Steven Kleene - US grants

Newly developed biophysical techniques allow study of the membrane processes responsible for excitability and transduction in olfactory receptor neurons (ORNs). Odorants are known to interact with the olfactory cilia and increase the activity of the enzyme adenylate cyclase. This increase is mediated by one or more GTP-binding proteins (G-proteins). The specific G-protein involved may be G-olf, a major stimulatory G-protein in olfactory cilia. However, there is no evidence that G-olf is required for olfactory transduction. The proposed experiments will determine whether G-olf mediates activation of the adenylate cyclase. G-olf in single cilia will be inactivated with specific antibodies, and the effects on the odorant response measured. cAMP produced by the cyclase is known to activate ciliary membrane channels, which allow a depolarizing receptor current to flow into the ORN. Preliminary studies suggest that divalent cations at the external surface of the ciliary membrane strongly inhibit this receptor current. The extent of inhibition by calcium and magnesium on the cAMP-activated current will be measured in single cilia. The cAMP-mediated depolarization is transient, and it is unknown how the odorant response is terminated. Preliminary studies indicate that calcium entering the cilia through the cAMP-gated channels activates a secondary chloride conductance. This calcium-activated chloride conductance may help to repolarize the neuron after the odorant response. Activation of the secondary chloride current will be demonstrated in single cilia. The hypothesis that this current repolarizes the neuron after an odorant response will bc tested in intact ORNs. Gigaseal patch-clamp recordings from isolated cilia will bc used to measure the ciliary membrane properties. Perforated-patch whole-cell recordings from the soma will measure the chloride current in intact ORNs without perturbing the normal intracellular chloride concentration. Pharmacological and immunochemical means will bc used to selectively control specific ionic currents implicated in ORN function. The olfactory sense has a major role in regulating body hormonal state, emotional disposition, hunger, and social behavior. The work proposed here will advance understanding of the molecular and cellular processes which subserve sensory function.

DESCRIPTION: Dr. Kleene and colleagues will develop patch clamp techniques to

Vertebrates detect many odorants with high sensitivity. An enormous amount has been learned about the chemical and electrical mechanisms underlying vertebrate olfactory transduction. A variety of GTP-binding proteins, enzymes, second messengers, and ligand-gated channels have been shown to function in transduction. There are 700 to 1000 genes that appear to code for odorant-receptor proteins. The remaining question is how this mass of transductory components is functionally organized across several million olfactory receptor neurons. The sheer number of components makes this question mathematically enormous and experimentally difficult. The nematode Caenorhabditis elegans presents an olfactory model that is tremendously less complex than the vertebrate system. Extensive evidence during the past five years has shown that the molecular components of olfaction in C. elegans closely parallel those of vertebrates. Such rapid progress has been enabled by the worm's small size and short generation time, and by the sequencing of most of its genome. However, in the absence of physiological studies, the exact functions of the molecular components of olfaction in C. elegans remain speculative. The proposed study will apply electrophysiological methods to the tiny identified chemosensory neurons of C. elegans. For the first time, the electrical basis of a sensory response in C. elegans will be demonstrated. The molecular functions implied by genetic and behavioral methods will be directly tested. The ability to record from these neurons will represent a significant enhancement of the existing methods and will broaden the usefulness of C. elegans as a model organism in neuroscience. C. elegans is a potential model for olfaction, chemotaxis, thermotaxis, mechanosensation, and responses to pheromones and light. The olfactory sense has a major role in regulating body hormonal state, emotional disposition, hunger, and social behavior. The work proposed here will advance understanding of the molecular and cellular processes which subserve sensory function.

The transmission of an odor stimulus to the brain begins in cilia, which are long thin processes that extend from the olfactory receptor neurons. These neurons are the first cells in the olfactory system that extend from the nose to the brain. The conversion of a chemical signal, the odor, into an electrical signal, appropriate for processing in the brain, is carried out by two sets of ion channels embedded in the membrane (surface layer) of the cilia. An interdisciplinary research group involving professors and graduate students in Mathematics and Experimental Neuroscience will develop procedures to determine the distributions of these channels through experimentation and the computer solution of a mathematical model. In a typical experiment, ligands for a specific channel type will be allowed to diffuse into a cilium, leading to a current after the molecules bind. The recorded current is the input data for the mathematical model. To determine the ion channel distribution at this stage requires the solution of an inverse or distributed parameter identification problem (DPIP) which is a nontrivial extension of more standard such problems. The main objectives of the work are the development of appropriate experimental procedures, mathematical models, and the solution of the resulting DPIPs. Both computational and analytical or perturbation approaches will be considered as will generalizations to other related situations.

Identification of detailed features in neuronal systems, such as the distributions of ion channels in cilia, forms an important challenge in the biosciences today. Although the properties of the olfactory channels have been determined, the distributions of the channels along the cilia are unknown. These distributions are crucial in determining the time course of the neuronal response. This work will have applications outside of olfaction in other areas in the brain such as the photoreceptor cells in the retina or the dendrites in the neocortex. It is also expected that this research will advance our understanding of the mathematics of inverse problems. Further, models of olfactory transduction are relevant to efforts to create mechanical-chemical "noses" for the detection of hazardous biological and chemical agents. The interdisciplinary nature of the research is also critical. It will enhance the infrastructure at the University of Cincinnati while providing interdisciplinary training to the students.

DESCRIPTION (provided by applicant): Most cells in the body possess a single primary cilium. These cilia are key transducers of chemical stimuli. Indirect evidence suggests that transduction of chemical stimuli by the primary cilium often depends on ion-conducting channels expressed in the ciliary membrane. However, the tiny size of the cilium has been a critical barrier to understanding its chemosensory functions. Existing studies of the ciliary channels have been indirect, using exogenous expression systems, artificial bilayers, and non-ciliary cellular compartments. Studies in the native ciliary membrane are virtually non-existent. To address this limitation, the applicants have developed a novel method that allows sensitive, repeatable, and stable electrical recordings from single primary cilia of cultured kidney cells. Recording begins the instant the cilium is plucked from the cell. The central goal of this project is to demonstrate that this is a highly efficient means of identifying mechanisms of electrochemical transduction in primary cilia. There are two specific aims. The first aim is to demonstrate that electrochemical transduction of external stimuli relevant to renal physiology can be effectively studied by recording from the cilium. The second aim is to demonstrate that ciliary recording is an efficient way to learn the effects of second messengers and voltage changes on ciliary transduction channels. This aim is proposed because preliminary studies by the applicants have identified ligand- and voltage-gated channels in the primary cilia. Because primary cilia are present on most mammalian cells, the significance of this work will be broad. In the kidney, mutations in ciliary proteins, including at least one channel protein, are hypothesized to cause cystic diseases that affect over 6,000,000 individuals worldwide. Validation of the novel method as proposed will reveal the functional physiology of the renal primary cilium in a way that has not previously been possible. Ultimately this will facilitate a new field of research: direct studies of the chemotransduction properties of the many primary cilia in the body. Such studies may identify targets for pharmacological intervention in treating diseases caused by defects in primary cilia.