Vincent Dionne - US grants

The control of membrane conductance by acetylcholine receptor at the garter snake neuromuscular junction and by extrajunctional receptors will be studied. Snake myoneural junctions in both twitch and slow fibers are anatomically similar to those in most higher vertebrates including man, and exhibit a host of essentially identical functional properties. Neuromuscular junctions will be located visually using Nomarski optics on twitch fibers and by induced response on slow fibers. The responses of junctional ecetylcholine receptors to various chemical agonists as a junction of temperature voltage and with different permeations will be recorded using the single channel method. In turn, these records will be processed and analyzed using single channel ensemble analysis to obtain information on receptor kinetic properties. Extrajunctional acetylcholine receptors will be studied similarly and their properties compared with those of junctional receptors. A significant portion of our knowledge of acetylcholine receptor mechanisms has been obtained by such studies. This work on the garter snake muscle is designed to continue the characterization of normal receptor mechanisms and to extent these studies to extrasynaptic receptors. Both the permanent population of extrasynaptic receptors near the tendinous insertion on the muscle and denervation induced receptors will be examined. Evaluation of receptor kinetic properties will allow a better understanding of the molecular mechanisms which control membrane conductance changes. This approach should be applicable to other chemical receptors.

Odors are transduced by processes that modulate ion channels in the membranes of olfactory receptor neurons. This study is aimed at identifying and characterizing these processes with the long-range goal of learning how the olfactory system detects, discriminates, and encodes odor information. The studies will be conducted with Necturus maculosus, a neotenic salamander that has been a valuable animal model for physiological investigations. Its large cells and aquatic habitat make it especially well suited for studying odor transduction. While specific features of odor transduction may not be shared by all animals, the principles and essential features that can be learned from Necturus may be universal. Patch-clamp electrophysiological methods, flash photolysis of caged compounds, calcium imaging, and molecular biological techniques will be used to address several long-standing questions about odor transduction. These include the characterization of a chloride channel that appears to be directly gated by intracellular cyclic AMP and cyclic GMP. This conductance, first detected in our studies, has a novel dependence on cyclic nucleotides that suggests it may be involved in odor transduction. A separate study will examine an odor transduction pathway that is stimulated by amino acids and involves the elevation of intracellular calcium in olfactory receptor neurons. The third part of the study focusses on the olfactory receptor gene family. In preliminary studies, we have cloned six olfactory receptor genes from Necturus.. Surprisingly in light of this animal's aquatic habitat, five of the six genes appear to be mammal-like while only one is fish-like. Using molecular methods, these and additional members of the olfactory receptor gene family will be studied-to learn about their functions in olfactory receptor neurons.

The long-range goal of this program are to learn how vertebrate olfactory receptor neurons detect and discriminate different odors. The study will use the mudpuppy, Necturus maculosus, an aquatic salamander with large cells that are well suited to patch-recording techniques. Receptor neurons will be isolated without enzymes to preserve their chemo-sensitivity, and studied using odorants which we have shown to stimulate the mudpuppy olfactory system in vivo. We have also shown that Xenopus oocytes injected with mRNA prepared from olfactory tissue can become sensitive to odor molecules. We will use this expression system and molecular biological techniques to identify and characterize receptors and other elements of olfactory transduction pathways. There are four specific aims for the next program period. 1. Characterize the cellular mechanisms used for detection of amino acids and aromatic compounds in dissociated olfactory neurons. 2. Examine the basis of chemo-sensory discrimination by evaluating the sensitivity exhibited by individual olfactory neurons. 3. Determine the origin of the receptor potential, identifying the ionic conductances which underlie it and how they are modulated by chemical stimulation. 4. Identify and characterize receptors and other components of the olfactory transduction pathways by expression in oocytes of mRNA from the olfactory epithelium. The suitability of the preparations and the feasibility of the methods have been demonstrated, providing strong assurance that the study will yield useful results.

Olfactory transduction is the physiological process of smell in which receptor cells transform their reception of an odorant into a nerve signal to send to the brain. The mechanisms involve modulation of ion-selective components of the membrane conductance in the olfactory receptor cell, to trigger excitation of a nerve impulse from the cell. Membrane properties often are studied in cells that have been dissociated and isolated in a dish, where they are easily accessible for physiological recording with micro- electrodes. This project uses a novel "slice" preparation of sensory epithelium, so that micro-electrode studies will allow characterization of the membrane properties of olfactory cells in a more normal environment, and determination of how the membrane conductances are modulated by odors. This preparation offers unique opportunities to study spatial interactions within the sensory epithelium, and the role of non-neuronal neighboring cells in modulating the neural response. Using patch-clamping techniques of electrophysiology, the chemosensory properties of two distinct morphological forms of olfactory cells are compared, along with the physiological properties of three classes of non-neuronal cell types in the epithelium. Results will have an impact on our understanding of how peripheral chemosensory function depends on the integrated activity within the receptor organ, as well as the properties of individual receptor cells. These results will be important to sensory neuroscience in general, in analyzing the capabilities for detecting, discriminating and encoding by a network system of neurons in a sheet of epithelium.

This study is directed at identifying and characterizing the physiological roles of gap junctions in the olfactory epithelium. New data have shown that olfactory receptor neurons are coupled to one another and possibly to non-neuronal cells in the olfactory epithelium of Necturus maculosus. Coupling was revealed by dye-transfer, by electrical measurements and by antibody labeling. Because gap junctions can be regulated by second messenger-dependent mechanisms, and because the same intracellular messengers that are elevated during odor transduction can activate these mechanisms, coupling of olfactory neurons to other cells in the olfactory epithelium is expected to be modulated during odor transduction. Changes in coupling could affect odor sensitivity. Studies are proposed (1) to examine the extent of intercellular coupling in the epithelium using dye-transfer with Neurobiotin(TM) and Lucifer yellow, (2) to identify the subtypes of gap junction proteins in the olfactory epithelium and their distributions by cloning and the use of molecular techniques, and (3) to evaluate the pathways that regulate olfactory gap junctions using dye-tracer and electrophysiological methods. Gap junctions are now known to be common throughout the nervous system and to have important functional consequences, but our understanding of their physiological roles is poor. These studies should provide greater knowledge of both the olfactory system and of gap junctions, a basic interneuronal feature that occurs throughout the central nervous system.

DESCRIPTION (provided by applicant): This proposal outlines the development and implementation of a multicellular recording method to study encoding of information by olfactory sensory neurons. Sensory information about odor is extracted by many different olfactory neurons simultaneously and distributed as trains of action potentials to glomeruli throughout the olfactory bulbs. The response of an olfactory sensory neuron to odor is usually described as excitatory, but in studies of isolated neurons, both excitatory and inhibitory odor responses have been reported, and the same odor can alter different membrane conductances in different neurons. The presence of such variability has raised questions about whether the responses are real or artifactual, and it has emphasized how limited our knowledge of the fundamental nature of the encoding process is. Present methods are not well suited for critically examining odor encoding. We propose to develop a new multicellular electrical recording technique to address the problem This technique will permit recording of odor- elicited electrical activity at the single-cell level simultaneously from a small population of olfactory sensory neurons. Such a capability would represent a technological breakthrough that could significantly advance our understanding of olfactory neural encoding. The method will utilize isolated olfactory epithelial tissue together with an array of independent glass microelectrodes, each recording extracellular action currents from the exposed cilia of olfactory sensory neurons. The neurons would remain in the intact tissue and not require degradative enzymatic treatment, thus allowing odor responses to be recorded under nearly physiological conditions. Because of its multicellular nature coupled with the possibility of long-term recordings, the method offers a unique, high-throughput physiological assay of odor responses. It should be ideal for studying the fundamental mechanisms of odor encoding with pharmacological tools, and it will offer an efficient means to study transgenic animals which are expensive to engineer.