Debra Ann Fadool - US grants

The broad, long-term objective of this research is to augment our understanding of the brain's ability to encode external information and to heighten our knowledge of the degree of sensory processing that takes place in the peripheral nervous system prior to its receipt at higher brain centers. Second messenger and related transduction cascades are a fundamental feature of electrical signalling, cell communication, and gene expression/regulation not only for cells that process sensory information but virtually for all cells across an array of systems. The specific aim of this research proposal is to discern the functional mechanism by which chemosignals will trigger an electrical response in the vertebrate vomeronasal receptor cell (VRN) membrane; the physiological basis for sensory transduction in the vomeronasal organ (VNO). Few studies have described the electrophysiological properties of vertebrate VRNs that must transduce chemical signals involved in the universal life processes of food finding, social interaction, and reproduction, and which are ultimately involved in the execution of species-typical behavior sand the initiation of neuroendocrine changes. This proposal will expand upon the body of biophysical properties known for these neurons, and apply single channel recording, whole-cell perfusion of G protein antibodies, and patch-cram recording techniques with biochemical and immunocytochemical verification of the VNO transduction components as a means for delineating the FUNCTIONAL odorant to electrical transduction in the vertebrate vomeronasal olfactory system. The proposal is designed to address: (1) Are there VNO specific stimuli? (2) Are GTP-binding proteins localized to the putative subcellular site of signal transduction? (3) Which GTP-binding proteins are physiologically involved in chemosignal transduction? (4) Can inositol phospholipids or cyclic nucleotides directly gate ion channels in the VNO? and (5) Can chemosignals evoke any second-messenger gated ion channel activity. This combined biophysical/biochemical/immunochemical approach for dissecting the molecular details of VNO transduction should provide new data concerning VNO function, which is currently unknown in humans.

DESCRIPTION (provided by applicant): The designed research is a multidisciplinary analysis of the modulation of potassium currents in granule and mitral cells of the olfactory bulb. The broad, long-term objective of this research is to elucidate how neurotrophins and growth factors can utilize ion channels as substrates for phosphorylation to give rise to short-term and long-term plastic changes in synaptic efficacy or to aid in the establishment of neural circuits in the olfactory bulb. Understanding the general principles governing these transduction cascades and the involvement of ion channels will provide information of how protein kinases and protein phosphatases contribute to the onset or severity of specific neuronal diseases, such as Alzheimer's, or how uncontrolled signaling of these enzymes leads to deregulated cell proliferation and diseases such as cancer and diabetes. Because of the unique trophic and regenerative capacity of neurons in the olfactory system, continual expression of neuromodulators could alter patterns of electrical excitability in addition to their well-studied roles in growth and differentiation. The specific aims of this proposal are to characterize using patch-clamp electrophysiology how receptor-linked tyrosine phosphorylation signaling in the olfactory bulb is altered by sensory experience, patterned electrical stimulation, and trophic factor infusion. By utilizing the cloned, olfactory bulb potassium channel Kv1.3 as a parallel model, combined biochemical measurement of kinase-induced tyrosine phosphorylation, co-immunoprecipitation, and molecular mutagenesis will elucidate the mechanistic details of how ion channels form molecular scaffolds with kinases and adaptor proteins through discrete protein-protein interactions at SH2, SH3, PDZ, and PTB domains. Gene-targeted deletions in Kv1.3 channel, insulin receptor kinase, and TrkB kinase will provide mechanistic details for the role for tyrosine phosphorylation signaling in olfaction and for neuromodulation in the CNS in general, as defined by loss of function experiments (behavioral, biochemical, electrophysiological) using knock-out mice strains. The proposal will provide new important information regarding the integration of signaling molecules by construction of protein-protein interactions with ion channels. Modulation of ion channel function would thus be dependent upon the repertoire of signaling proteins expressed in a given neuron, a background that could change with sensory experience or electrical patterning.

DESCRIPTION (provided by applicant): Potassium ion channels are traditionally viewed as the dampeners of neuronal excitability and are the predominant proteins that drive the resting membrane potential and spike firing frequency. It has been discovered that natural or disease driven changes in the sensitivity of the olfactory bulb are monitored at the level of a potassium channel and thus may contribute to the body's metabolic response to fat intake, hyperglycemia, or energy balance, in a brain region outside the traditional hypothalamic axis. The PRIMARY GOAL of this project is to discover molecules that can block the conduction of the potassium channel in the olfactory bulb leading to changes in metabolism. The influence of metabolic state on olfactory structure/function and energy expenditure at the level of the action potential will provide a knowledge base to extend to future directions of research at the interface between endocrinology and sensory systems. The latest statistics report that 65% of Americans are overweight while diet-induced or type II diabetes is becoming epidemic in our population, rising disproportionately in American children. The METHOD of this study will tackle an interdisciplinary approach whereby electrophysiology, anatomical (genetically identifiable) neuronal tracking, and systems physiology (metabolism, ingestive behaviors) will be applied to investigate how the olfactory bulb is designed as a metabolic sensor of body weight and energy homeostasis. The INNOVATION of the study will be that stable ion channel peptides will be delivered intranasally and via surgically implanted osmotic mini-pumps to assess increases in metabolism by blocking potassium channel conduction in the olfactory bulb. The SPECIFIC AIMS of the study are based upon three hypotheses: 1) blocking the lumen of the channel can differentially affect basal vs. activity-dependent metabolism, 2) energy availability affects the development of the olfactory sensory map and the function of the channel expressing post-synaptic targets, and 3) energy usage for action potential generation in the olfactory bulb is influenced by the metabolic state of the animal. This study will seek to elucidate the extent to which this particular potassium channel governs energy homeostasis to provide translational therapeutics for obesity and associated metabolic disorders while impacting the sensory physiology of chemoreceptors.

Summary This Chemosensory Training Program (CTP), operating within the interdisciplinary Program in Neuroscience at Florida State University, is a continuing application in its 25th year. The CTP Program is geared to train the next generation of researchers to become leaders in basic neural mechanisms of chemosensory systems interfaced with behavior. The program prepares 4 pre- and 2 post-doctoral trainees for research careers focused on olfactory and gustatory senses in context. One important context is the regulation of food intake and metabolic state, dysregulation of which can lead to obesity and diabetes, or anorexia. The powerful links between chemosensory systems and brain circuitry associated with emotional, motivational, and neuromodulatory processes requires a wide perspective for full understanding. The broad long- term objective is to provide the basic neuroscience platform upon which clinical understanding of chemosensory disease is built using a wide spectrum of experimental approaches including molecular neurobiology, neurophysiology, biophysics, psychophysics, and behavioral analysis. The strength of the CTP program that anticipates to provide 2 and 4 years of training for approximately five post- and fifteen pre-doctoral scholars, respectively, is the close guidance of trainees by expert faculty whom are accustomed to productive collaborations fostered from a wealth of historical chemosensory knowledge that shapes cutting-edge investigations for training. Trainees have access to state-of-the-art custom-designed chemosensory equipment, technical support staff, and new building infrastructure to perform their research. Value-added activities include ? 1) chemosensory tutorials (readings and lab practicum), 2) evening gather- ings (presentations, rigor and reproducibility training, and webinars), 3) chemosensory retreat (research progress/sharing and mentor/mentee career development), 4) structured oral, written, and analysis skill building and feedback, 5) depth of a continually evolving curriculum, and 6) an opportunity for alumni and speaker interaction that relays latest discoveries, allows career networking, and provides supplementary evaluation of the training program. The CTP Training Outcomes continue to be outstanding as reflected in published productivity, trainee extramural grants, and job placement (95%-predoc and 100%- postdoc in research-intensive and -related positions). Ten expert chemosensory trainers will shape the intellectual and scientific practice of trainees at two levels as they bridge to independent and externally-funded scientific research programs in chemosensory problems important for the quality of life and human health.

With this award, the Macromolecular, Supramolecular and Nanochemistry Program of the Chemistry Division is funding Drs. Hedi Mattoussi and Debra Fadool at the Florida State University to develop strategies to modify the surfaces of inorganic nanomaterials so that they can bind with biological systems. This will yield systems that can be used to probe specific biological species and monitor targeted biological processes without affecting the integrity of the host system. To meet this goal, new multifunctional polymers that strongly coordinate onto various nanomaterials will be designed, providing water-compatible nanoprobes that are easy to introduce into biological media/systems. The work at Florida State University provides a good example where nanotechnology is used to address important problems in biology. The present project is interdisciplinary in nature and it provides opportunities for training graduate and undergraduate students in areas such as growth and functionalization of nanocrystals, peptide design and synthesis, protein expression, and several other analytical techniques. Prof. Mattoussi and co-workers will also carry out outreach activities to undergraduate students, including students from underrepresented groups.

The project focuses on developing amphiphilic polymers as surface ligands that can tightly coordinate onto the surfaces of inorganic nanomaterials, including magnetic nanoparticles, luminescent quantum dots and gold nanoparticles. The chemical design exploits the effectiveness of the nucleophilic addition reaction to allow the simultaneous insertion of hydrophilic moieties, metal-coordinating groups and reactive functions on the same polymer platform. By combining multi-anchoring groups with hydrophilic and biologically-reactive functionalities in the same amphiphilic polymer, our design will enhance the ligand-to-nanoparticle binding, while facilitating their coupling to various target molecules. This approach will allow conjugation of QDs and magnetic nanocrystals to several target molecules, including dopamine and the margatoxin (MgTx) peptide. These resulting conjugates will be used to develop specific biosensors. In particular QD-dopamine conjugates will provide platforms that can target iron ions and cysteine-rich proteins in vitro and in cell cultures. Similarly, nanoparticle-MgTx conjugates will be used to control the current flow through the Kv1.3 potassium channels of mitral cells.