behavior. Control injections in both juveniles and adults produced specific patterns of projections from areas outside of HVC to areas outside of RA, illustrating an overall spatial organization of projections from HVC and neighboring cortical areas. Further, although neuronal connections of HVC are not topographic, projections of HVC, pHVC, and surrounding areas demonstrate a broad spatial organization of efferents to striatum and regions surrounding RA, thus defining a level of organization beyond that of individual song control nuclei. J. Comp. Neurol. 397:118–138, 1998.

Connections of a motor cortical region in zebra finches: relation to pathways for vocal learning.

The lateral magnocellular nucleus of the anterior neostriatum (lMAN) is necessary for both initial learning of vocal patterns in developing zebra finches, as well as for modification of adult song under some circumstances. Lateral MAN is composed of two subregions: a core of magnocellular neurons and a surrounding shell composed primarily of parvocellular neurons. Neurons in lMANcore project to a region of motor cortex known as robust nucleus of the archistriatum (RA), whereas neurons in lMANshell project to a region adjacent to RA known as dorsal archistriatum (Ad). We studied the axonal connections of Ad in adult male zebra finches. In contrast to RA, Ad neurons make a large number of efferent projections, which do not include direct inputs to vocal or respiratory motor neurons. The major efferent projections of Ad are to: (1) the striatum of avian basal ganglia; (2) a dorsal thalamic zone (including the song‐control nuclei dorsomedial nucleus of the posterior thalamus [DMP] and dorsolateral nucleus of the medial thalamus [DLM]); (3) restricted regions within the lateral hypothalamus (stratum cellulare externum [SCE]), which may also relay information to the same dorsal thalamic zone; (4) a nucleus in the caudal thalamus (medial spiriform nucleus [SpM]); (5) deep layers of the tectum, which project to the thalamic song‐control nucleus Uva; (6) broad regions of pontine and midbrain reticular formation; and (7) areas within the ventral tegmental area and substantia nigra (ventral tegmental area [AVT], substantia nigra [SN]), which overlap with regions that project to Area X, a song‐control nucleus of avian striatum. Inputs to Ad derive not only from lMANshell, but also from a large area of dorsolateral caudal neostriatum (dNCL), which also receives input from lMANshell. That is, lMANshell neurons project directly to Ad, and also multisynaptically to Ad via dNCL. Double‐labeling studies show that lMANshell contains two different populations of projection neurons: one that projects to Ad and another to dNCL. These results are exciting for two main reasons. The first is that some of these projections represent potential closed‐loop circuits that could relay information back to song‐control nuclei of the telencephalon, possibly allowing diverse types of song‐related information to be both integrated between loops and compared during the period of auditory‐motor integration. Because both auditory experience with an adult (tutor) song pattern and auditory feedback are essential to vocal learning, closed‐loop pathways could serve as comparator circuits in which efferent commands, auditory feedback, and the memory of the tutor song are compared in an iterative fashion to achieve a gradual refinement of vocal production until it matches the tutor song. In addition, these circuits seem to have a strong integrative and limbic flavor. That is, the axonal connections of Ad neurons clearly include regions that receive inputs not only from somatosensory, visual, and auditory areas of cortex, but also from limbic regions, suggesting that they may be involved in higher order sensory processing, arousal, and motivation. J. Comp. Neurol. 420:244–260, 2000.

Lesions of a Telencephalic Nucleus in Male Zebra Finches: Influences on Vocal Behavior in Juveniles and Adults

Male zebra finches learn to sing during a restricted phase of juvenile development. Song learning is characterized by the progressive modification of unstable song vocalizations by juvenile birds during development, a process that leads to the production of stereotyped vocal patterns as birds reach adulthood. The medial magnocellular nucleus of the anterior neostriatum (mMAN) is a small cortical region that has been implicated in song behavior based on its neuronal projection to the High Vocal Center (HVC), a nucleus that is critical for adult vocal production and presumably also plays a role in song learning. To assess the function of mMAN in song, ibotenic acid lesions of this brain region were made in juvenile male zebra finches during the period of vocal learning (40-50 days of age) and in adult males that were producing stable song (>90 days of age). Birds lesioned as juveniles produced highly abnormal, poor quality song as adults. Although the overall song quality of birds lesioned as adults was not highly disrupted or abnormal, the postoperative song behavior of these birds was discernibly different due to slight increases in variability of vocal production, particularly at the onset of singing. These results demonstrate that mMAN plays some important role in vocal production during the sensitive period for song learning, and is also important for consistent initiation and stereotyped production of adult song behavior.

Sexual Dimorphisms in the Neural Vocal Control System in Song Birds: Ontogeny and Phylogeny

Sex differences in the neural song system in oscine song birds develop in response to estradiol secreted during early periods of development. Estradiol produces sex differences in cell number and in the proportion of cells which are steroid targets. The pattern of development of these sex differences varies in different brain regions, suggesting that the mechanisms of estradiol regulation of neural development may also vary. The magnitude of sexual dimorphism in the neural song system varies across species, and is generally correlated with the magnitude of sexual dimorphism in vocal ability. Large species differences in neural structure can potentially be explained by small differences in the ontogenetic pattern of estradiol secretion, as is suggested by studies of neural development.

Localization of Met-Enkephalin and Vasoactive Intestinal Polypeptide in the Brains of Male Zebra Finches (Part 1 of 2)

An interconnected series of brain nuclei controls song learning and behavior in male zebra finches (Poephila guttata). This study examined the distribution of fibers, terminals, and somata immunoreactive for two neuropeptides, methionine-enkephalin (ENK) and vasoactive intestinal polypeptide (VIP), in song-control nuclei of adult males. In addition, the broad pattern of major regions of labeling throughout the forebrain and midbrain was determined. The telencephalic song-control nuclei MAN (magnocellular nucleus of the anterior neostriatum), Area X of the striatum, HVC (higher vocal center), and RA (robust nucleus of the archistriatum) contained abundant ENK immunoreactivity, including labeled fibers and somata. In addition, intensely labeled fibers and terminals were seen in the thalamic nucleus DLM (medial portion of the dorsolateral nucleus of the anterior thalamus). High levels of VIP immunoreactivity were also seen in MAN, HVC, and RA, but this label consisted of fiber and terminals only. Area X and surrounding striatum contained extremely sparsely distributed VIP-labeled processes. Somata positive for VIP were not seen throughout cortical regions such as the neostriatum and hyperstriatum but were abundant in the lateral striatum (paleostriatum augmentatum, PA) and may contribute to a dense field of terminal labeling seen in the globus pallidus. The apparent presence of a robust VIP-positive striato-pallidal projection is not typical of major basal ganglia pathways in vertebrates, raising the possibility that passerine birds have diverged from the typical amniote pattern.