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

Environmental O2 Availability:
Dissolved Oxygen in Trinity River, Ft. Worth
240
sea
level
2290
5280
Everest
9650
200
oxygen partial pressure (torr)
Dead Sea
equivalent altitude (m)
-2000
160
120
80
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 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
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.
From Lahiri et al., 2001
Histochemical Profiles of O2
Chemoreceptor Cells
Amphibians
Mammals
Birds
serotonin
dopamine
norepinephrine
acetylcholine
epinephrine
substance-P
enkephalins
nitric oxide
neuron-specific enolase
tyrosine hydroxylase
serotonin
substance-P
neuron-specific enolase
tyrosine hydroxylase
serotonin
enkephalins
neuron-specific enolase
tyrosine hydroxylase
Reptiles
Fish
serotonin
enkephalins
neuron-specific enolase
tyrosine hydroxylase
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.
X
IX
X
Fish
IX
X
Amphibians
IX
Birds
non-Chelonian
Reptiles
Mammals
HYPOTHESIS : BRANCHIAL NEUROEPITHELIAL
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 cardioventilatory 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
RESULTS
IMMUNOLABELING OF SEROTONIN
IMMUNOLABELING OF ACETYLCHOLINE
IMMUNOLABELING OF ACETYLCHOLINE
CONCLUSION
 Branchial
neuroepithelial of catfish
gills cells contain acetylcholine.
 These
data support previous reflex
and neural studies implicating
acetylcholine as the principal
neurochemical link between O2sensitve chemoreceptor cells and
primary sensory afferent nerves.
ACKNOWLEDGEMENTS

Thanks to:
Dr. Fuchs
 Dr. Turnbull
 David Oden

NIH
 Texas Parks & Wildlife
