Victoria Wright (Biology ’17). Poster Presentation. “Functional Genomics of Somatosensory Neuron Signaling and Morphology” (Advisor, Ian Woods). The Allied Genetics 2016 Conference, Orlando, FL. July 13-17, 2016.
- Support was provided by the H&S Student Travel and the Thomas J. '02 and David C. '05 Metzger Undergraduate Research Fund
Abstract: The somatosensory system detects mechanical, thermal and chemical stimuli; abnormalities in somatosensory signaling can lead to migraine and chronic pain. Neurons within the somatosensory system are specialized to detect different types of sensory stimuli, via expression of various cell surface receptors, development of diverse branching morphologies, and appropriate targeting to the central nervous system. To uncover the molecular mechanisms that generate these differences, we employed genome-wide transcriptional profiling in purified subpopulations of somatosensory neurons. From our list of differentially-expressed candidate genes, we are focusing on secreted peptides, transmembrane proteins, cytoskeletal regulators, and transcription factors for follow-up studies, as these gene families likely regulate development, morphogenesis, and function of somatosensory neurons. We are working to confirm enrichment of these genes in sensory neurons via fluorescence expression analyses, and to identify the neuronal subtypes in which these genes are expressed. Potential functions for the most promising candidates are being identified with genetic gain- and loss-of-function approaches. Specifically, neuronal outgrowth and branching morphology are quantified in embryos in which each candidate gene is overexpressed simultaneously with a fluorescent marker, driven by subtype-specific enhancer sequences. These embryos are also tested for changes in sensory responsiveness via high-throughput videotracking analysis. Similarly, CRISPR-generated knockouts will be analyzed for mutant phenotypes using assays of neuronal morphology and behavioral response to sensory stimuli of various modalities.
Andrew Rodenhouse (Biology ’16)
Presented, “Defining regions of CART gene expression in the larval zebrafish brain via monoaminergic landmarks”.NCUR. Asheville, NC
Katie Lee (Biology '15):
Oral presentation, "Comparative analysis of CART expression and function".NCUR. Eastern Washington University. April 2015.
Poster presentation, "Comparative analysis of CART expression and function" Whalen Symposium. Ithaca College. April 2015.
Haley Coleman (Biology '14). Oral presentation, "The Role of CART in arousal behaviors in larval zebrafish".NCUR. University of Kentucky. April 2014
Neuropeptidergic systems are important regulators of behavior in many organisms. We are interested in behaviors related to anxiety, appetite and addiction, and therefore study CART (Cocaine and Amphetamine Regulated Transcript) as it has proposed roles in each of these behaviors. Mammals have one copy of CART with pleiotropic functions and a complex pattern of expression in the brain. In contrast, zebrafish have at least 7 copies of the Cart gene, many of which have simple, discrete, and non-overlapping patterns of expression within the brain. We hypothesize that the multiple of Cart in mammals may be subdivided into discrete, yet analogous, functions in zebrafish. Thus larval zebrafish, due to their genetic tractability, optical clarity, and utility in high-throughput assays of behavior, are an ideal model system to study the individual roles of Cart signaling. We are taking two approaches to understand Cart function. First, we perform colocalization studies with landmark genes of known neurotransmitter systems, to determine which regions of the brain express each Cart transcript. We have optimized the in situ hybridization techniques and begun colocalization studies of genes in serotonergic, dopaminergic and histaminergic systems using this technique. Second, we examine behaviors induced by overexpressing each individual Cart transcript at the genetic level. For five of the cart genes, our lab has generated stable transgenic fish that allow inducible expression of this peptide. Understanding the function and location of each of the Cart genes using these techniques in larval zebrafish will help us determine potential functions of CART in different areas of the mammalian brain.
Elitsa Stoyanova (Biochemistry ’14) received a BBB Research Scholarship for her research project, “In vivo tracking of CART neuropeptide dynamics in Danio rerio”. The results of the research will be presented at a future convention and may be published in BIOS.
CART (cocaine- and amphetamine-regulated transcript) is a neuropeptide with functions in behaviors such as food intake, body weight maintenance, and reward. The aim of this research project is to elucidate the in vivo dynamics of the expression, release, transport and receptor localization of the CART peptide. We plan to accomplish this through four main steps, using Split GFP as a reporter of CART localization. First, we will generate plasmid reagents that will enable an inducible cart-gfp11 (hs-gfp-11) and a ubiquitously-expressed gfp1-10 (actin-gfp1-10). Second, we will test the functionality of the hs-cart-gfp11 via established behavioral assays. Third, we will create transgenic zebrafish for the hs-cart-gfp11 and actin-gfp1-10 construct, confirm their fluorescence in transiently-transgenic larvae, and generate stable lines for these transgenes. Finally, we will generate reagents for genome editing via CRISPR or TALEN technologies to introduce the gfp11 label to the CART gene on its endogenous locus. Taken together, these experiments will elucidate the dynamics of CART signaling in the context of a living, functioning brain.
Haley Coleman (Biology ’14) received research support from the H&S Dean's Office for her project, “The Role of CART in arousal behaviors in larval zebrafish".
I am specifically interested in how differences in behaviors are produced in the brain. Neuropeptides, small proteins that help neurons communicate, are implicated in behavioral regulation. Our lab studies a neuropeptide called CART (Cocaine-Amphetamine Regulated Transcript), which has proposed roles in anxiety, addiction, and arousal. In mammals, a single CART gene affects numerous diverse behaviors. Thus, elucidating the exact role CART plays in each distinct behavior is difficult. Larval zebrafish are a great system for studying the genetic and neuronal basis of behavior, as they develop transparently and it is relatively easy to manipulate their genes. Unlike mammals, five different CART peptides are encoded by zebrafish DNA. Based on discrete patterns of expression within the brain for these five CART genes, we hypothesize that the many roles of CART in mammals are subdivided by the multiple copies of zebrafish CART. The behavioral functions of CART in mammals, including appetite control, anxiety, locomotor activity, and reward-seeking behaviors, are united by their involvement in arousal within the brain. Thus we plan to dissect the role of CART in arousal behaviors in larval zebrafish Specifically, our research aims to: 1) Locate CART specifically within the zebrafish brain and compare it to other expression patterns of genes that possibly share a role in arousal pathways. This experiment requires the use of a specialized ‘confocal’ microscope that is not currently available at Ithaca College. 2) Analyze the behavior of transgenic fish that overexpress each of the 5 individual CART genes to study arousal behaviors given different stimuli.
Sadie Schlabach, Biology '15, received a Dana Internship to conduct research in the lab.
We are interested in studying how differences in behavior are generated in the brain. We, therefore, focus on the contributions of genes to behavior, and on the development of behaviorally-relevant regions of the nervous system. One gene that we have focused on recently is the CART (Cocaine and Amphetamine Related Transcript) neuropeptide. Since its discovery as a gene induced by cocaine exposure in rats, CART has been implicated in a broad spectrum of behaviors, including addiction, feeding, anxiety, and arousal. Studies of CART function in mammals are challenging because of its complicated pattern of expression in the brain and its widespread and varied roles in behavior. In zebrafish, cart function is partitioned between five different genes, each with a unique and relatively simple pattern of expression in the brain. This system thus provides a unique opportunity for fine-scale analysis of the broad variety of cart functions. This summer, Sadie will begin to analyze the behavioral roles of cart by pinpointing the functionally-defined brain structures in which they are expressed, and by analyzing behavioral deficits in zebrafish that either completely lack or overexpress each of the five individual cart genes. These studies will facilitate dissection of the molecular and neuroanatomical basis of arousal- and anxiety-related behaviors.
Rachel Noyes, Biochemistry ’13, received funding from the H&S Educational Grant Initiative and the Ithaca Fund to help support a loss-of-function analysis for one of the genes she is working on in my lab. In addition, she was awarded a Dana Internship to fund her research in the lab during the summer of 2012.
"Molecular Mechanisms of Somatosensory Development and Function"
The somatosensory system is a network of nerves that detects mechanical, thermal, and chemical stimuli. The trigeminal sensory ganglion detects stimuli to the head and sends this information to the spinal cord. In humans, this sensory circuit is activated in simple ailments such as headaches and toothaches, and also contributes to more serious conditions such as migraines and chronic pain. Neurons in this system are functionally diverse and include specific subtypes that respond to various innocuous and noxious stimuli.
While the different types of neurons are already well known, the manner in which each develops is largely mysterious. An improved understanding how these neurons develop would contribute to the study of neurogenesis and specialization, but more importantly, is crucial to uncovering treatments for people suffering from disorders of the nervous system. Zebrafish are ideal for this research because their transparent embryos allows for easy observation of the developing neurons. In addition, the zebrafish is amenable to both high-throughput behavioral analysis and genetic manipulation.
The purpose of my research is to determine the roles of specific genes in the development and function of trigeminal sensory neurons. These genes code for proteins that may regulate the shape of the neurons and the connectivity of the network. In previous work, my advisor, Dr. Woods, isolated genes that are expressed specifically in somatosensory neurons. Moreover, he has identified potential genes that varied between the types of somatosensory neurons.
I have been working to verify the presence of ten of these candidate genes in trigeminal sensory neurons to confirm that their expression varies between the stimulus-specific subtypes. After verification, I will identify two of the most promising candidates for further functional studies. The specific request of this funding proposal is to obtain materials necessary to knock out, or mutate, two candidate genes and thus to determine how these genes regulate the development and function of the trigeminal sensory system. By completing the verification stage of this research by the end of this summer and initiating the knockout studies, I will be poised to continue into the next stage of this project in the Fall, and with this opportunity I hope to obtain publishable results by the time I graduate.
This project will (1) introduce me to cutting-edge techniques and concepts in development, neurobiology, and behavior, (2) immerse me in the realities of academic research and its suitability as a possible career choice, (3) allow me to obtain sufficient results to present my research at regional and national conferences and (4) provide me the opportunity to potentially publish my work in peer-reviewed journals. I will work closely with my advisor Dr. Woods to fulfill all of these goals.