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Acetylcholine-Containing Neuroepithelial Cells in Fish Gills Support the Cholinergic Hypothesis of O 2 Chemoreception. Clinton Wanner , Mark L. Burleson Department of Biological Sciences, College of Arts and Sciences. Why is O 2 important?. Human survival: 3 weeks without food
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Acetylcholine-Containing Neuroepithelial Cells in Fish Gills Support the Cholinergic Hypothesis of O2 Chemoreception Clinton Wanner, Mark L. Burleson Department of Biological Sciences, College of Arts and Sciences
Why is O2 important? Human survival: • 3 weeks without food • 3 days without water • 3 minutes without oxygen
Everest Dead Sea Environmental O2 Availability: Dissolved Oxygen in Trinity River, Ft. Worth 240 -2000 200 sea level 160 equivalent altitude (m) oxygen partial pressure (torr) 2290 120 80 5280 9650 40 0
Discovery of O2 chemoreceptors • Corneille Heymans • Nobel Prize in Physiology or Medicine in 1938 • Chemoreceptors in cardio-aortic and carotid sinus areas • Showed that chemical changes in arterial blood elicited cardio-ventilatory responses. • Despite nearly 75 years of research, we still do not understand how O2chemoreceptors work!
What are O2 chemoreceptors? • O2chemoreceptors are specialized cells that detect changing levels of O2 availability and demand and initiate cardiovascular and ventilatory reflexes to maintain normal O2 uptake. From Lahiri et al., 2001
What are O2 chemoreceptors? Hypoxia (low oxygen) depolarizes the O2 chemoreceptor causing the release of a neurotransmitter. The goal of this study is to identify that neurotransmitter using a non-mammalian animal model. The branchialneuroepithelial cells of fish gills are the evolutionary precursors to mammalian O2 receptors. From Lahiri et al., 2001
Histochemical Profiles of O2 Chemoreceptor Cells Amphibians serotonin enkephalins neuron-specific enolase tyrosine hydroxylase Mammals serotonin dopamine norepinephrine acetylcholine epinephrine substance-P enkephalins nitric oxide neuron-specific enolase tyrosine hydroxylase Birds serotonin substance-P neuron-specific enolase tyrosine hydroxylase Fish serotonin enkephalins neuron-specific enolase tyrosine hydroxylase Reptiles serotonin enkephalins neuron-specific enolase tyrosine hydroxylase There is no consensus on the roles of any of these chemicals in the chemoreceptor control of ventilation in vertebrates!
phylogeny • Evolution of air-breathing was accompanied by a reduction and internalization of O2-sensitive chemoreceptors and their loci.
Hypothesis : Branchialneuroepithelial cells contain acetylcholine. • This hypothesis will be tested using immunohistochemistry and laser confocal microscopy
Criteria For Neurotransmitter: • Presence of the chemical within the cell. The chemical is either synthesized by the neuron or is taken up from other cells that release it.2 • Stimulus-dependent release. It is released in appropriate quantities by the neuron upon stimulation. • Action on postsynaptic cell. Exogenous application of the substance in appropriate amounts mimics the action of the endogenously-released substance on the postsynaptic cell or organ.Mechanism for removal. [Note, not always included as a criterion] A specific mechanism exists to remove the substance from the synaptic cleft, i.e., by degradation or reuptake.
Previous Studies: • Only acetylcholine consistently mimics the effects of hypoxia and cyanide (histotoxic hypoxia) on cardio-ventilatory reflex responses and neural activity in mammals and fish. • Furthermore, the effects are mediated by the nicotinic cholinergic receptor subtype.
Material and Methods • Channel catfish (Ictaluruspunctatus) obtained from TP&W Dundee Fish Hatchery • Maintained in 100 gal tanks. • On day of experiment, fish anesthetized (MS222), heparinzed, cannulated and gills exsanguinated. • Pieces of gill with filaments removed with scissors and fixed in buffered formadehyde • Processed, mounted, stained. • Observed using Zeiss 200M inverted optical microscope modified for confocal laser microscopy.
Immunolabeling of serotonin Results
Conclusion • Branchialneuroepithelial of catfish gills cells contain acetylcholine. • These data support previous reflex and neural studies implicating acetylcholine as the principal neurochemical link between O2-sensitve chemoreceptor cells and primary sensory afferent nerves.
Acknowledgements • Thanks to: • Dr. Fuchs • Dr. Turnbull • David Oden • NIH • Texas Parks & Wildlife