Todd F. Roberts, Ph.D. - US grants

DESCRIPTION (provided by applicant): It is not known if optimal coding strategies exist for learned motor behavior. Vocal learning is an excellent behavior in which this issue can be examined because all vocal learning species share similar constraints. This research will ask if parrots encode vocal representations in a manner similar to the vocal coding strategy previously described in songbirds. Birdsong is coded by a sparse-to-detailed temporal code between two premotor forebrain nuclei. This application asks if this coding strategy obtains in an analogous premotor circuit in the parrot forebrain. We will examine this issue using single cell chronic recordings during the production of learned vocalizations in an Australian parrot, the budgerigar. These studies will allow us to accurately assess the relation between motor output and premotor neural activity. If similar coding strategies are not evident among songbirds and parrots, it suggests that premotor representations for learned vocalizations are not highly constrained even among birds. Alternatively, if coding strategies are retained it will spur further investigation into other vocal learning species in order to establish a comparative data set for future cladistic analysis for coding strategies for learned vocalizations more generally.

How we learn from social experiences and during social interactions is poorly understood, but it is thought to involve intricate changes to nerve cells in the brain and the connections between these cells. Cells involved in social learning are intermingled and intertwined with cells that may have completely different functions. Because of this complexity, identifying and studying the specific cells and networks involved in social learning remain a major challenge, and new methods are required to address this needle-in-a-hay-stack problem. This research will build a new set of genetic tools that allow researchers to mark cells in the brains of mice and zebra finches that are specifically involved in learning during social interactions, and will apply cutting-edge imaging, physiological, and genetic methods to dissect how the marked cells change during learning. This research is of fundamental importance because it will shed light on the brain mechanisms involved in social learning and build a new set of genetic tools that can be used by the scientific community to study brain mechanisms involved in learning and memory. The research also is of importance because developmental disorders and head injuries can severely compromise circuits in the brain and individuals' ability to learn from social encounters and navigate complex social interactions. The tools and methodologies developed in this research will be made freely available to other scientists through the world-wide web (http://www.utsouthwestern.edu/education/medical-school/departments/neuroscience/index.html) and through the Addgene public repository (http://www.addgene.org/). Funding for this research will also be used to educate and train young scientists in novel genetic, molecular, imaging and behavioral methodologies.

The proposed research will identify neuronal mechanisms involved in social learning from olfactory and auditory cues in mice and zebra finches, respectively. The proposal takes a highly interdisciplinary, collaborative approach involving four independent laboratories. The researchers will fluorescently "tag" neurons in mice and zebra finches that are selectively activated by olfactory and auditory social experiences using novel genetic strategies and viral tools that leverage the immediate-early gene c-Fos. Within brain regions of interest (olfactory and vocal learning circuits), these viral tools will differentially label neuronal populations depending on cellular activity and the specific social cues animals experience. In vivo Ca2+ imaging will be used to identify and map populations of neurons involved in processing and learning from social encounters. Novel optical methods will be used to map synaptic connectivity among tagged neuronal populations in vivo. Electrophysiological and transcriptomic analyses will be used to identify physiological and genetic factors unique to each tagged population, and identify neural subtypes and subpopulations responsible for social learning. These combined approaches will help reveal the network-level plasticity induced by social experiences. This collaborative, high-risk/high-impact research will generate novel in vivo molecular tools that allow fine and selective dissection of the network components of social learning.

? DESCRIPTION (provided by applicant): Even brief social interactions can lead to long lasting memories that profoundly shape future behavior. For example, speech, language, and other culturally transmitted behaviors are learned from social experiences. Resolving how the brain forms and retains long-lasting memories of social experiences is an important goal in neuroscience because it can provide fundamental insights into how we learn from one another and how we communicate. Aside from human speech and language learning, song learning in birds provides one of the clearest examples of this. Following only brief tutoring from an adult bird, a juvenile songbird will establish an accurate, long-lasting memory of the adult model's song, as evidenced by the precise vocal imitation of this song many weeks, and in some species, months and years, later. A major challenge to identifying the neuronal circuits that encode and retain these lasting representations has been our inability to remotely monitor and manipulate neuronal activity on time scales congruent with social interactions and learning. Using optogenetic manipulation of conditionally targeted neurons, voice recognition software and optical imaging of neuronal activity, we have overcome these methodological road blocks. This research will identify the specific neurons that encode and store the memory of the tutor's song needed for vocal imitation using tutoring contingent optogenetic inhibition and two-photon imaging of neuronal activity in juvenile birds. Several lines of evidence have implicated the song premotor nucleus HVC in tutor song memory. However, it is not clear whether a single class of neurons in HVC or downstream of HVC, including those in the auditory forebrain, function to encode this memory. The objective of this proposal is to resolve this issue by identifying the specific class or classes of neurons that encode the tutor song memory, revealing how it is functionally represented in the juvenile brain, and examining how the song memory interacts with circuits important for evaluating singing performance. In the first aim of this proposal we wil use the conditional expression of an inhibitory light sensitive channel to transiently silence different classes of neurons in these brain regions to test their necessity in tutor song learning. In the second aim we will use in vivo imaging of neuronal activity to examine how sensory experience of the tutor's song is functionally and spatially represented in the brain. In the third aim we will use optogenetic inhibition during vocal rehearsal to identify how neurons encoding the tutor song memory interact with auditory feedback circuits. Through these aims we will provide fundamental insights into how the brain encodes and retains memories of vocal models and how these memories shape future behaviors.

Learning by observation and through social interaction is common in humans and other animals. Yet, the neural circuits involved in this complex form of learning are still poorly understood. The goal of this research is identify the brain circuits involved in learning from social experiences. This research is challenging because it requires methods for monitoring and manipulating neural circuit activity during social interactions, and ideally, during interactions that result in quantifiable changes in behavior. Songbirds and humans are among the very few animals that learn their vocalizations (song or speech) by imitating their parents or other social models. Juvenile songbirds memorize the song of an adult model through social experiences and use this memory to accurately imitate this song later in life. The PI is using optical methods for manipulating neural circuit activity in juvenile songbirds as they memorize the song of an adult model in order to identify the brain regions involved in this complex form of learning. Because the brain circuits involved in song and speech are analogous, this research can provide fundamental insights into the neural basis for complex human behavior, such as speech learning and social communication. Further, through designed outreach programs this research will help increase the quality of science education in the Dallas/Fort Worth region. The Principal Investigator and staff in his laboratory are working to train middle school and high school science teachers, and high school students through hands on systems neuroscience research. This training involves analytical, behavioral and biological methods as well as cutting edge behavioral and genetic methods.

Juvenile songbirds memorize the song of an adult model through social experiences and use this memory to accurately imitate this song later in life. The Principal Investigator has developed behavioral, and optogenetic methods for manipulating neural circuit activity in juvenile songbirds as they memorize the song of an adult model, thus providing the necessary methods to dissect the brain circuit involved in learning from social experience. This research will test if the memory of a vocal model is stored in motor circuits involved in producing the learned vocal behavior, as suggested by mirror neuron models of speech and language learning, or in auditory circuits, as suggested by auditory template models. Using optogenetic inhibition of neural activity in the juvenile brain, this research will test the necessity of individual cell types in the encoding of tutor song memory.

PROJECT SUMMARY The execution of learned sequential motor behaviors, like those involved in playing a well learned tune on the piano, are thought to be supported by precise sequences of neuronal activity in the brain. However, probing the ties between sequential neuronal activity, neuronal connectivity and behavior is challenging without methods for simultaneously observing and controlling neuronal activity with spatial and temporal precision. We propose to apply all-optical physiological methods, combining concurrent optogenetic manipulation, and population calcium imaging, to map the functional organization of circuits involved in a well-studied sequential motor behavior. This research will involve dissemination of cutting-edge optical methods central to the BRAIN Initiative and facilitate a better understanding of how patterns of neuronal activity underlying precise complex behaviors are generated in the brain.

The pace of scientific discovery is often dictated by the development of new tools and techniques that bring into focus previously imperceptible aspects of the matter under investigation. In the study of the brain, the development of new labeling methods, such as the Golgi stain in the 1870s or genetic expression of fluorescent proteins in the 1990s, continues to revolutionize our ability to better understand the organization of the brain and study how networks in the brain function to support behavior, learning and memory. The aim of this research is to identify and develop a novel set of genetic tools that allow brain researchers to label and manipulate brain circuits with increasing sophistication. In this project, expertise of virologists and neuroscientists is paired in order to design new, cutting-edge approaches for studying and manipulating brain circuits. These novel tools provide important methods for mapping the intricate connections between nerve cells that support learning and behavior. To facilitate broad adoption of the new, virus-based approaches, the tools generated through this research are made freely available to the scientific community, and on-line resources are constructed to help the research community search and request all the tools and protocols developed under this project.

Viruses have evolved over millions of years to evade detection and thrive in vertebrate hosts. Building on the evolutionary diversity of vertebrate viruses, this research examines the use of several, previously untested, viral species for neuroscience research. This innovative, high-risk project establishes a unique research partnership between virologists and neuroscientists, in order to identify new viral vectors for labeling and expressing molecular tools in large populations of neurons. The broader impacts of this proposal are many-fold. First, this research leads to the development of new viral tools for labeling and genetically manipulating neurons in diverse animal models. Second, by studying a diverse set of viruses in both birds and rodents, this research leads to a better understanding of virus-host interactions and how they can and should be considered when selecting and designing viral tools for neuroscience research. This research therefore provides valuable new information and tools for neuroscientists and virologists. Third, all genetic and viral tools generated through this research are made freely available to the research community. Fourth, an on-line resource describing results of all tested viruses is being generated in order to help guide other research labs when selecting and testing new viral tools.

PROJECT SUMMARY The molecular signaling pathways important to speech and language remain mostly unknown. FoxP2 is one of a few genes that has been directly linked to both speech and language in humans and learning and production of vocalizations in animal models (songbirds and mice). In humans and songbirds, striatal expression of FoxP2 is a key factor in the accurate development of learned vocalizations. This multi-PI grant will synthesize expertise in genomics and behavior to examine the role of FoxP2 during the sensitive period for vocal learning. We will examine the function of FoxP2 during vocal learning, and in adult animals, using a combination of novel viral methods for the reversible knockdown of FoxP2, and functional genomics, through two specific aims. In the first aim, we will use novel viral methods to test whether restoring normal FoxP2 expression levels in adults is sufficient to rescue vocal deficits caused by knockdown of FoxP2 expression during the sensitive period for vocal learning. In the second aim, we will use functional genomics to identify the expression and regulation of FoxP2 target genes during the sensitive period for vocal learning. The results of these studies will provide new insights into neurodevelopmental genetic disorders, including the identification of novel signaling pathways involved in learning of vocal behaviors, and how therapeutic intervention in adults can impact behavioral disorders acquired during developmental sensitive periods. These data can also be used for the future development of targeted therapeutics for the treatment of a range of neurodevelopmental disorders with speech and language phenotypes.

Project Summary Sequences of neuronal activity are thought to underlie planning, preparation, and production of voluntary skilled behaviors. Dissecting how these premotor and motor sequences are functionally and synaptically integrated to support fluent, context-appropriate performance of natural behaviors is a major research challenge and a central goal of the BRAIN Initiative. The songbird premotor cortical structure HVC (letters used as proper name) has emerged as a prominent model system for studying how sparse neural sequences underlie the production of a precise, ethologically relevant behavior - birdsong. A single class of projection neurons in HVC is necessary for acute performance of birdsong. Yet, technical limitations associated with cell- type selective monitoring and manipulation of these neurons has hindered the ability to study how their neural sequences are functionally and synaptically integrated to support the planning, preparation and execution of behavior. We have developed cell-type specific methods for population calcium imaging and optogenetic manipulations in HVC and demonstrate their value in dissecting the functional and synaptic organization of this circuit in singing birds. Our preliminary results support a new model for HVC functional organization that will be tested in the four aims of this proposal. We will use calcium imaging, electrophysiological recordings and optogenetic manipulations in freely singing birds to test how diverse neural sequences in HVC underlie the planning, preparation and production of song. In addition, we will use single-cell optogenetics, calcium imaging, and circuit mapping methods to test the functional, synaptic and areal organization of this circuit. Drawing on a variety of cutting-edge approaches and the combined expertise of three scientific groups, this research aims to provide a new functional model for how diverse neural sequences are synaptically integrated to support fluent production of a voluntary skilled behavior.

Project Summary It is commonly appreciated that cortico-basal ganglia circuits are involved in the volitional control, initiation and cessation of movements. Perhaps less well appreciated is the role of the basal ganglia in learning and adaptive modification of skilled motor behaviors. An emerging view is that cortico-basal ganglia circuits play a prominent role in trial-and-error learning of skilled behaviors by helping to optimize future performances. Yet, the role of the cortico-basal ganglia circuits in optimizing the performance of naturally learned skilled behaviors is still poorly understood. To provide a better understanding of the basal ganglia's role in learning motor skills, we will apply closed-loop optogenetic methods in the study of a well delineated cortico-basal ganglia pathway in the songbird. Zebra finches learn to produce a complex courtship song during development and practice extensively to maintain expert performance of their song in adulthood. Using song-contingent (closed-loop) optogenetic inhibition and excitation this research will dissect the functional contribution of striatal circuits and their cortical and subcortical inputs during learning and maintenance of song. This research will test the function of different inputs to the zebra finch vocal striatum in learning and examine how prominent models of basal ganglia function, developed through the study of externally reinforced behaviors in the laboratory, bear on the learning of a naturally produced skilled motor behavior.

Project Summary Mechanistically linking network connectivity and the dynamics of neural networks to variation in the behavior of individuals is an overarching goal of neuroscience. Here we address this goal using techniques from network science to calculate functional networks that summarize pair-wise and higher order interactions between all recorded neurons. Network activity will be assessed using sophisticated two-photon (2P) imaging of activity- dependent Ca2+ signaling optimized to maximize the rate of recording and the numbers of neurons recorded. Multineuronal interactions within the networks will be identified, giving rise to encoding models to predict the network activity. Techniques from statistical physics will be used to optimally couple data from intracellular recordings to biologically realistic Hodgkin-Huxley (HH) models representing the contributions of ion currents and other free model parameters of the individual neurons. Networks of HH neurons using model synapses will replace pair-wise correlations to delinate the interrelationships between the ion currents of individual neurons and network interactions and dynamics. Taking advantage of the birdsong learning model, in the proposed experiments these approaches will be applied to the cortical song system HVC nucleus, allowing us to link these scales of investigation directly to behavior. Recent results demonstrate that changes in the intrinsic properties (IP) (ion current magnitudes) of HVC neurons is related to each individual's song, implicating changes within neurons as well as at synapses and networks that are related to learning. Aim 1: fast 2P imaging will be made in brain slices containing HVC that express spontaneous network activity. Model building will be supported by extensive efforts at 3-cell and 4-cell whole cell patch recordings, to better characterize HVC connectivity. The hypothesis that network structure depends on learning will be tested by examining how models vary between individual birds who sang similar or different songs. Models will be extended to in vivo observations by fast 2P imaging in sleeping birds while eliciting fictive singing using song playback, and in singing birds using 1P imaging. Results from the other Aims will further constrain the network and HH model building of Aim 1. Aim 2: the predictive power of the models will be further tested by using cellular resolution 2P optogenetic inhibition of selected neurons in in vivo and in vitro preparations. Aim 3: the role of neuronal IP in shaping network dynamics will be tested by using genetic and viral techniques to transiently modify specific ion channels in specific classes of HVC neurons. Changes in birds' singing behavior will be compared against a predictive HH model relating song structure and ion channel efficacy. Fast 2P imaging in slice and multisite extracellular recordings in singing birds will help to define how IP contribute to network models. Aim 4: single cell gene expression techniques will be used to identify all the HVC cell classes, the ion channels they express, and assess individual variation by examining cohorts of related birds or those singing the same songs. The overall goals and the four Aims are also designed to align with a subsequent U19 application.

PROJECT SUMMARY People and other animals learn many of their complex and socially oriented behaviors by imitating more experienced individuals in their environment. Vocal imitation is one of the more striking and readily quantifiable examples of this type of learning, but the genetic basis of this complex trait is still poorly understood. The goal of this research is to determine the genetic basis of vocal imitation abilities by establishing the first mutagenesis screen in a vocal learning species and the genetic tools for independently testing the function of the identified genes by developing novel transgenic models using germline gene targeting technologies. Humans are the only primate and one of only a handful of mammalian species to have evolved the facility for vocal imitation. Aside from humans, songbirds, and in particular zebra finches, are the best studied vocal learning species and they provide the only practical platform for systematically identifying the genes involved in this important social behavior. Like speech, zebra finch song is a culturally transmitted behavior learned via imitation. Moreover, functional, genetic and molecular parallels underscore the use of zebra finch for identifying genes essential for vocal imitation. We hypothesize that a forward genetic dominant screen, followed by the detailed genetic mapping and manipulations developed through this proposal, will identify convergent and divergent genetic signatures for this polygenic trait. Establishing a forward genetic screen and the genetic tools for verifying gene function in zebra finches will provide a novel, comprehensive, and broadly impactful approach for trying to understand the genetic basis of vocal and social communication.