Ayako Yamaguchi - US grants

behavioral /social science research tag

DESCRIPTION (provided by applicant): The long-term goal of the proposed project is to understand how steroid hormones modify the functional properties of neurons and neuronal circuits to produce sexually distinct behavior patterns. In this project, the link between steroid hormones, neuronal function, and behavior will be investigated by using the vocal behavior of the African clawed frog as a model. Male and female frogs produce sexually distinct vocalizations that are regulated by steroid hormones. The vocalizations of male and female Xenopus are produced by the sex-specific firing rates of laryngeal motoneurons that control the vocal organ. Thus, in this system, the challenge of understanding the neural mechanisms of behavior can be reduced to a problem of understanding generation of stereotyped rhythmic spike patterns by motoneurons. The proposed research will investigate how biophysical and structural properties of laryngeal motoneurons and premotor neurons are modified by steroid hormones, and how these changes help shape the motor output that underlies sex-specific vocal production. Ultimately, understanding how steroids modulate the excitability of neurons will be important in unraveling the mechanisms of numerous human diseases including such common and important problems as epilepsy. Recent data suggests that certain steroids may be used as anti-epileptic drugs while in some patients, steroid hormones clearly increase the incidence of epileptic seizure. In addition, seizures can be linked to the menstrual cycle. However, there is very little known about how steroids can induce a sudden synchronous firing among neurons that causes seizure nor how they can be used to prevent such a storm of activity. By studying a well-defined neural system in which neuronal excitability is clearly regulated by androgen, as proposed here, we can better understand the cellular mechanisms that trigger bursts of neuronal activity. This more detailed understanding of cellular activity is necessary for further progress to be made in developing therapeutic approaches to epilepsy.

Animal behaviors are composed of movements that are coordinated across broad time scales. For example, human speech involves very rapid movements that control articulation of phonemes coordinated with slower movements of the respiratory musculature. The goal of this project is to understand how the temporal complexity of behaviors is generated by the nervous system. To this end, neuronal mechanisms underlying complex vocal behaviors of male African clawed frogs will be investigated. The central vocal circuit of Xenopus is an ideal model to achieve this goal because it remains functional in vitro (the brain can 'sing' in a dish), such that intensive physiological experimentation can be conducted during behavior, a feature that is not available in most other vertebrate species. The results of the study are expected to reveal cellular mechanisms by which the nervous system organizes and coordinates a functional sequence of motor programs. In addition, the project will provide opportunities for a graduate student and a postdoctoral fellow to be trained in the inter-disciplinary field of neuroscience, and outreach efforts will be made to recruit students from underrepresented groups and educators in local secondary institutions to participate in the research program.

The most salient output of brain function is behavior. However, how the nervous system produces behavior is not well understood, largely because most of the neural pathways underlying behavior are complicated. In this research project, vocal behavior of African clawed frogs is used as a model because their vocal neural pathways are simple and straight forward, and the pathways in action can be studied using techniques that were previously developed in the PI's laboratory. In addition to their simplicity and accessibility, the frog vocal pathways provide a unique opportunity to study how female and male brains function differently; male and female frogs produce sex-specific vocalizations during the breeding season, and the injection of male-specific hormones into an adult female results in male-like vocalizations within thirteen weeks. In this study, the focus is placed on one group of cells that are known to play a critical role in the operation of the pathways. A variety of experimental techniques will be used to understand where these neurons are, how these neurons function, and how they respond to male-specific hormones. The results of the study will not only provide us with the understanding of how behaviors are generated in the two sexes, but also provide us with an insight into how human brains generates rhythmic activity such as alpha and gamma waves, many of which are known to underlie cognitive processes, and known to be disrupted in diseased states.

A fundamentally important question in neuroscience is how neural networks function to generate motor programs that underlie behavior. Although analyses of a complete neural network that generates behavior is a formidable task, the relative simplicity of the Xenopus vocal network combined with the development of the fictive preparation (a "singing brain in a dish" preparation) and the application of behavioral, electrophysiological, anatomical, and newly developed optogenetic techniques allows detailed investigation of the dynamic organization of brain in action. The results of the proposed study will not only provide insight into the structure, function, and plasticity of the rhythm-generating neural network at the cellular levels, but also allow us to understand the logic of how a feedback loop should be engineered into a network to generate stable rhythms. Rhythmic neuronal activity is not limited to motor systems, but is prevalent across the entire CNS and is considered to underlie important functions such as perception and cognition. Thus, understanding the biophysical principles that govern rhythm generation using a simple neural network has a potential to elucidate mechanisms underlying neuronal oscillations in general. On a technical level, successful application of optogenetic tools to the Xenopus fictive preparation in vitro fills an important gap between research efforts conducted on genetic vs non-genetic model organisms. There are many non-genetic model organisms that present unique questions. The ability to express genetically encoded tools in non-model organisms represents a revolutionary change in the field of comparative neuroscience.

Amphibians (frogs and salamanders) are key indicator species for environmental change; many are threatened by habitat loss, rising sea levels and changing temperatures as they are “cold-blooded” and do not regulate body temperature. Some species, however, are resilient in the face of climate change both in physiology (e.g., temperature regulation), developmental requirements, and changes in behavior produced by the activity of nerve cells in the brain and spinal cord. African clawed frogs (Xenopus), though they live in fresh-water throughout life, can sequester in small chambers underground for very long periods when their environment becomes dry and hot. Xenopus used these resilience strategies to survive global extinction events. Spanish ribbed newts (Pleurodeles) can regenerate their entire nervous system, even as adults. To understand why these particular amphibians are so hardy, we need to find out how particular parts of their bodies work under stressful conditions. This project aims to develop “viral vectors”, non-infectious viruses that can be delivered to, and manipulate, genes in different parts of the body. These vectors can help test ideas about, for example, which parts of the brain are involved in resilience in frogs and how newts and salamanders regenerate whole parts of the body when they are injured. Also, the process of finding viruses that can infect amphibians will help investigators using other species such as birds and may reveal new ideas about how the ability of a virus to infect a different host species evolves, leaping from bats, for example, to humans. The project also includes training of undergraduate and graduate students, exposing them to international team science, as well as conferences and workshops, and sharing of protocols and non-infectious viruses on public databases to enable similar research by other investigators.

Viruses - natural multigene expression and delivery vehicles - evolved to target different species and tissues. Engineering Adeno-Associated Viruses (AAVs) for cold-blooded vertebrates (semi-aquatic or aquatic amphibians) is the focus of this EDGE project. Recombinant AAVs production enables a directed evolution approach for high-throughput selection and screening in two amphibians: the anuran Xenopus and the newt Pleurodeles. This research characterizes the blood brain barrier in both species to identify whether – or at what developmental stage – it forms. Leveraging the NSF-supported CLOVER Center at CalTech, researchers intravenously deliver an AAV serotype that transfects both species; they then harvest the animals’ central nervous system to produce, sequence, and bioinformatically analyze the resulting variants through two rounds of screening. Because of limits in the carrying capacity of AAVs, the project is developing transgenic cre lines that express specifically in neurons for both species. Using AAVs carrying floxed-CRISPR constructs and validated gRNAs, investigators knock out two native genes – rhodopsin and tyrosinase – in the eye via intraorbital delivery. Knocks outs are verified immunohistochemically using validated antibodies. AAVs are shared at cost with collaborators and deposited in Addgene. Results are shared via a US-based virtual conference, a hands-on US workshop, and an international conference. Protocols and validated results are rendered available to the broader research community via organism-based websites (e.g., Xenbase). All data and protocols are deposited in a publicly available data base and archived at Columbia University.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.