David F. Clayton - US grants

The aim of this research is to isolate and analyze genes that influence the generation and function of specific types of neurons. To accomplish this goal, special features of canaries are exploited: 1) continual production and replacement of neurons throughout the forebrain of adults; 2) well-characterized neuroanatomical loci in the forebrain underlying a learned behavior (song); 3) stimulatory effects of androgens on neurogenesis, song control neuroanatomy, and song learning/production. In preliminary work, the complexity of adult canary forebrain mRNA has been characterized, and cDNAs have been isolated for 8 rare mRNAs that are more abundant in the forebrain than in the nonforebrain. An improved in situ hybridization procedure shows that each of these cDNAs recognizes a different subset of brain cells based on anatomical distribution and morphology. Similar methods are now to be applied to seek clones of mRNAs that are preferentially expressed in cells during adult neurogenesis (i.e., precursors and migrating neuroblasts) or cells actively engaged in the song learning circuitry. To this end, tissues and mRNA will be analyzed and compared from the primary song control nucleus (HVc) including its overlying neurogenic ventricular zone, HVc of androgen-treated ovariectomized females and controls, and canary embryos. Identified clones will by characterized by nucleotide sequence analysis, in situ hybridization studies, and through production of antibodies to encoded peptides. Information and gene probes gained by this strategy may be applied to other species (including man) by isolating homologous sequences through nucleic acid hybridization. These studies may ultimately lead to therapies designed to modify the production or function of specific cell types in human brain.

This proposal arises out of a long-term effort by the PI to define the molecular mechanisms underlying behavioral plasticity, using songbirds as a model. Previous studies have shown that induction of a specific gene ("ZENK") occurs in specific parts of the songbird forebrain, in response to a normal perceptual experience -- hearing the sound of other birds singing. We have now found that this ZENK gene response is modified by recent experience: if a particular song is repeated for an hour the response to that song fades, but a new conspecific song from a different individual will immediately reinduce it. The aim of this project is to assess the functional significance of this experience- dependent change in gene expression in the brain. To this end, we will determine whether specific electrophysiological responses occur in response to conspecific birdsong in the caudomedial neostriatum ("NCM"), which is the primary site of ZENK gene induction and an avian homolog of mammalian neocortex. Experience-dependent physiological responses will be analyzed by counting the number of spikes elicited during auditory stimulus presentation, using "near-single-unit" recording techniques. In preliminary studies, we have successfully made such measurements in NCM in zebra finches, and have found evidence for a variety of song-selective changes in auditory responses. The specific aims of the proposed study are to: l) characterize the selectivity of auditory responses in NCM and related brain regions; 2) determine whether ZENK induction is anatomically correlated with sites of physiological plasticity; 3) determine whether production of ZENK protein is temporally correlated with consolidation of physiological change; 4) evaluate whether ZENK induction may have a role in song learning in juveniles; 5) evaluate whether ZENK induction is correlated with other specific behavioral changes in adults. A role for gene expression in guiding the brain's response to experience has long been hypothesized, and the songbird has unique advantages as a model system for testing this hypothesis. These advantages include a controlled perceptual stimulus (song), a defined neural circuit within parts of the brain homologous to the mammalian neocortex, and now the identification of specific gene responses within this circuitry. Clarification of the role gene expression plays in the brain's response to experience could have a major impact on our ability to analyze and treat numerous psychiatric, neurodegenerative and learning disorders.

DESCRIPTION: (Adapted from Applicant's Abstract): The goal of this proposal is to determine the normal function of synelfin, a neuronal protein abundant in the adult vertebrate telencephalon. Synelfin has been independently isolated from several species, including humans, where it has been referred to as NACP (the Non-Amyloid beta-Component Precursor) to indicate its relationship to a novel peptide recently purified from amyloid plaques in Alzheimer Disease (AD). Studies in songbirds suggest that this highly-conserved protein has a specific but yet-undefined role in the normal regulation of synaptic plasticity. To gain insight into the normal function of synelfin, three research aims are proposed: 1) conduct biochemical studies to test the hypothesis that synelfin has a functional relationship to apolipoproteins, as suggested by observation of conserved structural features. Normal and mutated forms of synelfin protein will be expressed in E.coli and analyzed for their interactions with phospholipids, using the well-characterized behavior of apoliopoproteins as a point of comparison. 2) determine the structural elements in the protein responsible for its accumulation at presynaptic terminals. Mutated DNA constructs will be introduced into primary cultured hippocampal neurons, and the encoded proteins will be localized by immunofluorescence. These studies may give insight into the apparent modular structure of the protein, and help in the design of experiments to identify other proteins with which synelfin interacts. 3) overexpress the synelfin protein in transgenic mice and assay the effect on neuroanatomy and neurological function. This will test the hypothesis whether a constitutive increase in synelfin will promote the development of Alzheimer-like neuropathology, or alter developmental or behavioral processes that depend upon neural plasticity.

DESCRIPTION (From the applicant's abstract): The long-term goal of this project is to define the normal and pathological roles of the synucleins, and in particular alpha-synuclein. Alpha-synuclein has emerged as a major focus of investigation because of an apparent (but poorly understood) role both in neurodegenerative disease and in normal synaptic plasticity and learning. The first aim for the next project period is to complete a formal biophysical characterization of human alpha-synuclein (halphaS), focusing on its ability to interact with membrane lipids and to self-associate. This aim will be conducted in collaboration with Dr. Seelig of the University of Basel. A second aim is to compare the properties of mutant synucleins linked to the development of Parkinson's disease, as well as a non-human (canary) alpha-synuclein, to gain insight into which features are specifically conserved and which may be more likely related to pathology. A third aim is to use site-directed mutagenesis to construct mutant forms of recombinant halphaS with altered lipid binding properties. This will provide insight into the functional organization of the sequence, and will generate mutant constructs potentially useful for probing synuclein's cellular functions. A fourth aim is to map the sites upon which synuclein is phosphorylated by the protein Casein Kinase II, and to investigate the structural and functional consequences of this modification on lipid binding and phospholipase D2 inhibition. A fifth aim will employ the various recombinant constructs produced and characterized in Aims 1-4, to probe the mechanisms by which synuclein can exert effects on cell function. In collaboration with Dr. Hyman of the Massachusetts General Hospital, the hypothesis that lipid binding is necessary for cell membrane association will be formally tested, and the necessity of lipid binding for both presynaptic terminal localization and PLD2 inhibition will also be tested. Collectively, these experiments test the hypothesis that synuclein's essential molecular function is related to its conserved structural features, which allow it to bind reversibly with intracellular membranes. Manipulation of these interactions could have uses in the development of therapies for age-related diseases including Alzheimer's and Parkinson's diseases.

Songbirds offer valuable opportunities for fundamental neuroscience research relevant to human health and disease. The Songbird Neurogenomics (SoNG) Initiative aims to advance this by aggressive, community- level application of emerging technologies in genomics and bioinformatics. In the previous period, >40,000 tags for genes expressed in songbird brain (Expressed Sequence Tags, ESTs) were created, from which a DMA microarray was produced for gene expression studies. 20 different research groups submitted propo- sals for experiments using these microarrays. The first aim now is to follow through with execution of these proposals. Collectively they address a broad range of fundamental research questions about functional relationships between brain and genome. >800 brain samples will be analyzed by microarray, following a novel "Community Collaboration" model whereby each individual research group provides brain samples (rigorously documented) to a collaborating core at Illinois, and the core executes all subsequent steps from RNA purification through microarray analysis. With common reference samples and techniques, the resulting expression data can be compared directly across all experiments. To facilitate analysis of results and sharing of data, an integrated, web-based suite of database, bioinformatics and statistical resources will be developed based on the NIH-supported "Beehive" project. Genomic information will be further enhanced through additional EST production, development of a Genome Browser track, and creation of an improved, next-generation microarray. These resources will then be focused to complete a deep profile of brain regions key to song learning, assessing how they compare to other regions including brain areas that correspond to parts of human brain (such as auditory cortex and cortical regions for the control of speech production). A series of community conferences will be held to promote awareness and use of these tools and data, and to focus attention on "next frontiers" for songbird neurogenomics. The songbird brain is of great interest for human neuroscience because it displays an unusual level of organization related to learning, adaptation, gender differences and vocal communication. Fundamental principles worked out first in songbirds have proven true for humans. Moreover, the approach here may serve as a model for doing science on a community scale, embracing individual initiative but coordinating efforts to achieve a larger synergy.

ABSTRACT NOT PROVIDED

Gordon Research Conference (GRC) on Genes and Behavior, 2008

Recent advances in genomics and molecular biology are making it possible for the first time to study the relationship between genes and behavior. To catalyze progress in this area, the Third Gordon Research Conference on Genes and Behavior is being organized (24-29 February 2008, at Barga, Italy). Funds received from NSF will be used to engage the active participation of a strong cohort of U.S. graduate and postdoctoral students in this conference by helping defray their travel and meeting costs. The Conference will focus on topics at the interface of animal behavior, neurobiology, genetics, molecular biology, ecology and evolutionary biology with an emphasis on the integration of molecular genetics, biotechnology and the behavioral sciences. Many of the speakers are current or past recipients of awards from the NSF Modulation program. All of the invited speakers and discussion leaders are highly regarded experts in their fields, and they were selected by the Conference Chair in consultation with a conference program committee and an ad-hoc committee of advisors. The Conference will provide a forum for the latest research linking genes to behavior, and should help identify gaps in knowledge and new strategies for promoting the integration of genomics and behavioral biology. Although graduate students and postdocs are the lifeblood of progress in science, they typically do not have access on their own to the funds required to attend a conference like this.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (01) Behavior, Behavioral Change, and Prevention and specific Challenge Topic, 01-GM-102: Model organisms for social behavior studies. Songbirds offer unique untapped advantages for integrative analyses of the genetic, biochemical, physiological, and environmental components of social behavior. Songbirds have complex nervous systems and are proven models for health-related brain research. They form complex and diverse social groups, and populate most ecosystems world-wide. The genome of one songbird, the zebra finch, has now been sequenced by the NIH. There remain two main barriers to the use of songbirds to investigate mechanisms of social behavior. The overall goal of this proposal is to break down these barriers and open a path for future research using songbirds as models for social behavior. The zebra finch is a gregarious colonial species that has been domesticated and is common in labs (and homes) around the world. Wild zebra finches are difficult to study in their native habitat (Australia) but the domesticated zebra finch has proven to be an exceptional experimental model. There have been careful descriptions of sociality in wild zebra finches, and focused lab investigations on mate choice and song learning - yet a formal scientific profile of the social behavior of the domesticated zebra finch does not exist. Moreover, how individuals vary in their social behavior is unexamined. To leverage the genomic investment in the zebra, these knowledge gaps need to be filled. Aim 1 will address this first barrier by constructing an "ethogram" to describe the social behavior in a zebra finch aviary across multiple generations. Additional experiments will evaluate how stable individual variation ("personality") affects the response to an acute social challenge (song playback). Results from these studies will inform Year 2 experiments comparing socially-driven brain gene expression in different contexts and individuals. Aim 2 addresses a complementary challenge: songbird diversity offers rich opportunities to compare related species that differ in social behavior and to study accessible species in their native environments, but only zebra finch gene sequence information is currently available. To overcome this barrier and enable the application of genomic tools to comparative analyses of social behavior, brain transcriptomes will be assembled for three key species: the violet-eared waxbill - a close relative of the zebra finch;and the song and white-crowned sparrows - major foci for North American behavioral ecology. These songbirds, in combination with the zebra finch, represent pairs of related species that display striking contrasts in social behavior (levels of social aggression) and therefore have tremendous potential for development as avian models for genomic studies of social behavior. PUBLIC HEALTH RELEVANCE: Many of the most important challenges for public health require a better understanding of how social factors impinge on the biology of the individual, and how variations in individuals influence social organization and function. Probing these links requires appropriate model organisms. This research aims to exploit natural advantages of songbirds as models for social behavior, leveraging recent progress with songbird neurobiology and genomics.