The purpose of the studies outlined in this proposal is to explore the neuroanatomy, mechanism of action and functional organization of respiratory CO2 sensitivity in terrestrial, pulmonate snails. The respiratory CO2 response in pulmonate snails resembles the CO2 response in mammals to an extraordinary extent. These investigators have isolated a discrete, CO2 sensitive location within the central nervous system of the snails. Focal stimulation of this region activates the respiratory mechanism and increases ventilation much as focal acid stimulation of the ventrolateral medulla does in mammals. Carbonic anhydrase increases the rate of response to CO2, and the animals are more sensitive to CO2 than to fixed acid at the same pH, just like mammalian central chemoreceptors. Within the chemosensitive area of the snail """"""""brain"""""""", these investigators have isolated individual cells sensitive to CO2. These cells are capable of duplicating the respiratory response to CO2 when stimulated electrically. Furthermore, when these cells are ablated, the CO2 response is destroyed. The CO2 response of these cells is not dependent on synaptic mechanisms. Having isolated respiratory CO2 chemoreceptor cells, the investigative team wants to know how they work. They can perform experiments that have been impossible in mammals because the actual CO2 sensitive cells are unknown in mammals. These investigators propose experiments to study two aspects of chemoreceptor function: the ionic basis of chemoreceptor responses and the actual stimulus to the chemoreceptor. The first set of experiments will use simple sharp electrodes to record single cell activity as well as current-clamp and voltage-clamp methods to examine the firing properties of chemoreceptor and non-chemoreceptor cells. The authors think that the sensor is a calcium channel, and have proposed a series of studies examining the role of Ca++ in the neural response to CO2. They will also begin to establish the morphology and projections of the chemoreceptor cells. Determination of the nature of the chemoreceptor stimulus is the second major focus of this proposal. Is it intracellular pH, extracellular pH, the transmembrane pH gradient, or molecular CO2? The proposed investigation aims to establish the stimulus-response profile of chemoreceptor cells in terms of the physiologically relevant stimulus.
