Eliot A. Brenowitz - US grants

neuroendocrine system; vocalization; voice;

Acoustic signals are important in understanding how members of the same species communicate with each other. Juveniles learn speech by listening to mature members of their species, and go through an initial phase of babbling before perfecting this behavior. Human speech and avian song have many similarities which allow bird song to be used as a model system for understanding the way in which the brain regulates communicaton behavior. An important component of communication is the ability to recognize signals produced by members of the same species, called conspecifics. In female birds, such recognition is essential in insuring that they mate only with conspecific males. This award to Dr. Eliot Brenowitz will investigate how females perceive song and the manner in which brain regions control recognition of mating songs produced by conspecific males. Selective inactivation of one brain region, the high vocal center, causes female birds to fail to distinguish between male canary song and the songs of males of other species. To determine if there is a pathway in the brain that is specialized for song recognition, brain regions connected to the high vocal center will be inactivated. The effects of such inactivation will be measured with a behavioral display performed by breeding females in response to presentation of songs sung by conspecific males. Specific elements of the song will be tested to determine which signals are most important. Additionally, each side of the brain will be testd separately to determine if the system is bilaterally asymmetrical as is the case for human speech. This work will contribute to understanding the way in which the brain controls recognition of signals critical for successful communication.

DESCRIPTION (provided by applicant): Seasonal growth and shrinkage of brain regions involved in control of birdsong provides a striking and unique opportunity to investigate the mechanisms regulating neuronal turnover associated with natural variation in steroids, and the functional consequences of this neural plasticity for sensorimotor learning. The aims in this proposal address fundamental issues of neural plasticity, and neuronal turnover in particular, in adult brains. These include the role of steroid hormones and neurotrophins in neuronal birth and death (Aims 1-2), the role of neurotrophins in neuronal activity that influences neurogenesis (Aim 3), and the coordinated expression of families of genes important in regulating functionally related processes of neurogenesis, neuronal protection, death, and activity (Aim 4). The birdsong system excels as a model for studies of neural plasticity; it is a well-defined and tractable neural circuit that shows extreme seasonal plasticity and regulates song, a learned sensorimotor behavior that is easily analyzed. This research will advance the field by 1) elucidating the mechanisms underlying the functional linkage between neuronal birth and death in adult brains (Aims 1,2); 2) providing the first evidence that neurotrophins influence the electrophysiology of neurons in the song system (Aim 3); 3) moving this field beyond the level of single gene analysis by opening up analysis of microRNA expression as a molecular mechanism for coordinating the expression of gene families that regulate the component cellular processes of adult neuroplasticity (Aim 4).

Social, Ecological and Genetic Variables in a Model Learning System

Michael D. Beecher

The use of elaborate acoustic vocalizations for communication is common in a wide variety of animal groups. In the oscine passerines (songbirds), such vocalizations are called song and have an additional, intriguing aspect: they are learned, with much of that learning occurring very early in life. Song learning in songbirds has been extensively analyzed in the laboratory and has become a major model system for studying the neurobiology of learning. Its value as a model system is enhanced by its many parallels with human language learning. These parallels include an early sensitive period, a perceptual filtering mechanism tuned to species communication signals, a crucial role for auditory feedback in normal development, a separation between sensory and motor learning, and an early subsong or babbling stage. Work on the neural basis of song perception and production in songbirds has revealed additional parallels between the neural centers for song in birds and those for language in humans. In this context, understanding the normal course of song development in songbirds becomes crucial if this model system is going to provide general insights into the development of vocal communication systems in general and human language in particular. Unfortunately, our understanding of normal song development is surprisingly incomplete, because most studies of song learning to date have been laboratory experiments in which essentially all social features have been removed. Four series of studies will be carried out to examine social, ecological and genetic variables in the song learning process for one particular species, the song sparrow. In the first series of experiments, singing interactions between tutor and tutee, and between tutors, will be manipulated and analyzed as a potential variable in song learning; this will be the first time this has been attempted in the laboratory. The setup will simulate four live song tutors, and both tutor-tutor and tutor-tutee singing interactions will be systematically varied; the general prediction is that birds will copy more songs from more interactive tutors. A second study will examine the role of genetic factors by comparing song learning by eastern and western song sparrows. On the basis of field studies, it has been hypothesized that birds in the two populations follow very different, genetically-based song-learning programs. This hypothesis will be tested by collecting birds from both populations and raising them in a common song-tutoring regime; according to the genetic hypothesis, the differences observed in the field should persist despite the common tutoring regime. In a third study, song learning will be directly examined in the field by radio-tracking young song sparrows through their first year and correlating the extent and timing of their interactions with potential song tutors and the degree to which the young birds copy their songs. In the fourth study, playback experiments in which the experimenter simulates a birds neighbor by playing recordings of the neighbor to the bird in a realistic simulation -- will be carried out to analyze how birds in both populations use their songs in territorial interactions with their neighbors. Field studies have suggested that the rules of communication in these two populations may be quite different, paralleling the presumed difference in their song learning programs. It is hoped that results from these different lines of research will combine to elucidate the social factors that shape the development of this model vocal learning system. In turn, such insights may shed light on human disorders such as autism that are characterized by a co-occurrence of language and social deficiencies.

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand the neuroendocrine basis of behavior used in communication. The birdsong system is a valuable model because song is learned, and its neural circuits are sexually dimorphic, hormone sensitive, and plastic in adults. Seasonal plasticity of the song control system provides an especially interesting model of plasticity in vertebrate brains, and a continuing goal of this research program is to understand the mechanisms and functional consequences of these seasonal changes. The immediate goal of this application is to receive training in methods of molecular biology that will enable the candidate to extend his studies of plasticity of the song system to a molecular level of analysis. Brenowitz will learn gene cloning, mapping and targeting, single and double-label in situ hybridization, PCR, and nonviral cRNA transfection methods in the laboratory of Robert Steiner at the University of Washington (UW). The candidate will be trained by Paul Neiman of the Fred Hutchison Cancer Research Center in Seattle in the use of eDNA microarrays to analyze patterns of gene expression, and in bioinformatic methods for data analysis. Brenowitz will take a course on microarray analysis at the UW Center for Expression Arrays. Scott Edwards of UW will train Brenowitz in DNA sequencing methods. The candidate will "apprentice" at the Institute for Systems Biology in Seattle to learn current methods of proteomics and bioinformatics. UW has a strong research program, with particular strengths in birdsong, endocrinology, neuroscience, molecular biology, animal behavior, and hearing research. The candidate's appointments in the Depts. of Psychology, Zoology, and the Bloedet Hearing Research Center provide access to shared facilities that benefit his research program. The proposed research will address mechanisms and functional consequences of seasonal plasticity in the song system. Aims 1 & 2, will test the hypotheses that social enhancement of seasonal growth is mediated by auditory cues and involves increased neuronal recruitment. Aim 3 will test the hypothesis that afferent innervation is necessary to maintain seasonally grown song nuclei. Aim 4 will test the hypothesis that seasonal growth of song circuits is mediated by estrogenic metabolites of testosterone. Aim 5 will use operant conditioning to test the hypothesis that seasonal plasticity of the song nuclei causes seasonal changes in song perception. Aim 6 will use in situ hybridization to test the hypothesis that expression of the gene for brain derived neurotrophic factor is upregulated when the song circuits are seasonally growing. Aim 7 will use subtractive suppressive hybridization to identify genes that are enriched in a song nucleus that grows seasonally compared with a nucleus that does not grow. Aim 8 will use a customized eDNA microarray to analyze global patterns of gene expression that are associated with seasonal growth of the telencephalic song control circuits. The results of the proposed studies will increase our understanding of the influences of steroid hormones and social stimuli on the nervous system, and the relationship between plasticity in the adult brain and learning.

Social insects are among the most ecologically dominant terrestrial animals. Their success is largely attributed to division of labor among the workers that make up their colonies. Individual differences in worker behavior are governed by physiological and anatomical changes in the nervous system, particularly in the brain. However, the dynamic properties of brain neurons that influence worker behavior are poorly understood. The goal of this project is to study how changes in brain neurons are associated with division of labor among social insect workers, focusing on neural plasticity in the mushroom bodies (MB). MB are structures in insect forebrains that are involved in learning and sensory integration, and the MB may play an important role in regulating division of labor.
As a first step toward understanding the evolution of MB effects on behavior, the relationships of MB neuroanatomy and worker behavior will be compared between a wasp species with small, simple societies (Mischocyttarus mastigophorus) and a species with larger, more complex colonies (Polybia aequatorialis). Three main approaches will be used. In the first study, MB neurons that are associated with individual differences in task performance will be identified. In the second study the effects of age on MB neuron plasticity and worker behavior will be measured. In the third study, colonies will be manipulated to induce changes in worker behavior, and associated neural changes will be measured.
The proposed projects include graduate and undergraduate training opportunities, and will provide inter-institutional training experiences. They will also promote education for Americans and local residents in Costa Rica, including biology guides (Monteverde Cloud Forest Reserve), and students on graduate and undergraduate field courses. Ongoing investigations in Monteverde will enhance the visibility of basic investigation at this important tropical research and conservation site. The research findings may also have relevance to the control/management of social insect pests and beneficial insects.

An intensive one-year field study will be carried out to examine social factors in bird song learning. Much of the rationale for this work comes from recent laboratory studies which have indicated, surprisingly, that a young bird's song learning is influenced at least as much by singing interactions he eavesdrops on as it is by direct interactions he has with adult song tutors. Because laboratory experiments always have limited ecological validity, these inferences need to be confirmed directly, in the field. Young song sparrows will be radio-tracked through their first year so that their movement patterns and the extent and timing of their direct and eavesdropped interactions with potential song tutors can be correlated with the degree to which the young birds copy the songs of these birds. Results from the field and laboratory lines of research can then be integrated to elucidate the social factors that shape the development of this model vocal learning system.

Song learning in songbirds has been extensively analyzed in the laboratory and has become a major model system for studying the ecology, neurobiology and genetics of learning. In addition, there are many important parallels between bird song learning and human language learning. Yet despite its broader significance, it is only recently that the significance of social factors in bird song learning has been recognized. Thus the proposed research could close a significant gap in our understanding of this widely studied model system and broaden the potential of applications to human behavior. Insights gained from research on this animal model system can ultimately be used to address autism and other developmental disorders pertaining to social learning and communication.

This research will examine a model system of vocal learning, bird song learning. The research will concentrate on the role of social factors, and will contrast several different hypotheses about these factors. According to the direct interaction hypothesis, young birds learn songs by interacting with older birds. According to the social eavesdropping hypothesis, the young bird learns primarily by extracting information from interactions among other birds that he observes or overhears. According to the social dominance hypothesis, the key information concerns the dominance relationships of those birds, while according to the singing rules hypothesis, the key information concerns which songs are appropriate replies to other songs. These hypotheses will be tested using the newly-developed 'virtual tutor' system which can both simulate interacting singers and interact directly with the subject. This system also captures many of the features of live counter-singing birds while permitting the investigator more experimental control than is possible with live birds. Parallel studies will be carried out in the field.

Song learning in songbirds has been analyzed extensively in the laboratory and, in part because of its many parallels with human language learning, it has become a major model system for studying the neurobiology and genetics of learning. It is only recently that another parallel with human language learning has been recognized; i.e., the role of social factors in vocal learning. This research will further develop the utility of this model system of learning. The research will support both graduate and undergraduate students. It will also play a role in public education, featuring the field research which takes place at Discovery Park, a 532-acre natural area park operated by Seattle Parks & Recreation.

LEARNING THROUGH EAVESDROPPING: FIELD EXPERIMENTS ON SONG LEARNING IN BIRDS

Eliot A. Brenowitz, P.I.
DDIG: Proposal #: IOS-0808562

The complex and beautiful songs of birds have always intrigued humans. For the Oscine passerines (songbirds), these songs are even more interesting because they must be learned. Song learning in birds has been extensively analyzed in the laboratory and has become a major model system for studying the ecology, evolution, neurobiology, and genetics of learning in animals. In addition, there are many parallels between the vocal learning of birds and humans, including the importance of social factors, making bird song one of the best models for studying human language learning. This research is an experimental field study investigating the role of social factors in bird song learning. Laboratory work of the last few years has suggested, surprisingly, that a young bird is more influenced by singing interactions he eavesdrops on than by direct interactions he has with adult song tutors. Recent fieldwork, using radio telemetry to follow juvenile birds in the wild and observe their interactions with adult tutors, also corroborates the importance of eavesdropping on singing interactions. The present study will use radio and heart rate telemetry to experimentally examine the behavioral and physiological response of juvenile song sparrows to different types of simulated singing. This research will investigate the importance of eavesdropping in song learning by assessing the response of juvenile birds to playback of different types of adult singing contests compared with solo adult singing. The results from these field experiments will compliment previous and ongoing laboratory research in elucidating the social factors that shape song development in this model vocal learning system. In addition to its scientific impacts, this work will be of considerable educational value, through dedicated training of undergraduates and through formal educational experiences and countless informal interactions with the general public at the field site (a busy urban park).

DESCRIPTION (provided by applicant): Seasonal regression of brain regions involved in the control of birdsong provides a striking and unique opportunity to investigate the mechanisms regulating neuronal degeneration and protection associated with naturally occurring variation in steroid hormones, and the functional consequences of neuroprotection for a learned sensorimotor behavior. The aims in this proposal address fundamental issues of hormones as neuroprotective agents in adult brains. These include the role of indirect genomic signaling pathways in hormonal neuroprotection (Aims 1-4), the role of kinase cascades in mediating transynaptic neuroprotective effects of hormones (Aim 3), and whether steroids can have neuroprotective effects in a manner independent of hormone receptors (Aim 2). The birdsong system excels as a model for studies of hormonal mechanisms of neuroprotection. It is a well-defined and tractable neural circuit that shows extreme seasonal patterns of hormone-regulated neuronal regression and protection. These processes of neural degeneration and protection occur with breeding-related hormonal cycles and thus can be studied in vivo without invasive manipulations. This research will advance the field by 1) providing the first evidence in the song system that kinase cascades are indirect genomic contributors to hormonal neuroprotection (Aims 1-4); 2) investigating the mechanisms by which hormones act transynaptically to have neuroprotective effects, and whether kinase cascades mediate this effect (Aim 3); and 3) determining whether the contribution of kinase cascades to the neuroprotective effect of steroids requires hormone receptor activation (Aim 2), an issue of continuing uncertainty.

Project Summary: The neural circuit that regulates birdsong, a highly precise, learned sensorimotor behavior, excels for study of fundamental mechanisms of adult circuit plasticity. The song system is a unique model of naturally occurring degeneration and compensatory regeneration in a behaviorally relevant neural circuit in adult brains. This circuit shows exaggerated seasonal degeneration and reconstruction via neurogenesis, in response to changes in circulating steroid hormone levels. Our long-term goal is to understand the fundamental mechanisms by which steroid hormones and neurotrophins interact to regulate plasticity of neural circuits and behavior. On a translational level, our goal is to understand how forebrain circuits can regenerate to support performance of complex learned motor skills. The central hypothesis of the proposed aims is that seasonal changes in hormones trigger changes in anterograde and retrograde trophic signaling that lead to remodeling of the HVC-RA circuit and changes in song behavior in adult birds.The goal of this application is to identify the trophic signaling pathways (molecular and electrophysiological) that regulate the the incorporation of newborn neurons to regenerate this circuit. This research will advance the field by elucidating fundamental issues of adult circuit plasticity. This topic is of translational relevance for exploiting endogenous or exogenous stem cells for therapeutic repair of injured or dysfunctional circuits in humans. These fundamental issues include whether new neurons added to adult circuits establish functional connections with efferent nuclei and restore behavior (Aim 1), the role of activity regulated genes in mediating retrograde trophic effects of neuronal activity on presynaptic adult neurogenesis (Aim 2), the role of calcium channels in mediating the transsynaptic neurotrophic regulation of postsynaptic activity (Aim 3), and the role of pre- and/or postsynaptic neuronal activity in maintaining a regenerated adult circuit (Aim 4).