TASTE CELL HETEROGENEITY AND GABA NEUROTRANSMISSION IN FACIAL AND VAGAL NERVE INNERVATED TASTE BUDS OF CHANNEL CATFISH, ICTALURUS PUNCTATUS by Mojgan Eram A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Physiology The University of Utah August 2004
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TASTE CELL HETEROGENEITY AND GABA NEUROTRANSMISSION
IN FACIAL AND VAGAL NERVE INNERVATED TASTE BUDS
OF CHANNEL CATFISH, ICTALURUS PUNCTATUS
by
Mojgan Eram
A dissertation submitted to the faculty of The University of Utah
in partial fulfillment of the requirements for the degree of
This dissertation has been read by each member of the following supervisory committee and by majority vote has been found to be satisfactory.
6 Uo!o1
C I/b/OL(-I I
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Chair: William C. Michel
�b� Ma T. Lucero �
� �� Kenneth W. Spitzer
THE UNIVERSITY OF UTAH GRADUATE SCHOOL
FINAL READING APPROVAL
To the Graduate Council of the University of Utah:
I have read the dissertation of Mojgan Erarn in its final form and have found that (1) its format, citations, and bibliographic style are consistent
and acceptable; (2) its illustrative materials including figures, tables, and charts are in place; and (3) the final manuscript is satisfactory to the supervisory committee and is
ready for submission to The Graduate School.
Date William C. Michel
Chair: Supervisory Committee
Approved for the Major Department
����--. -----�Fldone
Chair/Dean
Approved for the Graduate Council
d2�--. David S. Chapman
Dean of The Graduate School
ABSTRACT
Taste buds are comprised of group of 50-150 elongated taste cells grouped
together in a tulip-shaped structure predominantly located on the tongue and soft palate.
Tens of thousands of taste buds occupy the lingual epithelium of most vertebrates and
provide for the detection, selection and consumption of food. Catfish have a greatly
elaborated gustatory system with taste buds distributed over the entire body surface and
barbels, as well as the oropharyngeal cavity. These extra-oral taste buds are innervated
by the facial nerve and are important in the search for food. By contrast, oropharyngeal
taste buds innervated by vagal and glossopharyngeal nerves determine the acceptability
of food for consumption. Differences in the sensitivity of facial and vagal nerves to
amino acid stimuli have been demonstrated in several electrophysiological studies, which
lead us to their detailed examination for the underlying morphological differences. Early
studies classified taste cells into two to three types based on electron microscopical
properties, while recent studies with functional and histochemical markers point to a
more diverse taste cell population. The present project evaluates taste cell heterogeneity
using immunochemical staining, image analysis and multispectral classification
techniques to determine the distribution of several small molecular weight metabolites
including y-aminobutyric acid (GABA), glutamate, aspartate, alanine, taurine and
glutathione. Unique levels of expression are typical for each substrate in the facial
(FITBs) and vagal nerve innervated taste buds (VITBs). High levels of GABA occur in a
a prominent subset of taste cells, which suggests the possibility of GABAergic signaling
in catfish FITBs and VITBs. Collectively, the findings confirm the existence of basic
cytochemical and morphometric differences between the FITBs and VITBs, which could
account for differences in functional sensitivity and behavioral patterns. In particular, the
presence of GABA in diverse subsets of taste cells strongly suggests an important role for
GABAergic mechanisms in gustatory neuromoldulation of transduction-transmission in
the channel catfish.
v
To my parents
for inspiring me, encouraging me and giving me the wings to fly
TABLE OF CONTENTS
ABSTRACT ....................................................................................................................... iv
LIST OF FIGURES ........................................................................................................... ix
LIST OF TABLES ............................................................................................................. xi
ACKNOWLEDGMENTS ................................................................................................ xii
Morphology of Vertebrate Gustatory System ..................................................... 2 Innervation of the Gustatory System .................................................................. .4 Physiological Evidence for the Differences in Facial and Vagal Nerve Innervated Taste Buds .............................................................................. 5 Neurotransmission in Taste Cells ........................................................................ 6 Hypothesis and Specific Aims ............................................................................ 7 References ........................................................................................................... 9
2. MORPHOLOGICAL AND BIOCHEMICAL HETEROGENEITY IN FACIAL AND VAGAL NERVE INNERVATED TASTE BUDS OF THE CHANNEL CATFISH, ICTALURUS PUNCTATUS ......................................... 14
3. CLASSIFICATION OF FACIAL AND VAGAL NEREVE INNER V A TED TASTE CELLS OF CHANNEL CATFISH USING METABOLITE PROFILES ........................................................................... 52
Summary of Findings ...................................................................................... 128 Basic Morphometric Properties ....................................................................... 129 Taste Cell Heterogeneity ................................................................................. 130 Evidence for GABAergic Signaling in the Peripheral Taste System .............. 131 Future Directions ............................................................................................. 133 Hypothetical Model for the Role of GABA in Peripheral Gustatory System ............................................................................................................. 133 References ....................................................................................................... 136
VIl1
LIST OF FIGURES
Figure Page
2.1 Facial (FITBs) and vagal nerve innervated taste buds (VITBs) are found in high densities on the barbels and gill arches of channel catfish respectively ............................................................................................................ 23
2.2 The metabolite profile of a FITB ........................................................................... 27
2.3 The metabolite profile of a VITB .......................................................................... 29
204 Pixel intensity histograms reveal differences in the average metabolite profiles across nine FITBs (gray traces) and nine VITBs (Black traces) .............. 31
2.5 Morphological and metabolite characteristics of the basal cell, companion cell and nerve plexus ........................................................................... 32
2.6 The many distinctly colored cells in the color composite images indicate heterogeneous metabolite distributions in taste cells of both A,C: FITBs and B,D: VITBs ......................................................................... 37
2.7 The distribution of GABA in taste cell apical processes is shown in in registered overlays of electron micrographs (red) and images of GABA IR (blue) of non-osmicated A: FITB and B: VITB ............................. ..... 041
3.1 Longitudinal and cross sections through the mid portion of a FITB and VITB stained with anti-GAB A IgG ................................................................ 61
3.2 Cross sections through the apex and nerve plexus of a FITB and VITB stained with anti-GABA IgG ................................................................................. 63
3.3 Normalized pixel intensity distributions from the six FITBs (black) and six VITBs (gray) examined reveal significant differences in the overall staining profiles for some metabolites ................................................................... 66
304 The metabolite profile of mid portion of a facial nerve innervated taste bud (FITB) ............................................................................................................. 67
3.5 The metabolite profile of mid portion of a vagal nerve innervated taste bud (VITB) ............................................................................................................. 69
3.6 Cells in different clusters have a diverse metabolite profile .................................. 73
3.7 The metabolite profiles of the 15 cell clusters identified by the k-means cluster analysis are plotted ..................................................................................... 76
3.8 The radial distribution of taste cells in different clusters differs ........................... 83 4.1 Immunoblot analysis of epithelial samples from the maxillary barbel
confirms that antibodies for GAD65, the a3 subunit of GABAAR and GAT-2 recognize proteins of the correct molecular weight ................................ 105
4.2 GABA immunoreactivity (IR, blue) is superimposed on an electron micrograph (red) of a A: FITB and B: VITB ...................................................... 106
4.3 Both isoforms of glutamic acid decarboxylase (GAD) are found in FITBs and VITBs ................................................................................................. 1 07
4.4 Two isoforms of the GABAA receptor are differentially expressed in taste buds .......................................................................................................... 110
4.5 Three isoforms of the GABA transporter, A, B: GAT-I, C, D: GAT-2 and E, F: GAT-3, are expressed in FITBs and VITBs ........................................ 113
4.6 Patterns of co-localization of GAD65 (in red) with A: GAD67, B: GABAAR a1 subunit, C: GABAAR a3 subunit, D: GAT-I, E: GAT-2 andF: GAT-3 shown in green in FITB ................................................... 116
4.7 Patterns of co-localization of GAD65 (in red) with A: GAD67, B: GABAAR a1 subunit, C: GABAAR a3 subunit, D: GAT -1, E: GAT-2 and F: GAT-3 shown in green in VITB .................................................. 117
5.1 Diagram of one possible model for the action of GABA in taste cells ................ 135
x
LIST OF TABLES
2.1 Morphometric characteristics of facial (FITBs) and vagal nerve innervated taste buds (VITBs) of catfish ............................................................... 25
3.1 Significant differences among clusters based on pairwise comparisons (p>O.05) .................................................................................................................. 79
3.2 The distribution of FITB and VITB cells based on their GABA immunoreactivity pattern ....................................................................................... 81
ACKNOWLEDGMENTS
This project was funded by NIH DC01418, NS07938 and Willard L. Eccles
Charitable Foundation grants. Many people have contributed to this dissertation, and I
am greatly thankful to all. I would like to thank my committee, William Michel, Larry
Stensaas, Mary Lucero, Robert Marc and Ken Spitzer, for their advice and guidance
along the way. Many thanks to Mike Michel for teaching me to become a scientist and a
better thinker; Larry Stensaas for his critical review of my manuscripts, his brilliant ideas
and many stimulating talks; Mary Lucero as the graduate student adviser and her helpful
suggestions; Robert Marc for his vast knowledge of the field and technical assistance. I
greatly appreciate Ken Spitzer for inspiring and encouraging me to never give up.
I would like to thank Robert Marc and Signature Immunologics for the gift of
anti-GABA antibody, and other antibodies respectively, Nancy Chandler for her
assistance in electron microscopy, Kathleen Davis for her humor, friendship and cryostat
immunocytochemistry, and Ann Greig for her technical help with the Western blot
analysis. I thank David Lipschitz and Bryan Jones for their friendship and technical
support. I thank all the personnel at Michel's lab for their help. Additionally, I thank
everyone at the Department of Physiology for their support and friendship.
Above all, I thank my family for their love, support and sacrifices. I thank my
friends for keeping me busy on powder days on the slopes. I.specially would like to
thank my friend Steve Denkers for his support and guidance. Finally and foremost, I
would like to thank the Willard L. Eccles Charitable Foundation for supporting my
research project ... I am extremely gratefu1.
xiii
CHAPTER!
INTRODUCTON
2
Morphology of Vertebrate Gustatory System
The gustatory system is involved in the detection and evaluation of food in all
vertebrates. Specialized receptor cells for the detection of taste stimuli are typically
situated in the epidermal lining of the oral cavity, lips, tongue and pharynx in pear-shaped
structures known as taste buds. In some fish, taste buds are distributed over the entire
body surface in addition to the oral cavity (Hirata, 1966; Reutter, 1978; Reutter, 1982;
Reutter, 1986), and a large body of literature describes the comparative morphology and
structure of the gustatory system (review Herrick, 1901; Murray, 1971; Murray, 1973;
Kapoor et aI., 1975; Jakubowski, 1983; Reutter, 1986; Roper, 1989; Jakubowski and
Whitear, 1990). Taste receptors are elongated cells, which are exposed to the external
environment apically and make synaptic contact with primary afferent neurons near the
base of the taste bud. They are surrounded by perigemn1al cells laterally (Knapp et aI.,
1995) some of which may function as stem cells (Reutter, 1971), while the horizontal
basal cells are implicated in mechanosensory function (Hirata, 1966).
Two or three types of elongated taste cells have been identified in fish based on
their ultrastructure: light, dark and intermediate cells (for review see Royer and
and Kanner, 2002). Three types of GATs have been examined immunocytochemically in
rat taste buds (Obata et aI., 1997). GAT-2 was mainly present in nerve fibers beneath the
lingual epithelium, whereas GAT -3 was expressed in a subset of cells in the margin of
taste buds. The significance of such differences in expression are not known, but suggest
unique functional roles for each of these transporters (Schousboe and Kanner, 2002).
In the central nervous system, the action of GABA as an inhibitory
neurotransmitter is mediated mainly through type A (GABAA) postsynaptic receptors.
The GABAA receptor (GABAAR) is a ligand gated, anion permeable pentameric
structure, whose high affinity GABA binding site is located on the a subunit and is
essential for its proper function (review (Wisden and Farrant, 2002). Activation of the
receptor by GABA increases cr conductance and reduces cell excitability (Macdonald
and Haas, 2000). The properties of GABAAR are known to be modulated by
benzodiazepines and barbiturates.
Hypothesis and Specific Aims
The taste buds of vertebrates are complex sensory structures whose cellular
constituents regenerate. Behavioral studies in fish support the notion that facial and vagal
innervated taste buds are involved in different aspects of feeding (Atema, 1971) and have
different sensitivities to amino acid stimuli (Kanwal and Caprio, 1983; Kanwal et aI.,
1987; Kohbara et aI., 1992). This study investigates structural diversity and biochemical
heterogeneity among taste cells in FITBs and VITBs of the channel catfish, Ictalurus
8
punctatus, which has a well-developed system of taste buds located over the entire body
surface as well as in the oral cavity (Caprio et aI., 1993). Although morphological and
histochemical studies have revealed differences in the two taste cell types found in catfish
(Royer and Kinnamon, 1996), detailed characterization of taste cell heterogeneity
remains largely unexplored. Furthermore, there are no comparative studies of cell
morphology and metabolite taste cell heterogeneity in other fish species. We
hypothesize that the heterogeneity among the taste cells and associated facial and
vagal sensory axons are the major determinants of functional differences in the two
gustatory systems.
Some vertebrate taste cells have been shown to contain the inhibitory
neurotransmitter GABA (Jain and Roper, 1991; Obata et aI., 1997; Eram and Michel,
2001a). Our preliminary studies demonstrate significant differences in GABA
immunoreactivity between the FITBs and VITBs of channel catfish (Eram and Michel,
2001a). We hypothesize the existence of a GABA signaling pathway with a possible
inhibitory function in facial and vagal nerve innervated taste buds of catfish. To test
these hypotheses we propose the following specific aims:
In Chapter 2 we will determine whether immunocytochemical differences exist
among the taste buds and taste cells of in FITBs and VITBs by the standard
morphomertic analysis and immunocytochemical characterization of GABA, L
glutamate, L-aspartate, L-alanine, taurine and glutathione immunoreactivity. Electron
microscopy in conjunction with immunochemistry will be used to localize these
components to specific cells or regions in the taste bud.
9
In Chapter 3 we will characterize heterogeneity in the metabolic profiles of the
taste cells of facial and vagal nerve innervated taste cells utilizing plastic section
immunocytochemistry and cell classification based on metabolic profiles. Such
differences in the distribution of six metabolites mentioned above may shed light on
functional differences noted electrophysiologically in sensory axons of the two gustatory
nerves.
In Chapter 4 we will examine the distribution of components involved in GABA
signaling. In the first part, we quantitatively compare patterns of GABA
immunoreactivity. In the second part, we examine the presence and distribution of
components necessary for GABA neurotransmission including glutamic acid
decarboxylases (GADs), GABA transporters (GATs) and GABAAR subunits using
immunocytochemistry. We will further confirm the presence of these components by
Western blot analysis. The distribution of these key components of GABA signaling will
provide essential clues as to the mode of involvement of GABA in gustatory
neurotransmission and will be the first comprehensive study of potential structural
differences and biochemical heterogeneity in FITBs and VITBs of catfish.
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Erlander MG, Tillakaratne NJ, Feldblum S, Patel N, and Tobin AJ. 1991. Two genes encode distinct glutamate decarboxylases. Neuron 7:91-100.
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Fujimoto S and Yamamoto K. 1980. Electron microscopy of terminal buds on the barbels of the silurid fish, Corydoras paleatus. The Anat Rec 197:133-141.
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Grover-Johnson N and Farbman AI. 1976. Fine structure of taste buds in the barbel of the catfish, lctalurus punctatus. Cell Tissue Res 169:395-403.
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Knapp L, Lawton A, Oakley B, Wong L, and Zhang C. 1995. Keratins as markers of differentiated taste cells of the rat. Differentiation 58:341-349.
Kohbara J, Michel W, and Caprio J. 1992. Responses of single facial taste fibers in the channel catfish, Ictalurus punctatus, to amino acids. J Neurophysiol 68:1012-1026.
12
Legay F, Pelhate S, and Tappaz ML. 1986. Phylogenesis of brain glutamic acid decarboxylase from vertebrates: immunochemical studies. J Neurochem 46:1478-1486.
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13
Roper SD. 1993. Synaptic interaction in taste buds. In Simon SA and Roper SD, editors. Mechanisms of taste transduction. Boca Raton: CRC. p 275-293.
Roper SD. 1989. The cell biology of vertebrate taste receptors. Annu Rev Neurosci 12:329-353.
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CHAPTER 2
MORPHOLOGICAL AND BIOCHEMICAL HETEROGENEITY
IN FACIAL AND VAGAL NERVE INNERVATED
TASTE BUDS OF CHANNEL CATFISH,
ICTALURUS PUNCTATUS
15
Abstract
In catfish, the facial nerve innervates taste buds distributed over the entire body
including the barbels, while the glossopharyngeal and vagal nerves innervate
oropharyngeal taste buds. Facial nerve innervated taste buds (FITBs) are thought to be
involved in food detection and localization, while glossopharyngeal and vagal nerve
innervated taste buds (VITBs) evaluate the palatability of food prior to ingestion.
Physiological studies indicate that both oral and extra-oral taste buds detect sapid
substances such as an1ino acids and nucleotides, but the facial taste system is more
sensitive to some of these substances. The anatomical, molecular and/or physiological
mechanisms underlying functional differences in these two gustatory pathways remain to
be identified. In the current investigation we compare the basic morphological features
of FITBs and VITBs and the distribution of the following metabolites: y-aminobutyric
acid (GABA), glutamate, aspartate, alanine, taurine and glutathione. Vagal nerve
innervated taste buds are significantly longer and narrower than FITBs with fewer taste
cells and a smaller nerve plexus. Each of the metabolites examined was heterogeneously
distributed in taste cells with notably more GABA positive cells present in the VITBs.
Patterns of metabolite co-localization suggest the presence of several taste cell SUbtypes.
The morphological and metabolite differences noted between FITBs and VITBs provide
a potential anatomical basis for the previously noted higher sensitivity of the facial nerve
to amino acid stimuli.
16
Introduction
Catfish are sensitive to a wide variety of gustatory stimuli, such as amino acids
and nuc1eotides (Atema, 1971; Marui and Caprio, 1992; Caprio et aI., 1993). The catfish
facial taste system is distributed over the entire body sUlface and is primarily involved
with detection and localization of food (Bardach et aI., 1967; Atema, 1971). The vagal
and glossopharyngeal nerves, innervating taste buds in the oropharyngeal cavity and gill
arches, are primarily involved with the acceptance and ingestion of food.
Electrophysiological studies suggesting that the two gustatory pathways are
physiologically different demonstrated that vagal nerve innervated taste buds (VITBs)
have higher thresholds for the amino acids alanine and arginine than facial nerve
innervated taste buds (FITBs) (Kanwal and Caprio, 1983; Kanwal et aI., 1987). Potential
structural and molecular differences in VITBs and FITBs that might underlie the
observed physiological differences in sensitivity have not been examined.
Taste cell heterogeneity has been demonstrated genetically, histochemically and
physiologically in a variety of vertebrate species. Basic histology and electron
microscopy revealed the presence of two to three taste receptor cell types (Farbman,
1965; Murray, 1971; Murray 1973). Light cells are generally thought to be the taste
receptor cells while dark cells are presumed to serve a supporting role. Recent studies
using a variety of neuronal markers such as NCAM (Snlith et aI., 1993; Nelson and
Finger, 1993; Smith et aI., 1994; Takeda et aI., 1999; Yee et aI., 2001), calbindin
(Johnson et aI., 1992; Miyawaki et aI., 1996; Miyawaki et aI., 1998), enkephalins,
Substance P (Bensouilah and Denizot, 1991; Welton et aI., 1992; Huang and Lu, 1996),
neuron specific enolase (Ganchrow, 2000; Yee et aI., 2001), serotonin (Bensouilah and
17
Denizot, 1991; Jain and Roper, 1991; Welton et aI., 1992; Delay et aI., 1993; Kim and
Roper, 1995; Huang and Lu, 1996; Hamasaki et aI., 1998; Nagai et aI., 1998; Yee et aI.,
2001), protein gene product 9.5 (Yee et aI., 2001) and taste tissue specific markers such
as taste receptors (Montmayeur and Matsunami, 2002) and a-gustducin (Yang et aI.,
2000) suggest the existence of additional taste receptor cell subtypes. In mice expressing
OFP under the gustducin promoter, co-localization of OFP and the cell surface markers,
antigen H, antigen A and NCAM permitted live-cell identification of three cell types
(Medler et aI., 2003). Electrophysiological heterogeneity was found within a single class
of these taste cells. Although these studies have shown that taste cells are
histochemically and functionally heterogeneous, they do not yet provide a single, unified
classification system.
A recent alternative strategy to classifying cells is to examine the co-occurrence
of common small molecular weight compounds such as amino acids and peptides. Cell
classification based on such metabolite profiles has proven particularly useful in the
identification of retinal neurons (Marc et aI., 1995). Our preliminary studies have shown
that co-localization of a dozen or more substances is feasible in taste cells, thus providing
a detailed metabolite profile for use in cell classification (Eram and Michel, 2001 b). In
the current investigation, we first compare the basic morphology of FITBs, from the
maxillary barbel, and VITBs, from the 2nd through 5th gill arches. We then examine the
metabolite profiles of FITB and VITB taste cells to see whether the taste cell populations
of these two gustatory pathways are metabolically similar. The battery of metabolites
includes putative excitatory (glutamate and aspartate) and inhibitory (y-aminobutyric
acid, OABA) neurotransmitters, other amino acids (alanine and taurine) and a tripeptide
18
(glutathione). Our results not only reveal significant differences in the taste bud
morphology, including the numbers of taste cells, but they also delineate more metabolite
heterogeneity in channel catfish taste cells than has previously been reported.
Materials and Methods
Animal Care
Six juvenile catfish, lctalurus punctatus, (5-12 grams, 7-10 cm total length, sex
unknown) obtained from commercial sources, were held in recirculating 40-80 liter
aquaria (26-28°C) under a 12-hour light and 12-hour dark light cycle, and fed frozen
mosquito larvae daily. All experimental procedures have been approved by the
Institutional Animal Care and Use Committee of the University of Utah.
Tissue Preparation
Animals were decapitated, the maxillary barbels and gill arches dissected out in
cold fish Ringers solution (concentrations in mM: 137.0 NaCI, 2.0 KCI, 1.8 CaCh, 5.0
Hepes, 10.0 glucose, pH 7.4), and transferred to cold fixative (1 % paraformaldehyde,
2.5% glutaraldehyde, 3% sucrose, 0.01 % CaCh in 0.1 M phosphate buffer (PB), pH 7.4),
overnight at 4°C. All of the antibodies used in the current investigation were prepared
against small metabolites conjugated to bovine serum albumin by glutaraldehyde fixation
(Marc et aI., 1988; 1990; 1995), hence glutaraldehyde fixation of the tissue ensured
quantitative capture of the metabolites and generation of the appropriate antigenic sites.
Fixed tissue was dehydrated through graded (50%, 75%, 85%, 95%, and 100%) methanol
(or ethanol for electron microscopy) and 100% acetone (20 minutes each), agitated in
19
50% acetone and 50% Eponate Plastic (Ted Pella Inc., Redding, CA, USA) overnight,
transferred through two rinses of fresh 100% Eponate, oriented in embedding blocks and
cured overnight at 65°C. Ultra-thin (50-70 nm; visually silver-gray) serial sections were
collected using a Leica Ultracut T microtome (Leica Inc., Bannockburn, IL, USA) and
diamond knife (Delaware Diamond Knife, Inc., Wilmington, DE, USA). Individual
serial sections were either situated in wells of Teflon-coated spot slides (Erie Scientific,
Portsmouth, NH, USA) for light microscopy or placed on formvar-coated gold slot grids
for electron microscopy.
Immunocytochemistry
In accord with previously described post-embedding immunocytochemical
procedures (Marc, Wei-Ley, Kalloniatis, Raiguel, and Van Haesendonck, 1990), sections
were deplasticized with 25% sodium ethoxide (saturated sodium hydroxide in absolute
ethanol, 7 minutes), rinsed in 100% methanol (3 x 2 minutes each), rinsed in ultra-pure
water (5 minutes), and dried. Individual consecutive sections were incubated overnight at
room temperature in a humidified chamber with one of the following primary polyclonal
glutathione (GSH, 1:4000 dilution), anti-L-alanine (ALA, 1:8000 dilution), and anti-L
aspartate (ASP, 1 :2000 dilution) (Signature Immunologics Inc., Salt Lake City, UT,
USA) diluted in 0.1 M PB containing 1 % goat serum and 0.05% thiomerosal (pH 7.4).
Dot blot analysis indicates that the aspartate, GABA, glutamate and taurine antibodies are
at least 1000 fold less cross-reactive to other structurally-related antigens (Marc et aI.,
1988; 1990; 1995). Similarly low cross-reactivity is reported by the manufacturer for
GSH and alanine. Elimination of primary antibody and incubation of the tissues with
only the secondary antibody resulted in no immunoreactivity (IR). Following a rinse
20
with 0.1 M PB, sections were incubated in nanogold-conjugated goat anti-rabbit
secondary antibody (1 nm, 1:50 dilution; Amersham Corp., Arlington Heights, IL, USA)
for 1 hour at room temperature, rinsed with 0.1 M PB for 1 hour, and silver intensified for
3 minutes at 32°C using 0.14% silver nitrate in a hydroquinone (43 mM)/citrate buffer
(64 mM, citric acid; 141 mM sodium citrate) solution (Kalloniatis and Fletcher, 1993),
Following silver intensification the slides were cover slipped using cover glass and
Eponate plastic then cured at 65°C overnight.
Electron Microscopy
The aldehyde fixed tissue to be used for electron microscopy was osmicated in
2% osmium tetroxide (Sigma, St. Louis, MO, USA) (2 hours) at room temperature, and
rinsed with 0.1 M PB (3 x 20 minutes), except osmium tetroxide was omitted from tissue
prepared for use in both light and electron microscopy. lTltrathin serial sections were
stained with 3% uranyl acetate in distilled water (45 minutes) followed by Reynold's lead
citrate (20 minutes) and examined with a Hitachi H-7100 electron microscope (Hitachi,
San Jose, CA, USA) and photographed using Kodak 4489 film. Sixteen-bit, gray-scale
digital images of the transmission electron microscopy (TEM) negatives were obtained
using Microtek ScanMaker 5 scanner and Adobe Photoshop 6.0 software (Adobe
Systems Inc., San Jose, CA, USA).
21
Image Acquisition, Registration and Analysis
Taste buds, viewed with a Zeiss Axioplan2 microscope and a 100x oil immersion
lens, were captured as 8-bit gray scale digital images (1300 x 1030 pixels) using a CCD
canlera and Zeiss-Axiovision imaging software 3.0 (Thornwood, NY, USA). All the
images were captured using identical camera settings and illumination in a single session.
Each of the immunostained or TEM images of a taste bud was aligned with a reference
image of TAU IR using image analysis software (Geomatica software 8.0, PCI Remote
Sensing, Richmond Hill, Ontario, Canada). Sets of identical structures were selected to
seed a first order polynomial fitting algorithm that aligned and scaled (for the TEM
image) the images. A color composite image was formed by designating each amino acid
or TEM image as the red, green or blue (RGB) channels of an RGB image (Marc et aI.,
1995; Marc and Liu, 2000). For the co-localization of metabolites, the gray scale images
were inverted so that pixel intensities varied over the range of 0 (lowest) to 255 (highest),
respectively. For display, the raw gray-scale images were automatically contrast adjusted
by redistribution of their intermediate pixel values proportionately and portions of the
image not containing tissue were digitally cleaned-up by using Adobe Photoshop 6.0
(Adobe Systems Inc., San Jose, CA, USA).
Morphometric measurements were made using the Zeiss-Axiovision 3.0 or
Image-Pro Plus 4.0 software (Media Cybernetics Inc., Silver Spring, MD, USA) using
only those taste buds with an open taste pore and complete plexus and peduncle regions.
Taste buds meeting the above criteria were selected at random from the distal end of the
maxillary barbels and 2nd through 5th gill arches of six catfish. Taste bud length was the
distance measured fronl the pore opening to the basal cell; taste bud width was taken at
the widest point, typically just above nerve plexus. Total taste bud area was calculated
by tracing the perimeter of the entire taste bud including the nerve plexus and basal
cell(s). Nerve plexus area was measured similarly. A t-test for independent samples
(p<0.05; SPSS 11.0, SPSS Inc., Chicago, IL, USA) was used to establish significant
differences.
22
To quantify immunoreactivity in the nine FITBs and nine VITBs examined,
masks were generated using Image-Pro Plus 4.0 software (Media Cybernetics Inc., Silver
Spring, MD, USA) to restrict the analysis to cells within the taste buds. A histogram of
the number of pixels at each of the 256 pixel intensity levels was calculated for each
metabolite for each taste bud and exported to Microsoft Excel. For each histogram, the
bin counts for each pixel intensity level for each taste bud were normalized to the bin
with the highest count generating a proportion of maximum value ranging from 0 to 1 for
each bin. These normalized pixel counts were then averaged across the nine FITBs and
nine VITBs to produce an average pixel intensity distribution for each of the metabolites.
Results
General Morphometries of Facial and Vagal Nerve
Innervated Taste Buds
The shape of taste buds in the stratified squamous epithelium of maxillary barbels
or gill arches is generally similar (Fig. 2.1). A cross-section through the maxillary barbel
(Fig. 2.1A) reveals a central cartilaginous spine, several blood vessels, large anterior and
small posterior nerve bundles, and numerous taste buds. An FITB with an open taste
pore is shown at high magnification, which has facial nerve fibers entering the taste bud
23
Figure 2.1. Facial (FITBs) and vagal nerve innervated taste buds (VITBs) are found in high densities on the barbels and gill arches of channel catfish respectively. A: A crosssection through a maxillary barbel stained with toluidine blue reveals six taste buds (.), large nerve bundles and the cartilaginous spine. B: A gill raker on a gill arch also has six taste buds (.). C: FITB at high magnification (boxed area in A) is ovoid with a prominent nerve plexus (NP), single basal cell (*), and peduncle (P). D: VITBs are elongated and have a small nerve plexus and peduncle. E, epithelium; NB, nerve bundle; C, cartilage; R, gill raker; TC, taste cells; ., taste bud. Portions of the image not containing tissue were digitally cleaned up. Scale bars = 100 J.Lm in A and B, 10 J.Lm in C andD.
24
via a nerve peduncle or corium papillae, along with a well developed nerve plexus and
throughout the epithelium and around the nerve peduncle. Vagal innervated taste buds in
sections of gill rakers also had numerous taste cells, though large nerve bundles and
blood vessels were less common (Fig. 2.1C, D). The elongate VITBs had a less
pronounced nerve plexus and smaller peduncle.
Measurements from nine VITBs and nine FITBs (three taste buds from each of
three fish) revealed the tulip-shaped VITBs to be significantly shorter in width but
significantly longer than pear-shaped FITBs (Table 2.1, see Fig. 2.1). Although the
diameter of the pore openings and total areas of FITBs and VITBs were not significantly
different, the area of the nerve plexus of FITBs was significantly larger than VITBs (t
test, p<O.05). Cell counts, made from cross sections through six barbel and six gill arch
taste buds, revealed that there were significantly more taste cells in the FITBs than
VITBs (t-test, p<O.05).
Patterns of Metabolite Immunoreactivity
The distribution of several cellular metabolites was examined
immunocytochemically in VITBs and FITBs. The metabolites examined included the
putative neurotransmitters, GABA, glutamate and aspartate, two other amino acids,
alanine and taurine and the tripeptide glutathione. All of the metabolites were present in
both VITBs (Fig. 2.2) and FITBs (Fig. 2.3) but there were qualitative and quantitative
differences in their distribution patterns.
GABA exhibited the most diverse pattern of immunoreactivity of any of the
25
Table 2.1. Morphometric characteristics of facial (FITBs) and vagal nerve innervated taste buds (VITBs) of catfish. X is the mean, ±SD is the standard deviation, and N is the number of taste buds used for each measurement. * cell counts were made from cross section images of taste buds stained with anti-GABA antibody. TB, taste bud.
F acial-innervated Vagal-innervated
Taste Buds (FITBs) Taste Buds (VITBs)
- -X ±SD (N) X ±SD (N) Significance
Width (J.1m) 51.6 4.7 (9) 43.5 6.2 (9) p<0.05
Length (J.1m) 52.3 4.4 (9) 61.7 4.3 (9) p<O.Os
Pore opening
(J.11U) 10.1 3.6 (9) 8.5 3.3 (8) NS
Area (J.1m2) 1830.2 211.7 (9) 1686.9 296.9 (9) NS
Plexus area
(J.1m2) 269.9 33.0 (9) 159.2 51.4 (9) p<O.Os
Cell #fI'B 216.3* 34.0 (6) 135.0* 19.2 (6) p<0.05
26
metabolites examined in the taste bud (Fig. 2.2A and Fig. 2.3A). Individual taste cells
ranged from GABA negative to GABA positive; many had processes, which entered the
taste pore. Pixel intensity histograms of the average GABA IR of the nine VITBs and
nine FITBs examined revealed the presence of significantly more GABA-positive
structures in VITBs (Fig. 2.4A). In VITBs, the two peaks in the histogram, at pixel
intensity values of approximately 50 and 180, reflected the large classes of GABA
negative and GAB A-positive structures, respectively. The FITBs showed a similar
pattern of GABA-negative structures but far fewer GABA-positive structures (note the
small peak at ... 200). A broad shoulder in the FITBs histogram from 100 to 175
represented GABA-intermediate cells. Visual inspection of the images of GABA IR
confirmed that there are more GABA-rich taste cells in the VITBs and more GABA
intermediate taste cells in FITBs (Fig. 2.2A and Fig. 2.3A). In the basal half of the taste
bud, the more numerous GABA-negative taste cells were enveloped by GABA-rich
processes suggesting that the GAB A-positive cells are primarily supporting cells (dark
cells). Many fine processes in the peduncle and plexus showed GABA IR. Basal cells
(BC) in both FITBs and VITBs were GABA-rich.
A unique cell type was located just below the basal lamina in both FITBs and
VITBs peduncle (Fig. 2.5). We proposed the name "companion cell" (CC) because of its
close association with the basal cell. The companion cell had a flat shape, similar to the
basal cell, with processes running parallel to the basal cell and basal lamina (Fig. 2.5A
B). This cell had an oval flattened nucleus and abundant rough endoplasmic reticulum
(rER), resulting in a darker cytoplasm than the basal cell. Many mitochondria were also
present in the cytoplasm of the companion cell. Inspection of electron micrograph
Figure 2.2. The metabolite profile of a FITB. Six 50 nm thick serial sections through the mid-portion of a FITB compare immunoreactivity for A: GABA, B: glutamate (GLU), C: aspartate (ASP), D: alanine (ALA), E: taurine (TAU) and F: glutathione (GSH). Putative dying cells are designated with asterisks. BC, basal cell; CC, companion cell; other abbreviations as in Figure 2.1. Scale bar = 10 J,lm.
27
28
29
Figure 2.3. The metabolite profile a VITB. Six 50 nm thick serial sections through the mid-portion of a VITB compare immunoreactivity for A: OABA, B: glutamate (OLU), C: aspartate (ASP), D: alanine (ALA), E: taurine (TAU) and F: glutathione (OSH). Putative dying cells are designated with asterisks. BC, basal cell; CC, companion cell; other abbreviations as in Figure 2.1. Scale bar = 10 Jlm.
30
A B
D F
1.0 A.GABA B.GLU C.ASP 1.0 1.0
0.8 0.8
0.6 0.6
..... 0.4 0.4 0.4 s::: ::s 0 0.2 0.2 0.2 U
'"a) 0.0 :;..::
~ 0 50 100 150 50 100 150 200 250
] 1.0 D. TAU E.ALA F.GSH
~ 1.0 1.0
0.8 0.8 0.8
0 Z 0.6 0.6 0.6
0.4 0.4 0.4
0.2 0.2 0.2
0.0 0.0 0 250 0 100 150
Relative Concentration
Figure 2.4. Pixel intensity histograms reveal differences in the average metabolite profiles across nine FITBs (gray traces) and nine VITBs (black traces). The average normalized pixel counts and standard errors are plotted for A: GABA, B: glutamate, C: aspartate, D: taurine, E: alanine and F: glutathione. Differences in taste bud area were corrected for by normalizing the data for each metabolite for each taste bud to the bin containing the maximum pixel counts.
31
32
Figure 2.5. Morphological and metabolite characteristics of the basal cell, companion cell and nerve plexus. A: An electron micrograph image of an osmicated basal region of a FITB shows the relative positioning of a basal cell and companion cell. The companion cell lies parallel to the basal cell and basal lamina (BL, arrow). Both cells are diskshaped and oriented horizontally to the longitudinal axis of taste bud. B: The magnified (x 7000) portion of the basal cell and companion cell shown in (A, boxed area) shows no intrusion of companion cell into the basal lamina (arrow) and no direct contact between the basal cell and companion cell. C: An electron micrograph image of a non-osmicated FITB was registered to consecutive sections stained for OABA (light pink color) and OLU (purple color) IR to show the position of the companion cell relative to the basal cell and basal lamina (arrow). The basal cell has high OABA and lower OLU IR, whereas the companion cell has the opposite profile. The companion cell nucleus is small, oval and dark, whereas the basal cell nucleus is large, irregular and lighter. Each image was slightly contrast adjusted. Be, basal cell; ee, companion cell; NP, nerve plexus; BL, basal lamina; P, peduncle. Scale bar = 10 J..lm (A and B), 1 J..lm (e).
33
images revealed no intrusion of this cell through the basal lamina, and no direct cell-to
cell contact between the companion cell and basal cell (Fig. 2.SA, B). We did not
observe any sign of contact between this cell and the surrounding nerves or other
elements in the peduncle. In contrast to the basal cells, the companion cells had low
GABA lR (Fig. 2.SC).
34
Most taste cells had very low L-glutamate (GLU) and L-aspartate (ASP) levels
but occasional GLU- and ASP-positive cells, located in the basal half of the taste bud,
were noted (Fig. 2.2B, C and Fig. 2.3B, C). The highest levels of these amino acids were
found in the peduncle and nerve plexus. However, in all taste buds examined there were
more GLU lR than ASP IR structures in the plexus and peduncle regions. Basal cells
generally had low to intermediate GLU lR and intermediate ASP lR, while the
companion cell had high GLU lR and intermediate ASP lR. Glutamate IR in the plexus
and basal cell/companion cell region is shown in Figure 2.SC. Pixel intensity histograms
of the average GLU lR were similar in both FITBs and VITBs except for a shoulder
between ISO and 180 in VITBs (Fig. 2.4B), which presumably reflected the presence of
more GLU positive taste cells in VITBs (see Fig. 2.3B). Pixel intensity histograms were
similar for ASP lR in FITBs and VITBs (Figure 2.4C).
L-alanine (ALA) lR was intermediate in most taste cells and the companion cells
(Fig. 2.2D and Fig. 2.3D). However, many elongated taste cell processes extending to
the taste pore had high ALA lR, particularly in VITBs, and appeared to arise from a few
darkly stained cells. L-alanine positive processes were observed in nerve plexus. Basal
cells had relatively high ALA IR. A right shift in the ALA pixel intensity histogram of
35
VITBs, compared to the FITBs, indicated that the relative ALA IR was slightly higher in
VITBs (Fig. 2.4E). Most taste cells with high ALA IR also had high GABA IR.
SHghtly higher TAU levels were observed in VITBs than FITBs (Fig. 2.2E and
Fig. 2.3E). Most of the taste cells had high or intermediate levels of TAU IR, but a few
elongated cells with somas located in the basal region of taste buds, particularly in FITBs,
were TAU negative (Fig. 2.2E and Fig. 2.3E). Some of the TAU negative cells contained
significant levels of other metabolites but cells with low levels of TAU and also lacking
other metabolite IR were considered dying cells (asterisks). Taurine-positive processes
were found in the nerve plexus. The basal cells and companion cells had high levels of
TAU IR. Heterogeneous TAU IR was noted in the peduncle. In FITBs, the pixel
intensity histograms identified a peak at low concentrations (-50) and a larger peak at
higher concentrations (-180) (Fig. 2.4D). The relatively large error values associated
with each pixel intensity bin indicated a highly variable number of TAU-negative cells
across the individual FITBs. In VITBs, a large peak around 180 and a smaller peak at
220 represented elements with high TAU IR and the long shoulder represented the TAU
intermediate or negative structures.
Glutathione (GSH) IR was generally low throughout FITBs and VITBs (Fig. 2.2F
and Fig. 2.3F). A few cells located towards the center of the buds with elongated
processes reaching the taste pore had the highest GSH IR. In general, the nerve plexus,
basal cells and peduncle had low GSH IR. However, the companion cells, particularly in
FITBs, were GSH positive. Pixel intensity histograms of GSH IR were generally similar
in FITBs and VITBs with the small shoulder on the FITB distribution, perhaps reflecting
the companion cell labeling (Fig. 2.4F).
Comparison of Cellular Heterogeneity in FITBs and VITBs
Each of the six metabolites exhibited diverse patterns of immunoreactivity
suggesting that cells within channel catfish taste buds were chemically heterogeneous.
36
As each of the consecutive sections used for the immunocytochemistry was only 50-70
nm thick, it was possible to examine the patterns of metabolite co-localization in single
cells (average diameter - 6 /J.m) using precisely registered gray scale images mapped into
the individual channels of an RGB image. Full examination of all possible patterns of co
localization for the six metabolites would require inspection of 20 different RGB color
images. As a first approximation, we generated two sets of RGB color images from
inverted, registered gray scale images. In the first image, GABA, GLU and TAU IR
were mapped into the red, green and blue channels, respectively (Fig. 2.6A, B). The
second color image examined ALA (red), GSH (green) and ASP (blue) IR (Fig. 2.6C, D).
For each set of images, distinct colors represented unique patterns of metabolite co
localization. Since registration error might have generated a false impression of co
localization in extremely small structures, we restricted our evaluation of metabolite co
localization to taste cells, basal cells and companion cells.
Several relatively distinct patterns of GABA, GLU and TAU co-localization were
evident in taste cells (Fig. 2.6A, B). Taurine, as the most abundant amino acid in taste
buds, was reflected by the presence of many blue cells. Many of these cells were GABA
negative and the shades of blue, ranging from deep blue to aqua to green-blue, reflected
variation in the levels of TAU IR and GLU IR. TAU and GABA co-localization was
indicated with hues ranging from pink to purple. Pink cells contained high GABA and
Figure 2.6. The many distinctly colored cells in the color composite images indicate heterogeneous metabolite distributions in taste cells of both A,C: FITBs and B,D:
37
VITBs. Color composite images display metabolite co-localization patterns for GABA (red), GLU (green) and TAU (blue) IR (A, B) or ALA (red), GSH (green) and ASP (blue) IR (C, D). Similarly colored taste cells in both FITBs and VITBs suggest that both gustatory pathways contain cells with comparable metabolite profiles. Examples of putative dying cells are designated with asterisks. Gray scale images of each metabolite for these two taste buds are shown in Figure 2. Details concerning the production of these images can be found in the Methods. Portions of the image not containing tissue were digitally cleaned-up. BC, basal cell; CC, companion cell; TC, taste cell; NP, nerve plexus. Scale bar = 10 Jlm.
Green - Glutamate Blue - Taurine
Green - Glutathione Blue - Aspartate
38
39
intennediate TAU levels, whereas purple cells contained relatively higher TAU and
lower GABA levels. GABA and TAU co-localization was particularly noticeable at the
apical region of taste buds, where pink processes were mixed among the aqua-blue
primarily TAU IR processes. Yellowish cells, indicating co-localization of GABA and
GLU in TAU-negative cells, gave rise to a few yellow processes in the apical half of taste
buds. Green taste cells, representing primarily GLU IR cells, were generally absent.
Only one cell in all of the taste buds examined had GLU IR without either TAU or
GABA IR (data not shown). In VITBs, the perigemmal cells around the taste buds had
high levels of GABA, GLU and TAU IR and were white; perigemmal cells with similar
metabolite profiles were not observed in FITBs. The pale pink color of basal cells was
indicative of high GABA levels and slightly lower GLU and TAU levels (Fig. 2.6A, B).
The light blue companion cell with relatively high TAU and GLU levels and low GABA
IR had a pattern of co-localization that was most similar to the squamous epithelial cells.
When examining the codistribution of ALA, GSH and ASP, the abundant red
cells and red processes extending to the taste pore, particularly in VITBs, were an
indication of relatively higher ALA levels than either ASP or GSH (Fig. 2.6C, D). Co
localization of ALA and GSH was noted by the presence of a few purple cells. A few
green cells and numerous green apical processes were rich in GSH but lacked appreciable
ALA and ASP. Blue ASP rich cells were found in three different FITBs. In VITBs, the
ALA signal (red) was stronger and was expressed in more cells than in FITBs. An
abundance of yellow, orange and pink hues reflected variable levels of ALA and GSH
co-localization. A few, possibly dying cells had virtually no metabolites at all and as a
result were nearly black in both metabolite triplet images (asterisks). The pink
appearance of basal cells indicated that ALA was the strongest signal. Although the
pattern of metabolite co-localization of the companion cells generally matched the
squamous epithelial cells, significantly lower GSH and higher ASP levels in gill arch
tissues resulted in distinctly different patterns between FITBs and VITBs.
Association of GAB A Immunoreactivity with Cell Type
40
Hansen and colleagues (2002) have previously identified three taste cell types in
zebrafish based on microvillar structure. Receptor cells were described as having either a
single thick villus or several intermediate thickness microvilli (brush-like), while
supporting cells had numerous short microvilli. We examined GABA IR in the apical
region of taste cells in an attempt to determine if GABA IR in taste cells was associated
with one or more cell types. When images of GABA IR were registered to electron
micrographs we noted that GABA co-localized with supporting cells bearing short
microvilli and in some taste cells with only a single thick microvillus (Fig. 2.7 A, B). Not
all cells with a single thick microvillus were GABA positive further suggesting
heterogeneity within the taste receptor cell population. Too few cells with several
intermediate thickness microvilli were observed to draw any conclusions concerning their
GABAIR.
Discussion
Facial and vagal taste pathways of catfish subserve different behavioral functions
(Atema, 1971) and have physiologically distinct properties (Kanwal and Caprio, 1983).
In the current study, we have established that taste buds innervated by the facial branch of
41
Figure 2.7. The distribution of GABA in taste cell apical processes is shown in registered overlays of electron micrographs (red) and images of GABA IR (blue) of nonosmicated A: FITB and B: VITB. Dark cells with numerous small microvilli and some (arrow), but not all (arrowheads), light cells possessing a single large nlicrovillus were GABA IR. A cell with several intermediate microvilli was not GABA IR (A, asterisks). Insets show the GABA distribution in the entire taste bud. Portions of the image not containing tissue were digitally cleaned up. Scale bar = 10 f.lm.
42
the 7th cranial nerve, or the vagal nerve (10th cranial nerve) differ both in general
morphological features and metabolite composition. Although FITBs were, on average,
wider and shorter than VITBs, the overall morphological properties of taste buds
innervated by either cranial nerve were generally similar. Previously, three distinct taste
bud morphologies (I - III) were described in Xiphophorus helleri Heckel (Reutter, 1973),
the blind cave fish Astyanaxjordani and sighted river fish Astyanax mexican us (Boudriot
and Reutter, 2001). All the taste buds evaluated from channel catfish maxillary barbels
resembled the type II (slightly elevated) classification of Reutter (1973), Gill arch taste
buds were observed with short peduncles and apical openings either even with the
epithelial surface (type III) or very slightly elevated (type II). More significant
morphological differences between FITBs and VITBs included the area of the nerve
plexuses and the nUDlber of taste cells; both significantly greater in FITBs. In a
comparative study of blind and sighted fish of the same genus, the larger plexus area of
the blind cavefish Astyanax jordani compared to the sighted river fish Astyanax
mexican us was suggested to be a compensatory adaptation to cave dwelling (Boudriot
and Reutter, 2001). Thus, in channel catfish the enlarged FITB plexus area and higher
number of taste cells per taste bud might be adaptations for increased detection
capabilities and would be consistent with previous electrophysiological data suggesting
heightened sensitivity of the facial taste system (Kanwal and Caprio, 1983). The few
previous studies comparing fish oropharyngeal and external taste buds generally do so
without identification of the cranial nerve innervating the buds. Therefore, it is difficult
to determine if the morphological differences we observed are a general feature of fish
taste buds or are unique to the channel catfish. In the study of two loach species, (Cobitis
43
taenia and Misgumus jissilis), oropharyngeal taste buds located on the gill arches, which
we might assume to be innervated by either the glossopharyngeal or vagal nerves, were
found to be morphologically similar to external taste buds (Jakubowski, 1983) suggesting
that differentiation between these gustatory pathways may not be a general feature of
fish. However, further investigation is warranted.
The FITBs had significantly nlore taste cells and a larger plexus area than did
VITBs. Since the nerve plexus is the primary area of connectivity between taste cells
and afferent nerve fibers, the additional basal processes of FITB cells entering the plexus
would be expected to increase the plexus area. An increase in afferent nerve innervation
might also contribute to an increased plexus area. Within the nerve plexus intense OLU
IR (and to a lesser extent ASP IR) was noted in numerous small processes. In both
FITBs and VITBs of the channel catfish, the relative abundance of these OLU- and ASP
positive structures in the plexus and in peduncle and the presence of very few OLU- or
ASP-positive taste cells in the taste bud suggest that these metabolites are primarily
associated with afferent nerve fibers rather than taste cells. The high level of OLU is not
unexpected since these afferent nerves are likely to use glutamate as the excitatory
neurotransmitter in the facial and vagal lobes. In other species, OLU-positive nerve
is present (Caicedo et aI., 2000), and afferent gustatory nerve input to the brain has been
shown to be glutamatergic (Bradley and Orabauskas, 1998), including gustatory input to
the vagal lobe of goldfish (Smeraski et aI., 2001). The presence of more OLU-positive
facial nerve fibers entering the plexus of FITBs might also contribute to the increased
sensitivity of the facial taste system as measured by integrated nerve recordings (Kanwal
44
and Caprio, 1983). Whether glutamate also serves as a neurotransmitter in the channel
catfish taste bud, perhaps used by the taste cells with high glutamate levels, remains to be
determined.
GABAergic nerve fibers are present in taste buds of Necturus (Jain and Roper,
1991) and GABAergic fibers and taste cells are present in taste buds of mice (Obata et
aI., 1997). However, GABA IR has not previously been reported in fish. We note
GABA is present in several distinct cell types in both FITBs and VITBs. A few taste
cells with a single stout microvillar process, thought to be taste receptor cells (Hansen et
aI., 2002), and all basal cells had GABA IR, but GABA IR was most prominent in dark
cells whose processes enveloped and wrapped around light cells. Dark cells are
comnlonly thought to act as supporting cells (Hirata, 1966; Fujimoto and Yamamoto,
1980; Jakubowski and Whitear, 1990), but others suggest they serve a sensory capacity
(Reutter, 1978; Joyce and Chapman, 1978; Reutter, 1986). While its physiological role
in peripheral gustatory processing remains to be established, if GABA serves an
inhibitory role in taste buds, as it does elsewhere in the nervous system, the higher levels
of GABA in VITBs (see Fig. 2.3A) may contribute to the higher thresholds of afferent
nerves innervating these taste buds (Kanwal and Caprio, 1983). However, synaptic
connectivity within the taste bud remains poorly understood and further study is required
to determine if GABAergic mechanism(s) contribute substantially to the differential
sensitivities of the facial and vagal gustatory systems.
Basal cells, located below the nerve plexus and separated from companion cells
by the basal lamina, were present in both FITBs and VITBs of the channel catfish. All of
the fish species so far examined have basal cells on oropharyngeal taste buds, but some
45
lack basal cells on external taste buds (Jakubowski and Whitear, 1990). Several possible
functions have been suggested for the basal cell. Its Merkel cell-like properties lead to
suggestions that the basal cell has a mechanosensory function (Reutter, 1971). On the
other hand, its strategic location below the nerve plexus, high neurotransmitter levels
(Reutter, 1971; Toyoshima, 1989; Jain and Roper, 1991; Nagai et aI., 1998; Eram and
Michel, 2001a), and the presence of synaptic connections with afferent nerves and taste
cells lead to speculation that the basal cell may serve in neuromodulatory capacity
(Reutter, 1971). In addition to GABA, the relatively high GLU level we report in the
basal cell adds to the list of neuroactive substances, but offers little additional insight
concerning basal cell function. While the associated companion cell is evident in
electron micrographs of fish taste buds (Royer and Kinnamon, 1996); Boudriot and
Reutter, 2001), it has not been previously identified as a distinct cell type. Although the
term "companion cell" has been used in plant biology (Van Bel et aI., 2002) to describe
the inner most cell layer of outer root sheath of vertebrate hair follicles (Orwin, 1971; Ito,
1986; Ito et aI., 1986), we opted for this term due to the close association between the
basal cells, basal lamina and companion cells. The companion cell metabolite profile of
low GABA and high GLU levels was distinctly different from the basal cell, but was
similar to other cells found in the peduncle and to epithelial cells. The functional
properties of this cell remain to be determined.
Elongated taste cells have been classified by electron microscopy and
histochemical staining as either light or dark cells (Farbman, 1965; Murray, 1971;
Murray, 1973). While there is still insufficient information to definitively assign a
function to either cell type, the fact that processes of dark cells branch to envelop the
46
light cells suggests they serve a supporting role. We observed that the majority of dark
cells had intermediate to high GABA IR and similarly high ALA and TAU levels. In
contrast to the uniform metabolite profiles of dark cells, light cells, generally assumed to
be receptor cells, have heterogeneous metabolite distributions. The few GABA-negative,
GLU, ASP, GSH or ALA positive cells identify cell subtypes with distinct metabolite
profiles. Additional heterogeneity is evident when patterns of metabolite co-distribution
are considered. At minimum, five distinctly colored taste cells can be seen in the
composite metabolite image of GABA, GLU and TAU IR (Fig. 2.6A) suggesting
considerable heterogeneity in the taste cell population. Included in this heterogeneity are
cells with extremely low metabolite levels that are likely degenerating; dying cells have
previously been reported in taste buds (Crisp et aI., 1975). Whether the observed
heterogeneity represents distinct cell types each with specific functional properties or is
simply associated with the metabolic and/or developmental state of a functionally similar
population of cells is unknown but is currently under investigation. Our finding of
similar metabolite profiles in fish received and processed on different dates suggests that
cellular metabolite profiles are not random. However, it is also likely that progenitor
cells, immature receptor and supporting cells have different metabolite profiles from
those of mature or senescent cells.
Conclusions
In this study we investigated the patterns of metabolite distribution and
morphometric properties of the FITBs and VITBs of channel catfish. The larger nerve
plexus and higher number of taste cells per taste bud, and lower levels of GABA in the
47
FITBs may contribute to the higher sensitivity of these taste buds to amino acid stimuli,
compared to VITBs. Future physiological studies are needed to understand the
significance of the differences in metabolite patterns of FITBs and VITBs.
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Smith DV, Klevitsky R, Akeson RA, and Shipley MT. 1994. Expression of the neural cell adhesion molecule (NCAM) and polysialic acid during taste bud degeneration and regeneration. J Comp Neurol 347:187-196.
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CHAPTER 3
CLASSIFICATION OF FACIAL AND VAGAL NERVE
INNERVATED TASTE CELLS OF CHANNEL
CATFISH USING METABOLITE
PROFILES
53
Abstract
Input from the three gustatory nerves of vertebrates is used to evaluate the
nutritional quality of food. In some species, these cranial nerves are modified to
accomplish additional specific functions. The distribution of a highly sensitive facial
taste system over the entire body surface of catfish aids food search, and physiological
studies indicate that this external facial system is more sensitive to amino acids than
either the glossopharyngeal or vagal systems of the oral cavity. The current investigation
examines the heterogeneity of receptor elements in taste buds innervated by the facial
nerve and vagal nerve (VITBs) of the channel catfish, letalurus punetatus. The
distributions of five amino acid metabolites, alanine, aspartate, glutamate, 'Y-aminobutyric
acid (GABA), taurine and the tripeptide glutathione in taste cells were quantified
immunocytochemically and metabolite profiles of 2118 individual cells, subjected to k
means clustering, identified 15 classes of cells with quantitatively different patterns of
metabolite co-localization. About 9% of total cells, grouped into four classes, had high
levels of GAB A immunoreactivity. Two classes of GABA intermediate cells contained
17% of total cell population and the remaining nine classes had low GABA levels and
contained 74% of cells. Although there is significant heterogeneity among catfish taste
receptors, cells of similar metabolite profiles are found in both VITBs and FITBs.
Introduction
Vertebrates employ gustatory nerves to evaluate the nutritional quality of food.
Ontario, Canada). Taurine IR served as the reference image to which all other images
were registered. Sets of identical structures were selected in each image to seed a first
order polynomial fitting algorithm which aligned the images. To better evaluate the
correlation among the metabolite contents of the cells, triplet images were formed by
designating gray scale images of individual metabolites as the red, green or blue (RGB)
58
channel of an RGB image (Marc et aI., 1995; Marc and Liu, 2000). For the co
localization of metabolites, the gray scale images were inverted so that pixel intensities
varied over the range of lowest (0) to highest (255) concentration, respectively. For
display, the raw gray-scale images were automatically contrast adjusted by proportionate
redistribution of their intermediate pixel values using Adobe Photoshop 6.0 (Adobe
Systems Inc., San Jose, CA, USA).
Metabolite Quantification
To compare metabolite IR levels in FITBs and VITBs an area of interest (AOI)
was created for each taste bud that included the taste cells and perigemmal cells using
Image Pro Plus 4.0 software (Media Cybernetics Inc., Silver Spring, MD, USA) and the
pixel intensity histograms for each metabolite were exported to a spreadsheet (Microsoft
Excel). For each metabolite in every taste bud, the number of pixels in each of the 256
pixel intensity levels was normalized to the bin containing the largest number of pixels,
producing a normalized distribution ranging from zero to one. The normalized
distributions for each metabolite were averaged for the six FITBs and the six VITBs (±
standard error of the mean) and presented graphically. The final pixel intensity
distributions were inverted so that pixel intensity level varied with relative metabolite
concentrati on.
Taste Cell Classification
Precise registration of serial ultrathin (50 nm) sections allowed evaluation of the
six metabolites in individual cells over a 300 nm overall thickness. Quantitative analysis
59
of the metabolite profiles of individual taste cells employed a fixed-diameter circular AOI
over each cell using Image Pro Plus 4.0 software (Media Cybernetics Inc., Silver Spring,
MD, USA) to measure the average pixel intensity values of each of the six metabolites.
A k-means clustering analysis (SPSS 10, SPSS Inc., Chicago, IL, USA) was used to
group the 2118 taste cells obtained from the six maxillary barbels (FITBs, N=1303) and
six gill arches (VITBs, N=815) based on similarities in metabolite profiles. To
adequately describe the diversity in metabolite profiles, preliminary analyses with final
solutions of 6, 9, 12, 15 and 18 clusters were compared, and multivariate analysis of
variance (P<0.05) was performed to establish significant differences in metabolite
intensity for pairs of cell groups.
To find the spatial distribution of taste cells in each of the 15 cell clusters, and to
determine the relative location of cells with respect to the center of a taste bud, the center
and average diameter of each taste bud was determined using the center mass and
diameter mass functions in Image Pro Plus 4.0 software. With this information and the
location of the center of the AOI for each of the cells we calculated the radial distribution
of each cell. A value of zero indicates that the cell was located precisely at the center of
the bud while a value of one indicates its location at the periphery.
Results
We have previously shown that FITBs and VITBs contain heterogeneous
populations of taste cells based on small molecular weight metabolite immunostaining
(personal communication, Eram and Michel, in review). In the current study, horizontal
sections were used to formally classify cells to determine the relati ve abundance of each
60
cell type in FITBs and VITBs. The morphology of a typical catfish FITB and VITB in
longitudinal section stained for GABA and location of the analyzed sections is shown in
Figures 3.1A, B. In addition to numerous unstained taste cells elongated GAB A positive
taste and perigemmal cells were situated above the GABA positi ve basal cell (BC) and
nerve plexus (NP) containing GABA positive processes. The greatest number of taste
cells was seen in the central region (Fig. 3.1C, D) approximately 10 J..lm above the nerve
plexus in which elements unstained for GABA predominate. Sections through the apical
region (Fig. 3.2A, B) also contained numerous taste cells but the cross sectional area of
each cell was much smaller making analysis more difficult. In the nerve plexus near the
base of taste buds (Fig. 3.2C, D) a complex mixture of receptor cells and numerous small
processes of afferent nerve fiber were observed.
To illustrate how three levels of GABA IR could be delineated we Hposterized"
the image of a FITB and VITB (Fig. 3.1E, F) by setting pixels with intensity values of 90
or lower to white (0), values between 90 and 175 to gray (127) and values above 175 to
black (255). In general there were few GABA high cells (Fig. 3.1E, F, black cells)
interspersed among the GABA low and GABA intermediate cells. In contrast to the
round profiles of GABA high and GABA low cells, irregular thin GABA intermediate
processes (Fig. 3.1C, D, arrows) filled the space between the round profiles. Plots of the
normalized pixel counts for the six FITBs and six VITBs are shown in Figure 3.3, which
revealed a higher proportion of the total pixels with intermediate or high levels of GABA
in the VITBs (Fig. 3.3A). In VITBs, peaks in the pixel intensity distributions at
approximately 50 and 190 represented the two large classes of GABA low and GABA
high cells, respectively. GABA intermediate cells in VITBs ranged from 90 to 175. In
61
Figure 3.1. Longitudinal and cross sections through the mid portion of a FITB and VITB stained with anti-GABA IgG. A, B: Longitudinal sections through a FITB and VITB showing the location of sections (a, b, c) used for taste cell classification. Taste cells (TC), a prominent nerve plexus (NP) and a GABA-positive basal cell (BC) are noticeable in the images. C, D: Cross section through the wide midportion of a FITB and VITB contains the cell bodies and processes of most taste cells. Sections within the 10 Jlm distance from the NP (line marked as a in A and B) were collected for classification. E, F: three distinct levels of GABA IR are illustrated in "posterized" FITB and VITB shown in C and D in which the image was "posterized" by designating pixels intensity values (PIV) of 175 - 255 (GABA high), 90 - 175 (GAB A intennediate) and 90 or less (GABA low) to 255, 127 and 0 respectively. Note that the three level posterized images provide near identical representation of actual midlevel image of GABA IR. Scale bar = 10 JlM.
FITB A
F c=J 0-90
90-175
_175-255
62
VITB
63
Figure 3.2. Cross sections through the apex and nerve plexus of a FITB and VITB stained with anti-GABA IgG. A, B: Cross section through the apex of a FITB and VITB illustrates numerous GABA high processes wrapped around the GABA intennediate and GABA low taste cells. A few GABA high cells are noticeable in the cross sections. These sections were collected from the region above the line designated as c in Figure 3.1A, B. C, D: Basal cross section through the nerve plexus (NP), collected from the regions marked as b in Figure 3.1A, B, shows numerous GABA positive fibers in the center of the section and a few GABA high taste cells (TC) in the periphery. Scale bar = 10 JlM.
64
D
VITB
65
FITBs, the GABA low classes had the same pixel intensity value as the VITB cells, but
peaks representing the GABA intermediate and high cells were much smaller and located
around 175 and 200, respectively.
Taurine IR was heterogeneously distributed across the taste cell populations of
both FITBs (Fig. 3.4B) and VITBs (Fig. 3.5B). In contrast to GABA IR, the majority of
taste cells had high TAU IR with only a few cells devoid of TAU. The small populations
of TAU-negative cells were represented by the small peaks around 40 in the pixel
intensity distributions (Fig. 3.3B). The large peaks in the TAU pixel intensity
distributions at 160 and 190 for VITBs and FITBs, respectively, revealed that FITB cells
had higher TAU concentrations. The broad shoulders between the two peaks suggested
cells with a continuum of TAU concentrations.
The majority of taste cells had low levels of GSH IR. However a few taste cells
containing high levels of GSH were located centrally in both FITBs (Fig. 3.4C) and
VITBs (Fig. 3.5C). Examination of the GSH pixel intensity distributions in Figure 3.3C
did not reveal distinct peaks that might have represented unique subpopulations of GSH
IR cells in either FITBs or VITBs. The distribution for the FITBs was broad and
relatively symmetric with a peak centered around 80. The distribution for the VITBs
peaked at around 50 with a broad shoulder extending to higher GSH concentrations. No
distinct peaks were evident at higher pixel intensity values for either VITBs or FITBs.
Although a few ALA low and ALA high cells were noted, most FITB (Fig. 3.4D)
and VITB (Fig. 3.5D) cells had intermediate levels of ALA. The pixel intensity
distributions for ALA in FITBs and VITBs were similar with a single peak around 120
for VITBs and 130 for FITBs (Fig. 3.3D). A very small shoulder at about 175, in VITBs
Figure 3.3. Nonnalized pixel intensity distributions from the six FITBs (black) and six VITBs (gray) examined reveal significant differences in the overall staining profiles for some metabolites. The average value and standard error of the mean are plotted for A: OABA, B: TAU, C: OSH, D: ALA, E: OLU, F: ASP.
250
250
67
Figure 3.4. The metabolite profile of the mid portion of a facial nerve innervated taste bud (FITB). For each taste bud, serial 50 nm sections were probed with primary rabbit polyclonal antibodies for A: GABA, B: TAU, C: GSH, D: ALA, E: GLU, F: ASP. Each of the antibodies has a detection threshold of approximately 50 J..LM and saturates at an antigen concentration of 10-20 mM. Scale bar = 10 J,lM.
68
69
Figure 3.5. The metabolite profile of the mid portion of a vagal nerve innervated taste bud (VITB). For each taste bud, serial 50 nm sections were probed with primary rabbit polyc1onal antibodies for A: GABA, B: TAU, C: GSH, D: ALA, E: GLU, F: ASP. Each of the antibodies has a detection threshold of approximately 50 JlM and saturates at an antigen concentration of 10-20 mM. Scale bar = 10 J..lM.
70
VITB
pixel intensity histograms, revealed the presence of a small class of cells containing
higher ALA levels.
71
Intermediate levels of IR predominated in preparations stained for GLU or ASP
(Fig. 3.4E, F, and Fig. 3.5E, F). The pixel intensity distributions in Figure 3.3E, F
depicting GLU and ASP IR showed that taste cells had generally higher levels of GLU,
peaks at about 125 for FITBs, and 115 for VITBs, than ASP (-- 85 for FITBs and 65 for
VITBs). A small peak at high concentration was noted in the ASP pixel intensity
distribution (Fig. 3.3E). The GLU pixel intensity distribution for VITBs showed a
shoulder between 150 and 175, suggesting a class of cells with high GLU IR (Fig. 3.3F).
This shoulder was absent in the FITBs.
Taste Cell Classification
The use of registered ultrathin (50 nm) sections allowed examination of
metabolite co-localization in individual cells. The average metabolite levels of the 2118
taste cells examined from six FITBs (N = 1303 cells) and six VITBs (N = 815 cells) were
subjected to k-means classification to determine if groups of cells with unique metabolite
profiles could be identified. The appropriate number of clusters adequately describes the
heterogeneity in the taste cell population without pooling distinct cell types or splitting
cells with similar profiles into separate clusters but the number of clusters must be
specified before the analysis. To estimate the appropriate number of clusters, we ran the
analysis for 6, 9, 12, 15 and 18 clusters, and the average pixel intensities for each of the
metabolites for each cluster were calculated. Multivariate analysis of variance was used
to determine if the average metabolite concentrations of each cluster were significantly
72
different from the other clusters. For any pair of clusters to be considered significantly
different, only one of the six metabolites concentrations had to be significantly different.
The analysis yielding 15 clusters was adopted because it identified three additional
clusters that were significantly different from each other but were combined with other
clusters in 12-cluster analysis. The 18-cluster solution split several large clusters of
similar cells.
Sets of triplet RGB images of GABA (red), TAU (green) and GSH (blue) IR
(Figure 3.6A) and ALA (red), GLU (green) and ASP (blue) IR (Figure 3.6B) show cells
belonging to the clusters 1, 6, 9, 10, 13 and 15. These cells were selected because they
had average pixel intensity levels closest to the mean values for their entire cluster. Note
that although differences of colors in the two triplet images allowed some discrimination
of cell types, the quantification shown in figure 3.6C more clearly illustrated their
metabolite profiles. Each graph displays the average pixel intensities for a single
metabolite for each of the six cells; thus looking down a column provides the metabolite
profile for a cell. Although other metabolite differences existed, the relatively high
GABA IR (-200) of cell CI was sufficient to discriminate it from all other cells.
Likewise, cell C6 with intermediate levels of GAB A (120) could be discriminated from
cells C9, CIO, CIS and CI3, which had low GABA IR «80). Elevated GLU in cell CI
relative to cell C6 further discriminated these two cells. The four cells with low GABA
IR were discriminated by TAU IR (cell CI3 from cells C9, CIO, CIS) and GSH IR (cell
CIO from cells C9 and CIS). Finally, elevated levels of TAU, ALA and GLU IR in cell
C9 compared to cell CIS discriminated these two cells.
73
Figure 3.6. Cells in different clusters have a diverse metabolite profile. The FITB gray scale images shown in Figures 3.1C and 3.4 were used to form triplet images of A: R = GABA, G = TAU, B = GSH, and B: R = ALA, G = GLU, B = ASP. Each of the images was inverted and mapped into the appropriate channel of the RGB image but otherwise not manipulated. Six taste cells, belonging to clusters 1, 6, 9, 10, 13 and 15, are circled in both images. C: The average pixel intensity histograms of the six cells representing the clusters show a GABA high cell (Cl), GABA intermediate cell (C6) and GABA low cells (C9, 10, 13, and 15). Scale bar = 10 J,.LM.
K-means cluster analysis resulted in the classification of the 1303 FITB and 815
VITB cells into 15 clusters with significantly different metabolite profiles (p<0.05,
multivariateANOVA; Fig. 3.7). Each row presents the average pixel intensities for a
single metabolite for all 15 clusters. The average metabolite profile for a given cluster
can be seen by looking down a column. The top row, showing the average pixel intensity
values (API) of GABA IR, has been sorted from highest to lowest concentration.
Significant differences in GABA IR splited the 15 clusters into three main groups,
clusters 1, 2, 3 and 4, with GABA API values of about 170, represented GABA high
cells, clusters 5 and 6 with GABA API values of about 120 represented the GABA
intermediate cells and the remaining nine clusters (7-15) with GABA API values of :580
represented the GABA low cells.
Differences in the relative concentrations of other metabolites discriminated
clusters within these three main groups. Within the four GABA high clusters, significant
differences in TAU IR distinguished cells in Cluster 4 from cells in Clusters 1-3. Cluster
3 differed from Clusters 1 and 2 on the basis of high ASP and low ALA IR, Clusters 1
and 2 were distinguished on the basis of significant differences in TAU and GLU IR.
The two clusters containing cells with intermediate levels of GABA IR were
distinguished from each other on the basis of GLU IR. Significant differences in TAU IR
were critical for cluster forn1ation of the GABA negative cells. The low TAU IR of
Clusters 13 and 14 discriminated these two clusters from the other seven GABA low
clusters of cells. Significantly higher ASP IR separated cells in Cluster 14 from cells in
Cluster 13. High TAU IR separated Clusters 9 and 10 from the remaining five clusters
with intermediate TAU IR. Higher GSH IR distinguished cells in Cluster 10 from cells in
76
Figure 3.7. The metabolite profiles of the 15 cell clusters identified by the k-means cluster analysis are plotted. The metabolite profile of a cell cluster can be seen by looking down a column. The clusters were sorted according to GABA concentration. Quantification of immunoreactivity was done on inverted images so pixel intensity levels increased with increasing metabolite concentration. A total of 1303 FITB and 815 VITB cells (NTotal = 2118) were classified by the analysis.
Having established that metabolite profiling found quantitatively different clusters
of taste cells, we sought to examine the spatial distribution of cells within each cluster.
The distributions of taste cells within a cluster were examined by determining the
normalized distance from the center of the bud for each cell in each cluster. With a
normalized radius of 1, a radial distance of approximately 0.7 was required to divide a
taste bud into equivalent areas. Thus, distributions centered at 0.7 had cells distributed
throughout the bud, those centered below 0.7 were centrally located and those centered
above 0.7 as laterally distributed. Based on these criteria, cells in Clusters 1, 2, 4, 10, 11,
13, 14 and 15 were relatively uniformly distributed throughout the bud (Fig. 3.8). The
FITB cells of Clusters 6 and 12 were more centrally located, while these clusters in the
VITB cells were more uniformly distributed. Cells in Clusters 5, 7, 8 and 9 were found
more frequently along the margin of the taste bud. In Cluster 5 the bias was particularly
strong and found for cells in both FITBs and VITBs.
Discussion
The metabolites we examined perform diverse cellular functions in addition to
their role in intermediate metabolism. Glutamate generally is accepted to be an
excitatory neurotransmitter (Watkins, 2000), and alanine is present in some central
GABAergic neurons (Aiuchi et aI., 1976; Schousboe et aI., 2003) and may serve as a
precursor for the production of GABA via the GABA shunt (Schousboe et aI., 2003).
Taurine is an abundant, semi-essential amino acid that is found in variety of excitable
83
Figure 3.8. The radial distribution of taste cells in different clusters differs. The distribution ofFITB (open bars) and VITB (filled bars) cells in 14 of the 15 clusters (cluster 3 was not plotted because there were only two cells), No significant differences were noted in the distribution of cells in a given cluster between the two gustatory pathways.
Hansen et aI., 2002), but only two have been recognized in catfish (Royer and Kinnamon,
1996). Non-neuronal supporting cells have dark cytoplasm, an irregular nucleus, a large
87
number of free ribosomes, microfilaments, tonofilaments, an extensi ve supranuclear golgi
apparatus, a few mitochondria, and numerous, short, apical microvillar processes (Royer
and Kinnamon, 1996). Light cells, the presumptive sensory cells, have a single stout
apical microvillar process, a moderately electron-dense ovoid nucleus, an electron-lucent
cytoplasm containing many mitochondria, smooth endoplasmic reticulum, free
ribosomes, microtubules and intermediate filaments, typical of neurons (Royer and
Kinnamon, 1996; Boudriot and Reutter, 2001). The third taste cell type containing an
ovoid nucleus, numerous dense-core-vesicle cells has an apical surface, which terminates
in either small microvilli or undi vided thick villus and is similar to both light and dark
cells. The function of the third cell type is less clear, although the concentration of clear
and dense-core vesicles near the base suggests involvement in the sensory function of
taste bud (Boudriot and Reutter, 2001). These three cell types probably correspond to the
type I, II and III taste cells described in mammalian taste buds (Farbman, 1965; Murray,
1969; Murray, 1973; Kinnamon et aI., 1985). However, since neither the light/dark nor
the types I-III classification are used consistently, cross species comparisons are difficult
(Finger and Simon, 2000).
Though not widely used in fish, markers of certain macromolecules provide
evidence of taste cell diversity and, frequently, insight into function. T2R receptors for
bitter (Matsunami et aI., 2000; Adler et aI., 2000), and TIR receptors for sweet/amino
acid stimuli (Hoon et aI., 1999; Kitagawa et aI., 2001; Montmayeur and Matsunami,
2002) are expressed in distinct subsets of cells in the same taste bud. The taste cell
specific G-protein a-subunit, a-gustducin (Wong et aI., 1996; Chandrashekar et aI.,
2000) is also heterogeneously distributed in taste cells and co-localizes in some taste cells
88
with either T1R (sweet) or T2R (bitter) receptors adding further diversity to the
mechanisms of gustatory transduction (Hoon et aI., 1999). Rat type I cells are marked by
blood group H antigen (Pumplin et aI., 1999), a-gustducin labels a subset of type II cells,
while NCAM is specific to a subset of type III cells (Yang et aI., 2000; Yee et aI., 2001).
A recent electrophysiological study of these three types of taste cells found functional
diversity within cells sharing identical macromolecular marker profiles (Medler et aI.,
2003).
Conclusions
This study classified facial and vagal nerve innervated taste cells according to
their metabolite profiles using GABA, ALA, TAU, GSH, GLU and ASP. Fifteen unique
clusters were extracted using the k-means classification analysis, each of which had a
significantly different metabolite distribution. Although both facial and vagal nerve
innervated taste cells were present in the majority of clusters, their relati ve abundance
was different in clusters. GABA had the most di verse pattern of expression; four clusters
(9% of total cells) had high levels of GABA, whereas the majority of cells (nine clusters,
74% of total cells) had low GABA levels. This study clearly demonstrates that, based on
metabolite profiling, the taste cell population in catfish is far more heterogeneous that
previously thought.
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CHAPTER 4
GABAERGIC NEUROTRANSMISSION
IN THE CHANNEL CATFISH,
ICTALURUS PUNCTATUS
94
Abstract
Although the sense of taste is critical to survival, the mechanism(s) used to
communicate gustatory input from a heterogeneous population of taste cells to
appropriate afferent nerves remain poorly understood. We previously observed that y
amino butyric acid (GAB A) is highly expressed in certain cells of facial (FITBs) and
vagal (VITBs) nerve innervated taste buds of channel catfish, Ictalurus punctatus. In the
current immunocytochemical study we compare the expression of two isoforms of
glutamic acid decarboxylase (GAD65 and GAD67), the key enzyme for GABA
biosynthesis, with the distribution of three GABA transporter isoforms (GAT-I, 2 and 3),
and GABAA 0.1 and a3 receptor subunits. Each GABAergic signaling component was
expressed in both FITBs and VITBs, but differential expression was often noted. GAD65
and GAD67 are expressed in subsets of taste cells, but only GAD67 is expressed in basal
cells. There were more GAD positive cells in VITBs than FITBs. Each GABA
transporter is expressed in some taste cells and in the nerve plexus. Higher levels were
noted in FITBs. Both distinct puncta and diffuse GABAA receptor (GABAAR) 0.1
subunit expression was noted in taste cells and the nerve plexus. The 0.3 subunit is
diffusely expressed, mainly in the peduncle, in fiber-like structures. The 0.1 and 0.3
subunits have similar expression levels in FITBs and VITBs. The highly specific patterns
of differential distribution of GABA, GADs, GATs and GABAARs in catfish taste buds
suggest that GABA serves an important, probably modulatory, role in peripheral
gustatory signaling. Differences in expression levels in FITBs and VITBs may contribute
to previously noted differences in the sensitivity to taste stimuli.
95
Introduction
In fish, taste buds consist of a group of 50-150 elongated taste cells and a few
disk-shaped basal cells, which surround sensory nerve tenninals at the base of the taste
bud. A nerve plexus located just above the basal cells is the presumptive site of synaptic
interaction between taste cells and primary afferent neurons. Complex synaptic
interactions occur in taste buds, including afferent nerve/taste cell, taste cell/taste cell and
basal cell/taste cell synaptic communication, yet even the primary signaling mechanisms
among these cells remain poorly understood (Roper, 1993). Among the
neurotransmitters that have been implicated in gustatory neurotransmission and/or
neuromodulation are glutamate (Chaudhari et aI., 1996; Caicedo et aI., 2000a; 2000b),
serotonin (Kim and Roper, 1995; Hemess and Chen, 1997; Ren et aI., 1999),
cholecystokinin (Hemess et aI., 2002b), norepinephrine (Hemess et aI., 2002a), GABA
(Obata et aI., 1997), acetylcholine (Ogura, 2002), the neuropeptides substance P, VIP,
CGRP and many others (Welton et aI., 1992; Roper, 1993), To this date, the precise
mechanism of action(s) for any of these neurotransmitters has yet to be clearly elucidated.
y-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the
nervous system, and is present in a variety of sensory organs such as the retina
(Murakami et aI., 1972; Lam et aI., 1978) and inner ear (Mroz and Sewell, 1989), GABA
is synthesized from glutamate by the enzyme glutamic acid decarboxylase (GAD) and
two highly conserved isoforms (GAD65 and GAD67) have been cloned (Erlander et aI.,
1991; Bu et aI., 1992). The two isoforms of GAD are found in GABAergic neurons with
different subcellular distribution (Erlander and Tobin, 1991; Kaufman et aI., 1991), Of
the three classes of GABA receptors (GABARs), the GABAAR is a fast acting GABA-
96
gated CI- channel with multiple binding sites for GABA and other neuroactive agents
such as barbiturates, neurosteroids and benzodiazepines (review Wisden and Farrant,
2002). Combinations of 18 subunits (al-6, ~1-3, yl-3, S, €, 8, 1t, pl-3) form the
heteropentameric GABAAR channel whose functional properties and pharmacological
profiles are determined by subunit composition (Macdonald and Olsen, 1994; Sieghart,
1995), The GABAcRs are also ionotropic receptors, while the GABABRs are G-protein
coupled receptors. GABA neurotransmission is terminated by high affinity, sodium
chloride dependant GABA transporters (GATs). Four distinct types of GATs (GAT -1,
GAT-2, GAT-3 and BGT-1) have been identified in rat (Borden et aI., 1992; Durkin et
aI., 1995): GAT-1 in the brain and retina, GAT-3 in the brain and GAT-2 in brain, retina,
liver and kidney (Borden et aI., 1992), In neural tissues, GATs are mainly expressed by
presynaptic GABAergic neurons or astroglial cells (Schousboe, 1981; Kanner and
Schuldiner, 1987; Schousboe and Kanner, 2002).
GABA has been found in taste buds of Necturus (Jain and Roper, 1991), rat
(Obata et aI., 1997) and catfish (Eram and Michel, 2001a). GABA transporters are
expressed in rat taste buds (Obata et aI., 1997), but GABA transport could not be
demonstrated in taste cells or nerve fibers of Necturus (Nagai et aI., 1998). To our
knowledge there are no other studies of GABAergic function in taste buds. In catfish the
localization and detection of food occurs by means of external taste buds innervated by
the facial nerve (7th cranial nerve) and distributed over the entire body, including the
barbels. Oral taste buds, innervated by the glossopharyngeal (9th cranial nerve) or vagal
(10th cranial nerve) nerves, are important for the acceptance and ingestion of food
(Herrick, 1901; Herrick, 1904; Atema, 1971). A previous study showing high GABA
97
immunoreactivity (IR) in dark cells of catfish taste buds (Personal communication, Eram,
and Michel, in press) and higher levels of GABA IR in vagal nerve innervated taste buds
(VITBs) compared to facial nerve innervated taste buds (FITBs) suggests that
GABAergic modulation may contribute to the lower sensitivity of VITBs to certain
amino acid stimuli (Kanwal and Caprio, 1983; Kanwal et aI., 1987; Caprio et aI., 1993).
In the present investigation, we used immunocytochemical and Western blot techniques
to confirm the presence of GABA and to examine the expression of the two glutamic acid
decarboxylase isoforms (GAD65 and GAD67). Three GABA transporters (GAT-I, 2 and
3) and the al and a3 subunits of the GABAAR also occur in FITBs and VITBs of
channel catfish. We noted major differences in the expression levels of these GABA
signaling components, suggesting that GABAergic modulation may differentially affect
these two gustatory pathways.
Materials and Methods
Animal Care
Nine juvenile catfish, Ictalurus punctatus, (15-20 cm total length) were held in
recirculating 40-80 liter aquaria (26-28°C) under a 12-hour light and 12-hour dark light
cycle, and fed frozen mosquito larvae daily. Animals were used within three days of
their arrival. All procedures were approved by the Institutional Animal Care and Use
Committee (IACUC) of the University of Utah.
Immunocytochemistry
Tissue Preparation
98
Following decapitation, maxillary barbels and the 2nd through 5th gill arches were
dissected out in cold fish Ringers solution (concentrations in mM: 137.0 NaCI, 2.0 KCI,
1.8 CaCh, 5.0 Hepes, 10.0 glucose, pH 7.4). For plastic-embedded
immunocytochemistry and electron microscopy the tissues were transferred to a cold
GAT-2 (1:200), and rabbit anti-GAT-3 (1:200) (Chemicon International, Temecula, CA,
USA) and affinity-purified rabbit polyclonal anti-GABAAR a1 (1:200) and a3 (1:500)
subunits (Alomone Labs Ltd., Jerusalem, Israel) on a shaker for 2 hours at room
temperature, then rinsed 3 x 10 minutes with TBS with 1 % Tween-20. Detection of
immunoreactive bands was accomplished using the appropriate secondary goat anti
mouse antibody (1:3000) for GAD65 IgG or goat anti-rabbit antibody (1:3000) for the rest
of IgGs on a shaker for 1 hour at room temperature and a standard chemiluminesence
detection kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) according to the
manufacturer's protocol.
Image Acquisition and Registration
Light Microscopy
Ultrathin sections of taste buds stained for GABA were viewed with a Zeiss
Axioplan2 microscope and a 100x immersion oil lens; images were captured as 8-bit gray
scale digital files (1300 x 1030 pixels) using a CCD camera and Zeiss Axiovision
imaging software 3.0 (Zeiss Inc., Thornwood, NY, USA). Each TEM image of a taste
103
bud was aligned to the image of GABA IR using image analysis software (Geomatica
software 8.0, PCl Remote Sensing, Richmond Hill, Ontario, Canada). Sets of identical
structures were selected to seed a first order polynomial fitting algorithm which aligned
and scaled (for the TEM image) the images. A color composite image was formed by
designating the TEM image as the red and green, and the GABA IR image as blue (RGB)
channels of a RGB image (Marc et aI., 1995; Marc and Liu, 2000). For the co
localization of GABA and EM images, the GABA gray scale images were inverted so
that pixel intensities varied over the range of 0 (lowest) to 255 (highest), respectively.
Confocal Microscopy
A Zeiss LSM 510 confocal laser scanning microscope with a 63x/1.4 numerical
aperture objective lens was used to collect 1024 x 1024 resolution images of labeled taste
buds. A krypton-argon laser was used for 488 and 586 nm excitation wavelength. The
total laser power was set at 75%. To minimize photo bleaching, the transmission at the
excitation wavelength was set to 4% and 5% for the 586 and 488 nm bands, respectively.
For each antibody, the microscope settings were optimized for the brightest specimen,
and all other images from other taste buds were captured at the same settings. The
captured images were converted to Adobe Photoshop format for evaluation.
Results
The Western blot analysis of proteins extracted fronl maxillary barbel epithelium
revealed single bands for GAD65 , GABAAR a3 subunit and GAT -2 of the appropriate
molecular size (Fig. 4.1). Glutamic acid decarboxylase (GAD65) was detected as a single
104
band of approximately 65 kDa, while GABAAR 0.3 subunit was detected as a single thick
band of approximately 44 kDa. The GABA transporter GAT-2 was detected as a lightly
staining band of approximately 88 kDa. There was either insufficient protein for
detection, or the other antibodies were not monospecific, hence not recommended for
Western blot analysis (GAT-3).
GABA Distribution
A comparison of GABA IR in facial (FITBs) and vagal (VITBs) nerve innervated
taste buds (Fig. 4.2A, B) revealed the distribution of GABA positive taste cells and basal
cells. All basal cells appeared to be GABA positive. Consecutive sections processed for
GABA immunocytochenlistry and electron microscopy, allowed an image superposition.
A majority of GABA high and GABA intermediate cells had a dark cytoplasmic matrix
and irregularly shaped nuclei, a consistent feature of dark cells (Royer and Kinnamon,
1996; Grover-Johnson and Farbman, 1976). Cells without GABA IR had lighter
cytoplasm and prominent round nuclei characteristics of light taste cells (Royer and
Kinnamon, 1996; Grover-Johnson and Farbman, 1976).
Glutamic Acid Decarboxylase (GAD) Distribution
In both FITBs and VITBs, GAD65 IR (Fig. 4.3A-D) was stronger than GAD67 IR
(Fig. 4.3E, F). Basal cells were devoid of any GAD65 but GAD65 was highly expressed by
a few elongated taste cells where it was excluded from the nucleus (Fig. 4.3A-D). Many
small processes in the nerve plexus had high levels of GAD65 IR. In the peduncle, both
GAD65 (Fig. 4.3D, arrow) and GAD67 were present in some nerve fibers. Although
105
kDa
148.0-
64.0-
36.0-
GAD· a3 '",,65
Figure 4.1. Immunoblot analysis of epithelial samples from the maxillary barbel confirms that antibodies for GAD65 , the a3 subunit of GABAAR and GAT-2 recognize proteins of the correct molecular weight.
106
A
Figure 4.2. GABA immunoreactivity (IR, blue) is superimposed on an electron micrograph (red) of a A: FITB and B: VITB. Taste cells with GABA high and intermediate levels of immunoreactivity are primarily dark cells with irregular nuclei. GABA low immunoreactive cells have predominantly round nuclei. NP, nerve plexus; BC, basal cell; P, peduncle; TC, taste cell. Scale bar = 10 /lm.
107
Figure 4.3. Both isoforms of glutamic acid decarboxylase (GAD) are found in FITBs and VITBs. A-D: GAD65 IR is expressed at high levels in the nerve plexus and a few FITB and VITB taste cells; it is also expressed in some fibers in the peduncle (D, arrow) E, F: GAD67 IR is weaker than GAD65 IR. Note that both GAD65 and GAD67 are distributed in the taste cell populations; GAD67 expression is lower in the nerve plexus but present in the basal cell (arrows), while GAD65 expression is relatively high in the plexus but absent in the basal cell. Scale bar = 10 J.tffi.
108
109
GAD67 IR was stronger in the VITBs than FITBs, the overall level of staining was
relatively weak. The strongest GAD67 IR was seen in the basal cells of both FITBs and
VITBs (Fig. 4.3 E, F, arrows, and Fig. 4.6A and 7 A). Traces of GAD67 IR were noted in
the cytoplasm of a few taste cells and some processes in the nerve plexus region (Fig.
4.3E, F).
GABAA Receptor al and a3 Distribution
The patterns of al and a3 subunit expression in either FITBs or VITBs were
essentially nonoverlapping (Fig. 4.4A-D). The al subunit (Fig. 4.4A, B) was diffusely
disttibuted on taste cells and throughout FITB nerve plexus. In addition to the diffuse
labeling, punctate al subunit expression was most commonly observed on taste cell
processes towards the apex of taste bud and in the nerve plexus. The most notable
difference in the expression patterns of al subunit between FITBs and VITBs was the
reduction of al subunit expression in the nerve plexus region of VITBs to only a few
puncta. The perigemmal cells of FITBs had high levels of al subunit IR (Fig. 4.4A,
arrows), which was absent in the VITBs. In contrast to the al subunit, the majority of a3
subunit expression in FITBs and VITBs was concentrated in the nerve fibers of peduncle
(Fig. 4.4C, D). Only a few small fibers were a3 positive in taste buds, which may be the
processes of afferent neurons reaching into taste buds beyond the nerve plexus (Fig. 4.4C,
arrows).
110
Figure 4.4. Two isofonns of the GABAA receptor are differentially expressed in taste buds. A, B: The a1 subunit is primarily located on taste cells. Some perigemmal cells at the edge of FITBs express high levels of a1 (A, arrow). C, D: The a3 subunit is associated with nerves in the peduncle. Traces of a3 subunit expression is seen in the taste buds (arrows). Scale bar = 10 J,tm.
111
FITB VITB
112
GABA Transporters (GATs) Distribution
GABA transporter IR was in general higher in the FITBs than VITBs (Fig. 4.5A
F). Each of the three transporters was highly expressed in some taste cells and weakly
expressed throughout the taste bud. In GAT-1 positive taste cells labeling was seen
throughout the cell (Fig. 4.5A, B). The basal cells of FITBs also had high GAT-1
(Fig.4.5B). The majority of GAT-2 IR was located in the apical region of the taste bud
(Fig. 4.5C, D). The broad band of localization extended throughout the upper third of
FITBs but was restricted to the apical region in VITBs. Processes of only a few GAT-2
positive cells could be seen in the soma region suggesting that apical localization may be
due to extensive branching as has been previously reported for dark cells. A few taste
cells, the nerve plexus and the basal cells of FITBs showed high levels of GAT -3 IR (Fig.
4.5E), while each of these areas in VITBs showed generally low levels of GAT -3 IR (Fig.
4.5F). GAT -3 IR was the highest of the three transporters in the VITBs. Large GAT-1
and GAT-3 positive processes were observed in the nerve plexus, while the GAT-2
labeling in the nerve plexus was weak and diffuse. In general, GAT expression was also
low in the peduncle.
Patterns of Co-localization
We chose GAD65 to evaluate patterns of co-localization with each of the other
GABA signaling antibodies. GAD65 was the only antibody that was raised in mouse; the
rest were raised in rabbit. Since GAD65 is only expressed by GABAergic neurons, co-
113
Figure 4.5. Three isoforms of the GABA transporter, A, B: GAT-I, C, D: GAT-2 and E, F: GAT-3, are expressed in FITBs and VITBs. While GAT-2 expression is generally high throughout the taste bud, taste cells in FITBs have higher GAT -1 and GAT-3 expression levels than the VITBs. GAT -1 and GAT -3 expression is higher in the nerve plexus. It appears that only GAT-I is expressed in the basal cell of FITBs (A, arrow). Scale bar = 10 J.1m.
114
115
localization of GAD65 with any of the antibodies implies that these antigens are localized
in GABA positive cells.
Most of the GAD65 and GAD67 co-localization was focused in the fine processes
of the plexus (Fig. 4.6A and Fig. 4.7 A). A distinct green color beneath the nerve plexus
of both FITB and VITB showed basal cells that were only GAD67 JR. GAD65 co
localized to greater extent with GABAAR 0:1 subunit at the apical region of FITB (Fig.
4.6B) than in VITB (Fig. 4.7B). In FITBs, GAD65 and 0:1 co-localized in some fine
processes of nerve plexus, whereas this pattern was absent in the VITBs. There were
essentially no co-localization between GAD65 and GABAAR a3 subunit in the taste buds
(Fig. 4.6C and Fig. 4.7C). In most instances, the GAT positive taste cells were also
GAD65 positive (Fig. 4.6D-F, Fig. 4.7D-F), especially in the FITB taste cells, suggesting
that GAT isoforms expression may be overlapping. Co-localization of GAD65 with
GAT-l (Fig. 4.6D) and GAT-3 (Fig. 4.6F) was prominent in the processes of nerve
plexus in the FITB but was absent in the VITB (Fig. 4.7D, 7F).
Discussion
GABA has been reported to be present in taste buds of a several vertebrate species
(Jain and Roper, 1991; Obata et aI., 1997; Eram and Michel, 2001a). In the channel
catfish all basal cells and approximately 25% of taste cells had either high or intermediate
levels of GABA IR. It is likely that GABA serves a role in neurotransmission or
neuromodulation. In the nervous system GABA is synthesized by one of two isoforms of
glutamic acid decarboxylase (GAD65 and GAD67), which are specifically localized in
116
Figure 4.6. Patterns of co-localization of GAD65 (in red) with A: GAD67, B: GABAAR a.I subunit, C: GABAAR a.3 subunit, D: GAT-I, E: GAT-2 and F: GAT-3 shown in green in FITB. Scale bar = 10 ~m.
117
Figure 4.7. Patterns of co-localization of GAD65 (in red) with A: GAD67, B: GABAAR al subunit, C: GABAAR a3 subunit, D: GAT-I, E: GAT-2 and F: GAT-3 shown in green in VITB. Scale bar = 10 !-tm.
118
GABAergic neurons (Erlander et aI., 1991; Esclapez et aI., 1994). Considering that high
levels of GAD65 were observed in a subset of FITBs and VITBs cells and in the nerve
plexus, we suggest that GABA could be involved in synaptic regulation of taste cell
activity. On the other hand, high levels of GABA and GAD67 IR in basal cells may imply
that the synthesis of GABA in these cells may be regulated differently.
In neurons, GABAergic transmission is terminated by high affinity GABA
transporters, which are located at the presynaptic terminals and the surrounding
astrocytes and are responsible for the rapid reuptake of GABA from the synaptic cleft
(for review see Borden, 1996). Four distinct GABA transporters have been characterized
and cloned, each of which has a unique structure and pharmacological profile. The three
GABA transporters we examined (GAT -1, GAT -2 and GAT -3) are the predominant
forms in the central nervous system (CNS) where they are heterogeneously distributed
across neurons and glia (Krogsgaard-Larsen et aI., 1987; Guastella et aI., 1990; Lopez
Corcuera et aI., 1992; Borden et aI., 1992; Liu et aI., 1993; Itouji et aI., 1996; Schousboe
and Kanner, 2002). In brain, GAT-1 and GAT-3 are associated with axon terminals,
which is consistent with presynaptic activity (Radian et aI., 1990; Pietrini et aI., 1994),
while GAT -2 is mainly compartmentalized in the dendrite suggesting a postsynaptic
function (Borden et aI., 1992; 1995). We found expression of all three GABA
transporters in channel catfish taste cells. Co-localization of GAD65 with either GAT-1
or GAT-3 in the cytoplasm of a small subset of taste cells suggests a sensory function.
On the other hand, the diffuse apical GAT-2 expression is suggestive of a supporting role,
perhaps in the maintenance of homeostasis in the presence of a dilute aquatic
environment. The fact that GAT-l and GAT-3 also co-localize with GAD65 in some taste
119
cells and are robustly expressed in the plexus region, where synaptic communication is
thought to occur, is consistent with GABA having a role in the modulation of afferent
nerve activity.
Released GABA is detected by ionotropic GABAARs and GABAcRs, and by
metabotropic GABABRs receptors. In the current study our examination was limited to
two of 18 GABAAR subunits. In the catfish peripheral taste system expression patterns
of the a1 and a3 subunits were distinctly different. GABAARs containing the a1 subunit
were largely restricted to taste cells. The presence of both dense punctata, and
widespread diffuse labeling suggests that GABAARs may act in two distinct ways. The
dense punctata are suggestive of synaptic localization and are likely to involve rapid,
transient GABA-mediated changes (Pirker et aI., 2000). On the other hand, diffuse
labeling is consistent with extrasynaptic localization which in the CNS typically mediates
slower and more persistent cellular responses (Pirker et aI., 2000). Expression of the a3
receptor by fibers within the peduncle, most of which are likely to terminate in the nerve
plexus, suggests that GABA release by taste cells would decrease activity of any a3
subunit expressing cells and decrease gustatory sensitivity. Lack of colocalization of a1
and a3 subunits in catfish taste buds suggests expression of different GABAAR
heteropentamers.
Light, dark and intermediate taste cell types have been described in fish taste buds
based on electron microscopy and morphological studies (Kapoor and Ojha, 1973; Crisp
et aI., 1975; Kapoor et aI., 1975; Grover-Johnson and Farbman, 1976; Reutter, 1978;
1986; Tucker, 1983; Reutter and Witt, 1993). Complex synaptic interactions between
120
taste cells, basal cells and/or afferent neurons have been reported (Reutter, 1978; 1986;
Delay and Roper, 1988; Delay et aI., 1993; Ewald and Roper, 1994). We confirm
previous findings that most GABA positive cells are dark cells (Bram and Michel, 2003)
and propose that specific subpopulations of GABA positive cells may not only serve to
modulate the activity of other taste cells via synaptic and extrasynaptic mechanisms, but
may also regulate synaptic communication between taste cells and afferent nerve fibers.
Significant differences were observed in the levels of GABA, GAD65, GAT -1 and
GAT -3 between taste buds innervated by the facial and vagal nerves. Consistent with our
observation that there were more GABA positive cells in VITBs, we noted significantly
more GAD65 positive VITBs cells. With greater synthetic capability and reduced
transport function, ambient GABA levels in VITBs might be expected to be generally
higher. With similar levels of GABAAR a1 subunit expression, it might be expected that
GABA would exert a larger modulatory role in VITBs. Although the specific function of
the GABAergic modulation ren1ains to be determined, a number of possibilities come to
mind. First, GABA may serve to tune the overall sensitivity of taste buds. The vagal
taste system is reported to be less sensitive to amino acid stimuli than the facial taste
system (Kanwal and Caprio, 1983; Kanwal et aI., 1987) and may be explained by greater
tonic GABAergic modulation as previously described. Reduced GABAergic modulation
of the facial taste system may thus provide an advantage during food search.
GABA may playa role in gustatory mixture interactions. The bitter substance
quinine is reported to reduce the sensitivity of facial taste fibers to amino acid stimuli
(Ogawa et aI., 1997). Mixture interactions were originally proposed to occur at the level
of transduction cascades; however, recent studies indicate that receptors for bitter and
121
amino acid stimuli are expressed by distinct populations of taste cells requiring
intercellular interactions (Adler et aI., 2000; Montmayeur and Matsunami, 2002).
Release of GABA by bitter sensitive cells might potentially decrease the sensitivity of
GABAAR expressing neighboring cells or directly modulate synaptic efficacy at the
amino acid-sensitive taste cell/afferent nerve fiber synapse. Physiological studies to
better understand the role of GABA in peripheral taste function are clearly warranted.
Conclusions
We investigated the GABA signaling components, GABA, GAD65, GAD67, GAT-
1, GAT-2, GAT3, GABAAR al and GABAAR a3 subunits, in the FITBs and VITBs of
channel catfish. Each of the components tested in this experiment was expressed in both
FITBs and VITBs, although there were some striking differences. FITBs had lower
levels of GAD65 and higher levels of GAD67, higher levels of transporters, and similar
levels of GABAAR al and GABAAR 0.3 subunits, compared to VITBs. This is the most
comprehensive study of GABAergic signaling in the taste bud to date, and the first
comparative study of GABAergic signaling in facial and vagal nerve pathways in any
species.
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CHAPTERS
CONCLUSIONS
Summary of Findings
In this study we address two main hypotheses:
1) Taste cell heterogeneity is a major determinant of functional differences in the
facial and vagal gustatory systems.
128
2) A GABA signaling pathway is present in facial and vagal nerve innervated taste
buds of catfish.
Our hypotheses are supported by the findings in Chapters 2, 3 and 4. The following is
the summary of our findings.
In Chapter 2, we investigated the morphometric properties of facial (FITBs) and
vagal (VITBs) nerve innervated taste buds of channel catfish, and showed that since
FITBs have a larger nerve plexus and more taste cells per taste bud, such differences
could account for the higher sensitivity of the facial systen1 in detecting certain amino
acid stimuli (Kanwal and Caprio, 1983; Kanwal et aI., 1987; Michel and Caprio, 1988;
1991; Kohbara et aI., 1992; Caprio et aI., 1993). We used immunocytochemistry and
image analysis to demonstrate differences in the y-aminobutyric acid (GABA), glutamate,
aspartate, alanine, taurine and glutathione profiles of FITBs and VITBs. Of these
metabolites, GABA had the most diverse expression pattern with more GABA-positive
cells in VITBs than FITBs. We noted a unique cell type in connective tissue of the
peduncle adjacent to and parallel with the overlaying basal lamina of the taste bud.
Because of its close association to near by basal cells of the taste bud, we named this
element "companion cell". The function of the companion cell is unknown, but we
suggest that it may participate in the mechanosensory function of basal cells, where it
serves as an anchor to the basal cell.
129
In Chapter 3 we classified FITB and VITB taste cells of channel catfish according
to levels of expression of the six metabolites identified in Chapter 2 using a k-means
analysis. Of the 15 clusters of cells extracted, each had a unique metabolite signature
significantly different than the others. Several clusters contained cells predominately
from either FITBs or VITBs.
In Chapter 4 we investigated the synthetic enzymes, receptors and transporters
involved in GABA neurotransmission in FITBs and VITBs using immunocytochemistry
and Western blot analysis. By comparison with VITBs, FITBs generally had lower
glutamic acid decarboxylase (GAD6S) and higher GAD67 levels, higher GABA
transporters (GAT-I, 2 and 3) levels and similar levels of GABAAR al and a3 subunits.
We confirmed the presence of GAD6S, GABAAR al subunit and GAT-2 with Western
blot analysis.
Collectively, these studies demonstrate significant differences in the
morphometric and metabolite distribution of FITBs and VITBs, which may account for
the functional differences reported previously. We also provide, for the first time, strong
evidence for the existence of GABAergic signaling in catfish gustatory system. The
following sections consider our results in the context of current literature dealing with
this field of study.
Basic Morphometric Properties
By contrast to oropharyngeal taste buds innervated by vagal and glossopharyngeal
nerves, the abundance of taste buds distributed over the entire catfish body surface and
with high densities in the barbel epithelium (innervated by facial nerve) makes this
130
species an ideal model organism for studying the gustatory system (Herrick, 1901). The
facial taste system is essential in detection and localization of food, while oropharyngeal
taste pathways are important for final acceptance and consumption of food (Atema,
1971). Gustatory responses from catfish facial (Michel and Caprio, 1988; 1989; 1991;
Kohbara et aI., 1992) and vagal (Kanwal and Caprio, 1983) nerves have been recorded
electrophysiologically, and significant differences in their responses to amino acid stimuli
have been documented ..
Although differences among taste buds have been reported previously (Reutter,
1971), this dissertation provides the first comparative morphometric examination
specifically related to the facial and vagal nerves. The morphometric differences we note
may be significant to the functional properties of FITBs and VITBs, especially with
regard to the putative neurotransmitters GABA and glutamate. Since efficient food
search and selection is critical for survival, the higher number of taste cells and larger
nerve plexus of FITBs may provide a selective advantage.
Taste Cell Heterogeneity
The identification of taste cells in early studies was essentially limited to
morphological observations and electron n1icroscopy (Farbman, 1965; Hirata, 1966;
1978; Grover-Johnson and Farbman, 1976). Considerable heterogeneity in taste cells is
based on the distribution of macromolecular markers such as neural cell adhesion
molecule (NCAM) (Takeda et aI., 1992), a-gustducin (Wong et aI., 1996; Chandrashekar
et aI., 2000), neuron specific enolase, serotonin (Yee et aI., 200 1), the blood group
carbohydrate epitopes antigen A and antigen H (Pumplin et aI., 1997).
Our method of classification using metabolite profiling demonstrates additional
diversity among taste cells and reveals 15 clusters; the greatest diversity of taste cells
types reported in any species. It remains to be determined whether significant functional
differences exist among these cell types. Our approach (Marc et aI., 1995) to
classification according to patterns of co-localization of metabolites provides the
advantage of compatibility with electron microscopic analysis, and although a battery of
only five amino acids and the peptide GSH were employed as markers, many more could
have been used if other useful reagents had been identified.
Evidence for GABAergic Signaling in the Peripheral Taste System
In a sensory system, the paramount goal is to understand the generation of action
potentials: namely the process by which the individual activation of receptor elements is
transmitted to a responsive afferent neuron. In the taste system, many putative
neurotransmitters and modulators have been proposed and a variety of second messenger
pathways have been suggested to be involved (Roper, 1993; Yamamoto et aI., 1998;
Herness and Gilbertson, 1999; Gilbertson et aI., 2000). Yet, even the most basic
neurotransmission mechanisms in the taste bud remain to be confirmed. The large
number of proposed transmitter candidates, along with a multiplicity of transduction
132
pathways is consistent with the notion of a diverse and heterogeneous population of taste
cells each of which helps with essential survival skills of food selection and consumption.
In detennining the metabolite profile of channel catfish taste cells, we noted high
level of the inhibitory neurotransmitter GABA in a subset of taste cells, which led us to
suggest that GABA plays a role in modulating gustatory neurotransmission. Our
experimental results confinn that components essential to GABAergic signaling are
present but are differentially expressed in FITBs and VITBs. We now propose a
hypothetical model by which GABA may selectively modulate the sensitivity of taste
cells innervated by these two gustatory nerves, which would account for the lower
sensitivity of VITBs to amino acid stimuli (Kanwal and Caprio, 1983; Kanwal et aI.,
1987; Kohbara et aI., 1992; Caprio et aI., 1993).
Although GABA has previously been reported in taste buds (Jain and Roper,
1991; Obata et aI., 1997; Nagai et aI., 1998), ours is the first comprehensive study to
examine the other components necessary for GABAergic signaling and to compare their
distribution in FITBs and VITBs. Like GABA, there were more GAD65 positive cells in
VITBs. Although FITBs had more GAD67 positive cells, its overall level of expression
was lower than GAD65. Higher levels of all three GABA transporters (GAT-I, 2 and 3)
in FITBs suggest that GABA modulation in FITBs is primarily regulated by GABA
uptake. Fast removal of GABA by high affinity transporters is therefore expected to
lessen the potential inhibitory effects of GABA in FITBs and thus contribute to a higher
sensitivity to amino acid stimuli (Kanwal and Caprio, 1983; Kanwal et aI., 1987; Kohbara
et aI., 1992; Caprio et at, 1993). It seems unlikely that patterns of receptor expression
are the principal detenninant of functional differences between FITBs and VITBs,
133
although GABAAR al and a3 subunits have similar, though non-overlapping expression
patterns. Further analysis of the subunit pattern of expression of other GABAAR (Pirker
et aI., 2000) may reveal other aspects of synaptic modulation.
Future Directions
Future experiments compatible with our current analysis of metabolite profile
involve the use of the cation channel permeant organic molecule agmatine (AGB), which
was first employed in the identification of retinal cell types bearing AMP AlKA and
NMDA receptors by Robert Marc (l999a; 1999b). This activity marker has been
invaluable in determining the role of ionotropic glutamate receptors in odor-stimulated
activation of olfactory bulb neurons in fish (Edwards and Michel, 2002). Since two
classes of high affinity receptors for L-alanine and L-arginine have been identified
(Kalinoski et aI., 1989; Caprio et aI., 1993; Kumazawa et aI., 1998), future studies using
AGB as an activity-dependent probe will identify arginine-sensitive taste cells and
determine whether such arginine sensitive belong to light or dark cell types utilizing
registered electron microscopic images.
Hypothetical Model for the Role of GABA in
Peripheral Gustatory System
Quinine is a bitter substance with the ability to decrease the response of single
facial fibers to certain amino acid stimuli (Ogawa et aI., 1997). We speculate that this
response suppression may be brought about by the inhibitory action of GABA.
According to this view, quinine activation of GABAergic cells leads to GABA release,
134
which then binds to GABAARs on neighboring amino acid sensitive taste cells (see Fig
5.1). GABA may mediate either phasic and tonic inhibitory responses depending on the
activation of synaptic or extrasynaptic GABAARs (Saxena and Macdonald, 1996). Tonic
inhibition results from activation of high affinity extrasynaptic GABAARs, whereas the
phasic GABAergic inhibition is brought about by the activation of low affinity, rapidly
desensitizing synaptic GABAARs (Richerson and Wu, 2003). If our speculation is
correct, the application of a GABAAR agonist such as muscimol is expected to reduce the
magnitude of amino acid evoked responses and eliminate bitter suppression. In contrast,
a GABAAR antagonist such as GABAzine is expected to either not affect or enhance
amino acid evoked responses, but should also eliminate bitter suppression.
We further speculate that GABA transporters are active participants in
GABAergic modulation of the peripheral gustatory response. GABA transporters also
regulate extracellular GABA concentration thus establishing a level of tonic inhibition
(Richerson and Wu, 2003). Resting membrane potential, the transmembrane gradients
for sodium and chloride and the intracellular GABA concentration determine the
extracellular GABA concentration. Under normal physiological conditions, GABA
transporter expressing cells with high intracellular GABA concentrations (2.5 mM),
normal ionic composition and a resting potential of approximately -60 mV would
establish in an extracellular GABA equilibrium concentration of 0.1 11M. This
extracellular GABA concentration is sufficient to activate high affinityextrasynaptic
GABAARs (Saxena and Macdonald, 1996). Hyperpolarizing membrane potentials favor
GABA transport into the cell by forward transport mechanism, while at depolarizing
membrane potentials GABA is transported out of the cell by reverse transport action.
Arg/Pro Bitter Ala
Bitter receptor GAD65 GABA
GABAAR GATs
A. Quinine
B. Amino acid
C. Amino acid + Quinine
D. Amino acid + Quinine + GABAzine
«) +
Arg or ++ Arg/Pro taste fiber
++ Ala taste fiber Bitter taste
fiber ??
135
II
I" II 111111111111 I I
-GABAAR
- GABAzine block
- Excitation (more pulses trigger)
- Inhibition
Figure 5.1. Diagram of one possible model for the action of GABA in taste cells. GABA may modulate quinine suppression of the single fiber response to amino acid stimuli. Release of GABA from the GABAergic cells stimulated by quinine results in inhibition or decrease of neighboring sell response to amino acid stimuli through GABA receptors.
136
At synaptic sites, during GABA release, high extracellular GAB A concentrations favor
forward transport regardless of the membrane potential. At extrasynaptic sites the
transporters expressed by the same cell recover synaptic GABA but release extrasynaptic
GABA. If, as we propose, the bitter sensitive cells are the GABA positive cells
depolarization may potentially initiate synaptic GABA release via a classical vesicular
release process and extrasynaptic GABA release via reserve transport.
In summary, this project has provided the initial evidence for the presence of
GABAergic signaling in catfish taste buds. Still, the functional role of GABA in the
peripheral gustatory system requires further attention. Electrophysiological nerve
recordings comparing catfish facial and vagal nerve responses following systemic
GABAAR agonist or antagonist application are required to determine the functional
significance of the heterogeneous distribution of GABAergic signaling components in
these two gustatory pathways. Single fiber recordings are required to determine if
specific gustatory modalities are affected. Additional pharmacological studies will be
required to determine if classical synaptic signaling, transporter mediated signaling or a
combination of both predominate.
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