Clinton Wanner , Mark L. Burleson

ACETYLCHOLINE-CONTAINING NEUROEPITHELIAL CELLS IN FISH GILLS SUPPORT THE CHOLINERGIC HYPOTHESIS OF O2 CHEMORECEPTION

Clinton Wanner, Mark L. BurlesonDepartment 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

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Environmental O2 Availability:Dissolved Oxygen in Trinity River, Ft. Worth

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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 O2 chemoreceptors work!

WHAT ARE O2 CHEMORECEPTORS? O2 chemoreceptors 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

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 branchial neuroepithelial cells of fish gills are the evolutionary precursors to mammalian O2 receptors.

Histochemical Profiles of O2 Chemoreceptor Cells

Mammalsserotonindopaminenorepinephrineacetylcholineepinephrinesubstance-Penkephalinsnitric oxideneuron-specific enolasetyrosine hydroxylase

Birdsserotoninsubstance-Pneuron-specific enolasetyrosine hydroxylaseReptilesserotoninenkephalinsneuron-specific enolasetyrosine hydroxylase

Amphibiansserotoninenkephalinsneuron-specific enolasetyrosine hydroxylaseFishserotoninenkephalinsneuron-specific enolasetyrosine hydroxylase

There is no consensus on the roles of any of these chemicalsin 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.

IXX IXX X IX

Fish Amphibians Birdsnon-ChelonianReptiles

Mammals

Fig. 1

HYPOTHESIS : BRANCHIAL NEUROEPITHELIAL CELLS CONTAIN ACETYLCHOLINE.

This hypothesis will be tested using immunohistochemistry and laser confocal microscopy

•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.

CRITERIA FOR NEUROTRANSMITTER:

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 (Ictalurus punctatus)

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.

GILL ANATOMY

IMMUNOLABELING OF SEROTONINRESULTS

IMMUNOLABELING OF ACETYLCHOLINE

IMMUNOLABELING OF ACETYLCHOLINE

CONCLUSIONBranchial neuroepithelial 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