Acetylcholine-Containing Neuroepithelial Cells in Fish Gills Support the Cholinergic
Hypothesis of O2Chemoreception
Author: Clinton Wanner
Faculty Mentor: Mark L. Burleson, Department of Biological Sciences, College of Arts and
Sciences
Department and College Affiliation: Department of Biological Sciences, College of Arts and
Sciences
Special thanks to Dr. Jannon Fuchs for technical assistance and the use of her laboratory and
equipment. Dr. Lon Turnbull played a critical role in this research for his part in operating the
laser confocal microscope and obtaining photomicrographs. David Oden provided important
technical support in preparing tissues and operating the sliding microtome. This research was
supported by The National Institutes of Health (NIH) Heart, Lung, and Blood Institute (NIH
Grant HL076205, Dr. Mark L. Burleson, P.I.). Texas Parks and Wildlife Dundee Fish Hatchery,
in Wichita Falls, provided the catfish for the research.
Acetylcholine-Containing Neuroepithelial Cells in Fish Gills
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Bio:
Clinton Wanner, a Dean’s List student, born in Oklahoma is graduating from the College of Arts
and Sciences at the University of North Texas with a Bachelor of Arts degree in Biology and a
minor in Chemistry and Psychology. Clinton has a particular interest in the natural sciences
where he won first place in 2011 for a paper submitted during the University Scholar’s Day at
the University of North Texas. After obtaining his bachelor’s degree Clinton aspires to pursue
his dream of attending dental school to provide dental care to people in need. Clinton’s future
career goals consist of continuing research in the natural sciences while obtaining his
certification for oral surgery.
Acetylcholine-Containing Neuroepithelial Cells in Fish Gills
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Abstract:
The neurochemical link between O2 chemoreceptors and afferent nerves that carry information
about O2 levels to cardio-ventilatory centers in the brain has yet to be determined. This study
examines the roles of two candidate neurotransmitters thought to be involved in O2
chemoreception, using channel catfish, Ictalurus punctatus. Fish gills are the evolutionary
progenitors of arterial arches (aortic and carotid) of mammals where O2 chemoreceptors are
located. Neuroepithelial cells (NECs) containing serotonin (5-HT) and acetylcholine (Ach) were
confirmed in the first gill arch using immunohistochemistry and laser confocal microscopy. 5HT-containing NECs were aggregated around the efferent branchial artery, near tips of filaments
and lamellae, ACh-containing NECs at the distal tips of filaments. Preliminary co-localization
experiments indicate separate 5-HT and Ach-containing cells. This is the first demonstration of
ACh-containing NECs and results of this study support pharmacological studies suggesting that
ACh is the primary neurochemical involved in O2 chemoreception in vertebrates.
Acetylcholine-Containing Neuroepithelial Cells in Fish Gills
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Introduction
Oxygen is the most vital element for the existence of life. Initially, the atmosphere did
not contain O2; it was not until photosynthesis evolved that atmospheric O2 levels rose to
approximately 21%. To illustrate the importance of O2, the average human being can survive
about 3 weeks without food, 3 days without water, but only 3 minutes in the absence oxygen. In
water, where vertebrates first evolved, O2 levels vary both daily and seasonally. This is attributed
to the production of O2 from photoautotrophs and its consumption by nearly all living organisms.
Also, O2 demand varies with activity and temperature. The constant changes in O2 availability
and demand likely shaped the evolution of chemoreceptors to detect these changes and induce
compensatory responses.
Corneille Heymens was awarded the Nobel Prize in Physiology and Medicine in 1938 for
the discovery of O2-sensitive chemoreceptors in the carotid bodies. His experiments showed that
chemical changes in arterial blood going to the carotid bodies elicited cardio-ventilatory reflex
responses that increase O2 uptake from the environment. Specifically, O2 chemoreceptors detect
O2 availability, demand, and initiate regulatory responses in the cardiovascular and ventilatory
systems to maintain normal O2 uptake in the face of changing environmental levels and internal
demands.
Phylogenetically, the artery supplying blood to the first gill arch of fish is homologous to
the carotid artery where the carotid body, the primary peripheral chemoreceptive organ in
mammals, is located. The carotid body and first gill arch are innervated by the glossopharyngeal
(cranial nerve IX) nerve (Sundin and Nilsson, 2002; Gonzalez et al., 1994). The branchial
neuroepithelial cells of fish gills are the phylogenetic precursors to glomus (O2-sensing cells of
the mammalian carotid body. Thus, studies on the more primitive receptors may provide insight
Acetylcholine-Containing Neuroepithelial Cells in Fish Gills
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into mechanisms and evolution of oxygen chemoreception. Figure 1, illustrates the evolution of
the central cardiovascular region of vertebrates. It shows how the innervation was conserved
during the evolution of terrestrial vertebrates and the internalization and reduction of gill
(vascular) arches.
There is not a direct connection between the O2-sensing cells and the brain. The O2sensing cells synapse with primary afferent neurons (Figure 2) which transmit information
regarding internal and environmental O2 levels to cardio-ventilatory control regions in the
brainstem. The nature of the chemical link between the O2-sensor and primary afferent has been
the subject of intense research and debate.
A variety of different neurochemicals have been identified in glomus cells. These include
acetylcholine, dopamine, epinephrine, norepinephrine, serotonin and substance P. The effects of
various neurotransmitter agonists and antagonists have been tested. R.S. Fitzgerald proposed the
―Cholinergic hypothesis‖ (reviewed in Fitzgerald, 2000) which postulates that glomus cells in
the carotid body release a transmitter, acetylcholine (ACh), which binds to postsynaptic
cholinergic receptors on primary sensory afferent fibers of the carotid sinus nerve creating action
potentials sent to the cardio-ventilatory control centers in the nucleus tractus solitarii of the
brainstem.
The glomus cell, or type I cell, has been described as functioning as the presynaptic unit
involved in chemoreception. The glomus cell has one or more sensory nerve fibers with cell
bodies located in the petrosal ganglion and inserting into the nucleus tractus solitarii. These
structural components were identified by De Castro in 1928. In hypoxic conditions, the glomus
cell is somehow depolarized (entire mechanism not completely understood), causing exocytosis
of a transmitter. The transmitter then diffuses across the synaptic cleft and binds to post synaptic
Acetylcholine-Containing Neuroepithelial Cells in Fish Gills
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receptors in the (afferent) sensory neuron. This causes a depolarization of the afferent neuron and
propagation of an action potential. The action potential arrives in the cardio-ventilatory control
centers in the brain which relay another action potential to the effector muscles altering cardioventilatory performance. Much evidence supports this model and many early investigators
proposed acetylcholine as the primary transmitters involved in this mechanism. To date, the
putative O2 sensing cells in fish have been identified to contain serotonin (5-HT), enkephalins,
neuron-specific enolase, and tyrosine hydroxylase (Milsom and Burleson, 2007). ACh containing
cells have been identified in the carotid body and pulmonary neuroepithelial cells of mammals,
however, not in fish. The identity of the primary neurochemical involved in this process is
currently unresolved. Any given chemical must meet the following requirements to be
considered a neurotransmitter: presence of the chemical within the cell, stimulus-dependent
release, action on postsynaptic cell, and a mechanism for removal. Thus, the purpose of this
experiment is to examine the presence and roles of two candidate neurotransmitters, 5-HT and
ACh, thought to be involved in O2 chemoreception.
Materials and Methods
Animals
Adult channel catfish, Ictalurus punctatus, of either sex were obtained from the Texas
Parks & Wildlife Dundee Fish Hatchery. They were maintained indoors at 25°C in aerated 100
gallon tanks equipped with gravel biological filters, aerators, and were fed a constant diet of
commercial animal food. Fish used for tissue analysis were randomly selected and euthanized
using MS-222 (3-aminobenzoic acid ethyl ester) dissolved in dechlorinated water. All procedures
were reviewed and approved by the University of North Texas Institutional Animal Care and Use
Committee (protocol #0411).
Acetylcholine-Containing Neuroepithelial Cells in Fish Gills
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Tissue Preparation
The fish were heparinized and the ventral aorta occlusively cannulated in order to
exsanguinate the gills with cold saline before extraction. The first gill arch was surgically
extracted and rinsed in Cortland Saline. Gill tissue was stored in buffered 4% paraformaldehyde
at 4°C overnight. Tissues were then rinsed in Cortland Saline and stored in a 30% sucrose
solution overnight at 4°C. Tissues were segmented in halves, embedded using OCT (Sakura
Finetek Tissue-Tek O.C.T. Compound) and frozen using dry ice before being stored at -80°C
until the blocks were sectioned transverse to the gill filament at 20-25µm using a sliding
microtome. Sections were cryoprotected and stored at 4°C until mounted on adhesive AmFrost®
Amino-Silane Charged slides for immunohistochemistry.
Immunohistochemistry
Slides were washed in TBS and stored in 3% normal goat serum for 1 hour Primary
antibodies were diluted in a permeabilizing solution (TBS, 0.2%, 3% normal goat serum, 03%
Triton-X 10%) and set on slides to incubate overnight at room temperature. Following
incubation, slides were rinsed with TBS and incubated in blocking solution again for 30 minutes.
Slides were then incubated in labeled secondary antibodies diluted in TBS for 2 hours in
darkness at room temperature. Following a final wash with TBS, slides were mounted with
Vectashield (Vector Laboratories, Burlington, ON, Canada) to prevent photobleaching, and the
edges of the cover slips were sealed with nail polish. Control experiments were performed in
which primary antibodies were excluded to control for non-specific binding of the secondary
antibody. Slides were stored at 4°C until viewed by a Zeiss 200M inverted optical microscope
modified for laser confocal microscopy.
Acetylcholine-Containing Neuroepithelial Cells in Fish Gills
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Results
Immunopositive cells were found in the primary branchial epithelium along the filaments
and clustered at the tips. These cells were not labeled in control preparations which consisted of
treatment with only the secondary antibodies. Figure 3 and Figure 4 show the results from the
experiment. This is the first demonstration of acetylcholine in the branchial neuroepithelial cells
of fish.
Discussion
In support of the cholinergic hypothesis applying to all vertebrates, not just mammals,
previous experiments in rainbow trout (Burleson and Milsom, 1995) showed that of all candidate
neurotransmitters, only acetylcholine elicited reflex and neural responses similar to hypoxia and
cyanide (histotoxic hypoxia). Furthermore, their data suggest that it is the nicotinic subtype of
cholinergic receptor that mediates the response. Other neurochemicals likely function as
modulators of activity. Their effects on cardio-ventilatory variables and neural activity are
modest compared to acetylcholine and nicotine. These other modulatory neurochemicals may
serve to adjust chemoreceptor activity and/or sensitivity in response to various environmental
and physiological conditions. These adjustments play a vital role in fine-tuning cardioventilatory performance to maintain optimal gas exchange with minimal energy expenditure in
response to highly variable environmental and physiological variables.
The data in mammals, however, is not completely clear as some ACh antagonists do not
completely block the response to hypoxia. We believe that given the importance of oxygen
chemoreception that multiple/overlapping mechanisms likely exist in the mammalian carotids.
Studies on the more primitive receptors may provide insight into mechanisms and evolution of
Acetylcholine-Containing Neuroepithelial Cells in Fish Gills
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oxygen chemoreception. The data presented here adds one more piece to the puzzle and provides
further support to the cholinergic hypothesis.
Acetylcholine-Containing Neuroepithelial Cells in Fish Gills
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References
Bailly, Y., Dunel-Erb, S. and Laurent, P. (1992). The neuroepithelial cells of the fish gill
filament: indolamine-immunocytochemistry and innervation. Anat. Rec. 233, 143-161.
Burleson, M.L., Milsom, W. K., (1995) Cardio-ventilatory control in rainbow trout: 1.
Pharmacology of branchial, oxygen-sensitive chemoreceptors, Respir. Physiol 100, 231238.
Burleson, M.L., Milsom, W. K., (2003) Comparative aspects of O2 chemoreception: anatomy,
physiology, and environmental adaptations. In: S. Lahiri, G.L. Semenza and N.R.
Prabhakar, Editors, Oxygen sensing: Responses and adaptation to hypoxia, Marcel
Dekker, New York, pp. 685–707.
De Castro F. (1928). Sur la structure et l'innervation du sinus carotidien de l'homme et des
mammifères. Nouveaux faits sur l'innervation et la fonction du glomus caroticum. Trav.
Lab. Rech. Biol. 25, 331–380.
Fitzgerald, R.S., (2000) Oxygen and carotid body chemotransduction: The cholinergic
hypothesis - a brief history and new evaluation. Respir Physiol. 120(2):89-104.
Gonzalez, C., Almarez, L., et al. (1994). Carotid body chemoreceptors: from natural stimuli to
sensory discharges. Physiol. Rev. 74, 829-896.
Milsom, W.K., Burleson, M.L., (2007) Peripheral arterial chemoreceptors and the evolution of
the carotid body, Respir. Pysiol. Neurobiol., doi:10.1016/j.resp.2007.02.007.
Sundin, L. and Nilsson, S. (2002). Branchial innervation. J. Exp. Zool. 293, 232-248.
Acetylcholine-Containing Neuroepithelial Cells in Fish Gills
Table 1. Details of primary and secondary antibodies used for immunohistochemistry
Antiserum
Dilution Antigen
Host
Source
Secondary
Wavelength
antiserum
(nm)
FITC
—
Primary
5-
1:100
Serotonin
Rabbit Abcam
HT†
1:500
Acetylcholine Rabbit Genway FITC’
ACh‡
1:100
Rabbit IgG
Goat
Abcam
—
488
Secondary
1:500
Rabbit IgG
Goat
Abcam
—
488
FITC
FITC’
†Monoclonal Antibody
‡ Polyclonal Antibody
—
11
Acetylcholine-Containing Neuroepithelial Cells in Fish Gills
Figure1. Schematic diagram illustrating the distribution of O2 sensitive
chemoreceptors in different vertebrate groups. Note that evolutionarily, vascular
arches and their chemoreceptor groups were reduced and internalized among the
species. (Fig from Burleson and Milsom, 2003)
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Acetylcholine-Containing Neuroepithelial Cells in Fish Gills
Figure 2. Simple schematic diagram of the Reflex Arc. The O2 chemoreceptor
cell is stimulated in response to changing levels of O2. An electrical signal is
relayed to the CPG/CRG centers in the brain where a signal is sent to the
effector muscle to initiate compensatory actions of the cardio-ventilatory
system.
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Acetylcholine-Containing Neuroepithelial Cells in Fish Gills
Figure 3. Immunolabeling of serotonin (5-HT) in Ictalurus punctatus along a single gill
filament with multiple lamellae at 20x (top left), single lamella on gill filament at 40x
(top right), and a single filament distal tip at 20x (bottom left) and 64x (bottom right).
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Acetylcholine-Containing Neuroepithelial Cells in Fish Gills
Figure 4. Immunolabeling of acetylcholine (ACh) in Ictalurus punctatus at the distal
tips of a single gill filament at 20x (top), along the gill filaments and lamellae with
localization in distal tips at 5x (bottom left), and along the proximal end of multiple
gill filaments and lamellae at 5x (bottom right).
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