Acetylcholine-Containing Neuroepithelial Cells in Fish Gills Support the Cholinergic Hypothesis of O2 Chemoreception

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. 5-HT-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.

Table of Contents: 

    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 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 O2-sensing 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 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 cardio-ventilatory 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).

    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.

    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 cardio-ventilatory 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 oxygen chemoreception. The data presented here adds one more piece to the puzzle and provides further support to the cholinergic hypothesis.

    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, 231-238.
    • 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.

    Table 1: Details of primary and secondary antibodies used for immunohistochemistry

    Antiserum Dilution Antigen Host Source Secondaryantiserum Wavelength(nm)
    Primary5-

     

    HT†

    ACh‡

    Secondary

    FITC

    FITC’

    1:100

     

    1:500

    1:100

    1:500

    Serotonin

     

    Acetylcholine

    Rabbit IgG

    Rabbit IgG

    Rabbit

     

    Rabbit

    Goat

    Goat

    Abcam

     

    Genway

    Abcam

    Abcam

    FITC

     

    FITC’

     

    488

    488

     

    †Monoclonal Antibody         ‡ Polyclonal Antibody

    Figure 1: 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)

    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.

    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).

        

        

    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).