Johannes Reisert - US grants

DESCRIPTION (provided by applicant): This collaborative research project will combine electrophysiological recordings from olfactory receptor neurons (ORN) of genetically modified mouse lines with computational modeling of the (slow) signal transduction cascade and (fast) action potential generation. In doing so, the researchers aim to develop a refined mathematical description regarding the molecular basis for olfactory transduction and adaptation within vertebrate ORNs. This is an important goal not only because olfaction shapes animal behavior and social ecology but also because similar molecular mechanisms are implicated in olfactory disease. One of the current challenges in neuroscience research is bridging the temporal scales of biochemical and neural processes. Olfaction provides a convenient model system for studying how single neurons can leverage the timing of molecular events to process information. Subsequent to odor presentation, a G-protein-coupled signal transduction cascade is activated within the ORN cilia leading to current influx. Ultimately, ORNs generate action potentials, which are the primary information transmitted to the olfactory bulb. A successful model of the ORN must, therefore, be able to illustrate and predict the molecular processes underlying both the slow (transduction) and fast (action potential) currents during stimulation. The applicants'model is unique among the existing models in its ability to accurately predict the adaptive responses of ORNs to repetitive stimuli, the oscillatory responses during sustained stimulation, and the desensitization following a single brief odorant pulse. This model has recently been expanded to include the generation of action potentials. Mutant mouse lines, which are currently available and will be created, will be used to probe the relation of these components to transduction activity through computational modeling of electrophysiological recordings of the mutant ORNs.

DESCRIPTION (provided by applicant): Animals live in an environment of constantly changing and complex odorous signals, which are delivered by the inhaled air to olfactory receptor neurons (ORNs) in the nasal cavity. ORNs recognize odorants and convert odorant stimulation into action potentials to be conveyed to the first relay station in the brain, the olfactory bulb. This is achieved by activation of odorant receptors, leading to cAMP generation via a G protein-coupled cascade and the opening of ion channels present on the olfactory cilia and subsequent depolarization. Odorant specificity is provided by the expression of only one type of odorant receptor in a given ORN out of ~1000 different receptors in mice and 350 in humans. Most major components of olfactory signal transduction have been identified. Our goal is to determine what limits and controls the kinetics with which olfactory transduction components interact, how this controls action potential generation and coding and what the behavioral implications are for, in particular, odorant discrimination and initiation of sniffing. Using electrophysiological techniques, we will investigate how mouse ORNs transduce odorant stimulation. Using rapid, repetitive stimulation designed to simulate high-frequency, sniffing-driven odorant delivery, we will establish whether ORNs merely report these rapid changes in odorant concentration or if in fact they themselves actively process this information in a stimulation-frequency-dependent manner. We will determine the functional role in olfactory transduction kinetics of olfactory marker protein (the function of which has not been found since its discovery in 1972) as well as determining the role of different odorant receptors in shaping the time-course of the odorant-induced response. The importance of fast and precise olfactory transduction will be studied using behavioral testing on genetically altered mice to investigate speed-accuracy tradeoff in odorant identification. Monitoring the breathing frequency during active olfactory exploration will allow us to establish the contribution of ORN kinetics and peripheral-versus-central influence on controlling changes in breathing and sniffing rates. PUBLIC HEALTH RELEVANCE: The proposed work will address the importance of both precise timing and fast transduction of odorous signals by G protein-coupled receptors in olfactory receptor neurons from the single-cell to the complex-behavioral levels such as tracking a food source or avoiding a predator. The work has broader implications in that the results will yield fundamental insights into how members of the G protein-coupled receptor family (which comprise a large part of the genome) and neurons that express them, control time-dependent cellular processes ranging from heart beat regulation to conveying hormonal signals.

DESCRIPTION (provided by applicant): The Monell Chemical Senses Center is a unique multidisciplinary institute devoted to investigating the science of the chemical senses. Currently there are 21 participating faculty members representing disciplines ranging from molecular biology and genetics to psychophysics and nutrition and conducting research in both basic and clinical aspects of olfaction and gustation. The Monell Center has enjoyed a successful Interdisciplinary Training Program in the Chemical Senses for over 30 years. The long-term goal of the training program is to provide a pool of scientists well-trained in the chemical senses who are capable of becoming independent scientists. Trainees from a wide variety of scientific backgrounds both within and outside of the chemical senses area are recruited to the program. The Postdoctoral Training Program consists of didactic courses, research training and research experience, grant writing as well as training in the ethical principals of scientific research. Trainees are assigned a mentoring team to ensure completion of required training components and obtainment of scientific goals such as publications and grant submissions. This proposal requests funds to defray the costs of training four postdoctoral fellows per year.

Summary Our senses convert environmental stimuli into electrical signals that are ultimately interpreted by the brain to guide our behavioral decisions. The conversion of stimuli relies on the ion channels expressed in sensory cells, and their properties thus determine how we perceive our environment. Olfactory receptor neurons (ORNs) in the nasal cavity recognize odorants and, unlike other sensory neurons such as photoreceptors and hair cells, are in direct contact with the external environment, protected only by a thin mucus layer. Olfactory cilia, the cellular compartment that contains the machinery that transduces odorants, must survive in this environment while remaining functional, adding extra demands on membrane integrity and function. The initial event of an odor molecule binding to an odorant receptor in the ciliary membrane leads, via activation of adenylyl cyclase, to opening of the olfactory cyclic-nucleotide gated (CNG) channel that allows Ca2+ influx, which in turn activates an excitatory Ca2+-activated Cl- channel, further depolarizing the neuron. This two-tiered sensory transduction mechanism based on one cationic and one anionic channels, is unique to ORNs and highly conserved across all vertebrates. Both the reason why ORNs use this two-stage ion channel system in general and why a combination of cation and anion conductances in particular is used to perceive odorants are unclear, as is the role of the Ca2+-activated Cl- channel. Only in 2009 was the molecular identity of the olfactory Ca2+-activated Cl- channel determined to be anoctamin 2 (Ano2), and despite a knockout model being available, the roles of Ano2, and therefore also of the CNG channel, remain unclear. We propose to use an Ano2-knockout mouse, electrophysiological and molecular approaches to define how these two ion channels shape the odorant-induced response. We will characterize which specific aspects of the response (adaptation, response reliability, action potential coding, etc.) are determined by a single ion channel or jointly by both. In addition, because the two channels must function in the constraints of the ciliary membrane, we will investigate how the channels rely on membrane constituents for their function and how altered membranes leads to detrimental olfactory function. By examining how the two-tiered sensory transduction mechanism of a cationic and an anionic ion channel operates seamlessly as a dual-component system, we will address fundamental questions in olfaction that have remained unanswered for the past 25 years. The long-term goal of this proposal is to establish how ORNs use their signal transduction in general and their ion channels in particular to reliably encode odorant stimuli, how transduction functions within the constraints of the ciliary membrane, and how this ultimately determines how odorants are perceived.

Project Summary Olfactory receptor neurons (ORNs) are located in the olfactory epithelium in the nasal cavity and are the primary neurons that convert odorous information into nerve signals to be conveyed to the brain. As such, they drive a variety of behaviors including food localization, friend and foe recognition, and evaluation of reproductive status. Cilia that emanate from ORNs into the mucus that covers the epithelium are the site of olfactory transduction, and cilia from neighboring ORNs form an entangled mesh. Upon activation by odorants, a cAMP and Ca2+-based transduction cascade generates a ciliary transduction current that depolarizes the ORNs and leads to the generation of Ca2+-driven action potentials in the cell body. But little is known about the Ca2+ kinetics in cilia and the rest of the ORN, or how cilia from different ORNs might interact and influence each other within the environment of the mucus. The goal of this NIDCD Research Career Enhancement Award for Established Investigators proposal is to enhance the applicant?s current research program to learn and adopt cutting-edge lattice light sheet microscopy approaches to monitor Ca2+ responses in ORNs in the intact olfactory epithelium using genetically-encoded Ca2+ indicators. The long-term goal of the proposed mentored research experience is to understand how ORNs function in their polarized environment, how Ca2+ contributes to olfactory signaling and how the interaction of cilia in the mucosal environment contributes to coding and signaling of olfactory information.