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

Copyright © Mojgan Eram 2004

All Rights Reserved

THE UNIVERSITY OF UTAH GRADUATE SCHOOL

SUPERVISORY COMMITTEE APPROVAL

of a dissertation submitted by

Mojgan Eram

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

? ..

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

1. INTRODUCTION ......................................................................................................... 1

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

Abstract ............................................................................................................. 15 Introduction ....................................................................................................... 16 Materials and Methods ...................................................................................... 18 Results ............................................................................................................... 22 Discussion ......................................................................................................... 40 Conclusions ....................................................................................................... 46 Referencess ........................................................................................................ 4 7

3. CLASSIFICATION OF FACIAL AND VAGAL NEREVE INNER V A TED TASTE CELLS OF CHANNEL CATFISH USING METABOLITE PROFILES ........................................................................... 52

Abstract ............................................................................................................. 53 Introduction ....................................................................................................... 53 Materials and Methods ...................................................................................... 55

Results ............................................................................................................... 59 Discussion ......................................................................................................... 82 Conclusions ....................................................................................................... 88 References ......................................................................................................... 89

4. GABAERGIC NEUROTRANSMISSION IN THE CHANNEL CATFISH, ICTALURUS PUNCTATUS ......................................... 93

Abstract ............................................................................................................. 94 Introduction ....................................................................................................... 95 Materials and Methods ...................................................................................... 97 Results ............................................................................................................. 103 Discussion ....................................................................................................... 115 Conclusions ..................................................................................................... 121 References ....................................................................................................... 121

5. CONCLUSIONS ........................................................................................................ 127

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

Kinnamon, 1996; Kapoor et aI., 1975; Tucker, 1983; Reutter, 1986; Caprio, 1988;

Boudriot and Reutter, 2001; Hansen et aI., 2002). Light cells are the presumed primary

receptors cells, which have electron lucent cytoplasm, a round or oval nucleus, a single

large microvillus and basal synaptic connections (Royer and Kinnamon, 1996; Reutter,

1971; Reutter, 1978; Reutter, 1986). The dark taste cells (Grover-Johnson and Farbman,

1976), sometimes termed supporting cells (Hirata, 1966; Fujimoto and Yamamoto, 1980;

Jakubowski and Whitear, 1990), have dark cytoplasm, many short apical microvilli, an

3

irregular, elongated shape nucleus and lateral processes that wrap around other cells

(Hirata, 1966; Fujimoto and Yamamoto, 1980; Toyoshima et aI., 1984). They are

considered by some to be sensory cells since they sometimes make synaptic contact with

basal cells and primary afferent neurons (Reutter, 1982; 1986). The third type is

identified by many dense-core vesicles in the basal cytoplasm have an apical process

covered by medium size microvilli, which are presumed to be sensory in function (Roper,

1989; Reutter and Witt, 1993; Boudriot and Reutter, 2001). In mammals, taste cells

classified according to electron microscopic criteria also fall into three groups (type I, II

and III), which correspond to the dark, light and intermediate cells of fish (Murray, 1971;

Murray, 1973; Reutter and Witt, 1993; Finger and Simon, 2000).

Unlike the radially elongate chemosensory elements, basal cells like the Merkel

cells of mammals are thought to respond to mechanical stimuli (Hirata, 1966). Disk­

shaped basal cells are located just above the basal lamina and below the nerve plexus. As

in other vertebrate spike-like and presumed mechanosensory processes extend from the

upper surface between which synaptic contacts are formed with nerve fibers in the

overlaying nerve plexus (Royer and Kinnamon, 1996). Some investigators regard basal

cells as intemeurons (Reutter, 1971) due to the presence of GABA and serotonin

immunoreactivity (Jain and Roper, 1991; Eram and Michel, 2001a).

Despite the variety of histochemical, electron microscopic and functional

techniques that have been used to delineate subsets of taste cells (Finger and Simon,

2000), no universally acceptable classification scheme has as yet been proposed that

elucidates the functional properties of these cells. One of the primary goals of this

project is to investigate the heterogeneity among catfish taste bud cells using a metabolite

profiling technique (Marc et aI., 1995). This powerful immunocytochemical approach

uses registered ultrathin serial sections to quantitatively analyze metabolite composition.

Pioneering studies in the retina (Marc et aI., 1995) allowed identification of unique cell

types based on the differential distribution of glutamate, GABA and glycine, and

subsequent metabolic profiling of the olfactory epithelium in our laboratory (Michel,

unpublished data) has shown that it is an invaluable approach in determining cell

heterogenei ty.

Innervation of the Gustatory System

4

Taste buds are innervated by facial (VII), glossopharyngeal (IX) and vagus (X)

nerves in all vertebrates, but the pattern of innervation differs in various species (Reutter

and Witt, 1993). In mammals, the facial nerve innervates fungiform papillae of the

anterior tongue and soft palate, the glossopharyngeal nerve projects to folliate papillae of

the posterior tongue, and the vagus nerve supplies taste buds in the upper esophagus (for

review see (Sn1ith and Davis, 2000). All mammalian gustatory nerve fibers project to the

nucleus of the solitary tract in the brainstem. In fish, facial and vagal nerves project to

separate facial and vagal lobes (Herrick, 1901; Herrick, 1905; Finger, 1978). The catfish

facial nerve innervates taste buds on the body surface, lips and barbels, while taste buds

located in the oral cavity are innervated by the vagus and glossopharyngeal nerve. Atema

(1971) showed that the facial nerve is responsible for localization and selection of food,

whereas the vagus nerve is involved with food acceptance and ingestion. Differences in

the metabolite profile of facial nerve innervated taste buds (FITBs) and vagal nerve

innervated taste buds (VITBs) are expected to provide novel information not only about

the heterogeneity of taste cells, but may also reveal metabolic and morphological

correlates to known functional differences distinguishing the two gustatory pathways.

Physiological Evidence for the Differences in Facial and Vagal

Nerve Innervated Taste Buds

5

Mammalian gustatory neurons respond to a variety of taste stimuli that are

categorized, in human, as sweet, sour, bitter, salt and umami (glutamate) taste sensation.

In fish, gustatory nerve fibers have high sensitivity to L-amino acids.

Electrophysiological recordings from catfish facial (extra-oral taste buds) and vagal (oral

taste buds) nerves reveal significant differences in their response to various amino acids

(Kanwal and Caprio, 1983; Kanwal et aI., 1987; Kohbara et aI., 1992). Since taste buds

in the oral cavity have a higher threshold for L-alanine, L-arginine and L-proline than

those located externally, the two gustatory pathways may be involved with distinct

aspects of feeding (Atema, 1971). Similar studies in mammals reveal differences in the

response of chorda tynlpani (CT) compared to superior laryngeal (SL) nerves suggesting

different functional roles in coding the four basic taste stimuli (Frank, 1973; Frank et aI.,

1988; Smith and Hanamori, 1991). In the hamster, the relative sensitivity of the CT

nerve was sucrose>NaCI> HCI with little or no response to bitter stimuli, while

sensitivity in SL nerve was observed to HCI>NaCI>quinine>sucrose (Smith and

Hanamori, 1991). Furthermore, while single taste fibers show high sensitivity to a

specific stimulus, they also respond to other stimuli indicating they are in fact multi modal

sensory units. Based on these considerations the present project seeks to examine the

6

underlying structural and metabolic differences between catfish FITBs and VITBs, which

could account for physiological differences in sensitivity and behavior.

Neurotransmission in Taste Cells

Gustatory signaling starts at the apical microvillar sutface of a receptor cell with

the initiation of a generator potential. When transmitted to the basal process, it activates

chemical synapses, which engage the temlinals of afferent gustatory neurons (Roper,

1993). A variety of putative transmitters and modulators (Yamamoto et aI., 1998) may

be involved with gustatory signaling, including glutamate (Chaudhari et aI., 1996;

Caicedo et aI., 2000a; Caicedo et aI., 2000b), GABA (Obata et aI., 1997), acetylcholine

(Ogura, 2002), serotonin (Kim and Roper, 1995; Hemess and Chen, 1997; Ren et aI.,

1999), cholecystokinin (Hemess et aI., 2002b) and norepinephrine (Hemess et aI.,

2002a). It should be added, however, that no single definitive transmission mechanism(s)

has as yet been confirmed.

y-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter widely

distributed in central and peripheral neurons. In brain, GABA is produced by

decarboxylation of L-glutamate by the enzyme glutamic acid decarboxylase (GAD)

specifically localized in GABAergic neurons (Erlander et aI., 1991; Esclapez et aI.,

1994). Two highly conserved GAD isoforms (GAD65 and GAD67) have been cloned

(Legay et aI., 1986; Erlander et aI., 1991; Bosma et aI., 1999). Although the presence of

GAD in a subset of taste cells would strongly suggest that GABA has a role in

neurotransmission, to date, there are no such data.

7

GABA neurotransmission is terminated at the synaptic cleft by four high-affinity,

sodium coupled GABA transporters (GATs) (Borden, 1996; Kanner, 2000; Schousboe

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.

References

Atema J. 1971. Structures and functions of the sense of taste in the catfish, Ictalurus natalis. Brain Behav Evol 4:273-294.

Borden LA. 1996. GABA transporter heterogeneity: pharmacology and cellular localization. Neurochem Int 29:335-356.

Bosma PT, Blazquez M, Collins MA, Bishop JD, Drouin G, Priede IG, Docherty K, and Trudeau VL. 1999. Multiplicity of glutamic acid decarboxylases (GAD) in vertebrates: molecular phylogeny and evidence for a new GAD paralog. Mol BioI EvoI16:397-404.

10

Boudriot F and Reutter K. 2001. Ultrastructure of the taste buds in the blind cave fish Astyanaxjordani ("Anoptichthys") and the sighted river fish Astyanax mexicanus (Teleostei, Characidae). J Comp Neurol 434:428-444.

Caicedo A, Jafri MS, and Roper SD. 2000a. In situ Ca2+ imaging reveals neurotransmitter

receptors for glutamate in taste receptor cells. J Neurosci 20:7978-7985.

Caicedo A, Kim KN, and Roper SD. 2000b. Glutamate-induced cobalt uptake reveals non-NMDA receptors in rat taste cells. J Comp NeuroI417:315-324.

Caprio J. 1988. Peripheral filters and chemoreceptor cells in fishes. In Atema J, Fay RR, Popper AN, and Tavolga WN, editors. Sensory biology of aquatic animals. NY: Springer-Verlag. p 313-338.

Caprio J, Brand JG, Teeter JH, Valentincic T, Kalinoski DL, Kohbara J, Kumazawa T, and Wegert S. 1993. The taste system of the channel catfish: From biophysics to behavior. Trends Neurosci 16:192-197.

Chaudhari N, Yang H, Lamp C, Delay E, Cartford C, Than T, and Roper S. 1996. The taste of monosodium glutamate: Merrtbrane receptors in taste buds. J Neurosci 16:3817-3826.

Eram M and Michel WC. 2001a. Anlino acid immunoreactivity of channel catfish peripheral taste system. Chern Sens 26(8), 1125.

Erlander MG, Tillakaratne NJ, Feldblum S, Patel N, and Tobin AJ. 1991. Two genes encode distinct glutamate decarboxylases. Neuron 7:91-100.

Esclapez M, Tillakaratne NJ, Kaufman DL, Tobin AJ, and Houser CR. 1994. Comparative localization of two forms of glutamic acid decarboxylase and their mRNAs in rat brain supports the concept of functional differences between the forms. J Neurosci 14:1834-1855.

Finger TE and Simon CM. 2000. Cell biology of taste epithelium. In Finger TE, Sil ver WL, and Restrepo D, editors. The neurobiology of taste and smell. New York: Wiley-Liss. p 287-314.

Finger TE. 1978. Gustatory pathways in the bullhead catfish. II. Facial lobe connections. J Comp NeuroI180:691-705.

Frank M. 1973. An anal ysis of hamster afferent taste nerve response functions. J Gen PhysioI61:588-618.

Frank ME, Bieber SL, and Smith DV. 1988. The organization of taste sensibilities in hamster chorda tympani nerve fibers. J Gen Physiol 91:861-896.

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.

11

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.

Hansen A, Reutter K, and Zeiske E. 2002. Taste bud development in the zebrafish, Dania reria. Dev Dyn 223:483-496.

Hemess S and Chen YS. 1997. Serotonin inhibits calcium-activated K+ current in rat taste receptor cells. Neuroreport 8:3257-3261.

Hemess S, Zhao FL, Kaya N, Lu SG, Shen T, and Sun XD. 2002a. Adrenergic signalling between rat taste receptor cells. J Physiol 543:601-614.

Hemess S, Zhao FL, Lu SG, Kaya N, and Shen T. 2002b. Expression and physiological actions of cholecystokinin in rat taste receptor cells. J Neurosci 22:10018-10029.

Herrick CJ. 1901. The cranial nerves and cutaneous sense organs of the north american siluroid fishes. J Comp Neuroll1(3): 178-249.

Herrick CJ. 1905. The central gustatory paths in the brains of bony fishes. J Comp Neurol 15:375-455.

Hirata Y. 1966. Fine structure of the terminal buds on the barbels of some fishes. Arch Histol Jpn 26:507-523.

Jain S and Roper SD. 1991. Immunocytochemistry of gamma-aminobutyric acid, glutamate, serotonin, and histamine in Necturus taste buds. J Conlp Neurol 307:675-682.

Jakubowski M. 1983. New details of the ultrastructure (TEM, SEM) of taste buds in fishes. Z Mikrosk Anat Forsch-62.

Jakubowski M and Whitear M. 1990. Comparative Morphology and cytology of taste buds in teleosts. Z Mikrosk Anat Forsch 104:529-560.

Kanner B.L 2000. Cytoplasmic membrane transporters for GABA. In Martin DL and olsen RW, editors. GABA in the nervous system: the view at fifty years. Philadelphia: Lippincott Williams & Wilkins. p 43-49.

Kanwal JS and Caprio J. 1983. An electrophysiological investigation of the oro­pharyngeal (IX-X) taste system in the channel catfish, lctalurus punctatus. J Comp Physiol A 150:345-357.

Kanwal JS, Hidaka I, and Caprio J. 1987. Taste responses to amino acids from facial nerve branches innervating oral and extra-oral taste buds in the channel catfish, lctalurns punctatus. Brain Res 406:105-112.

Kapoor BG, Evans HE, and Pevzner RA. 1975. The gustatory system in fish. Adv Mar Biol 13:53-108.

Kim D-J and Roper SD. 1995. Localization of serotonin in taste buds: A comparative study in four vertebrates. J Comp Neurol 353:364-370.

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.

Marc RE, Murry RF, and Basinger SF. 1995. Pattern recognition of amino acid signatures in retinal neurons. J Neurosci 15:5106-5129.

Murray RO. 1971. Ultrastructure of taste receptors. In Beidler LM, editor. Handbook of sensory physiology. Volume IV. Chemical senses. Part 2. Berlin: Springer­Verlag. p 31-50.

Murray RO. 1973. The ultrasturcture of taste buds. In Friedman I, editor. The ultrastructure of sensory organs. Anlsterdam: North-Holland Publishing Co. p3-37.

Obata H, Shimada K, Sakai N, and Saito N. 1997. OABAergic neurotransmission in rat taste buds: immunocytochemical study for OABA and OABA transporter SUbtypes. Brain Res Mol Brain Res 49:29-36.

Ren Y, Shimada K, Shirai Y, Fujimiya M, and Saito N. 1999. Immunocytochemical localization of serotonin and serotonin transporter (SET) in taste buds of rat. Brain Res Mol Brain Res 74:221-224.

Reutter K. 1971. The taste-buds of Amiurus nebulosus (Leseur) Morphological and histochemical investigations. Z Zellforsch 120:280-308.

Reutter K. 1978. Taste organ in the bullhead (Teleostei). Adv Anat Embryol Cell BioI 55:1-98.

Reutter K. 1982. Taste organ in the barbel of the bullhead. In Hara TJ, editor. Chemoreception in fishes. Amsterdam: Elsevier Scientific Publishing Company. p 77-91.

Reutter K. 1986. Chemoreceptors. In Bereiter-Hahn J, Matoltsy AO, and Richards KS, editors. Biology of the integument: Vol. 2 Vertebrates. New York: Springer­Verlag. p 586-604.

Reutter K and Witt M. 1993. Morphology of vertebrate taste organs and their nerve supply. In Simon SA and Roper SD, editors. Mechanisms of taste transduction. Boca Raton: CRC. p 29-82.

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.

Royer SM and Kinnamon JC. 1996. Comparison of high-pressure freezing/freeze substitution and chemical fixation of catfish barbel taste buds. Microsc Res Tech 35:385-412.

Schousboe A. and Kanner B. 2002. GABA transporters: functional and pharmacological properties. In Egebjerg J, Schouboe A., and Krogsgaard-Larsen P., editors. Glutamate and GABA receptors and transporters. Structure, function, and pharmacology. New York: Taylor and Francis. p 337-349.

Smith DV and Davis BJ. 2000. Neural Representation of Taste. In Finger TE, Silver WL, and Restrepo D, editors. The neurobiology of taste and smell. New York: Wiley­Liss. p 353-394.

Smith DV and Hanamori T. 1991. Organization of gustatory sensitivities in hamster superior laryngeal nerve fibers. J NeurophysioI65:1098-1114.

Toyoshima K, Nada 0, and Shimamura A. 1984. Fine structure of monoamine-containing basal cells in the taste buds on the barbels of three species of teleosts. Cell Tissue Res 235:479-484.

Tucker D. 1983. Fish chemoreception: Peripheral anatomy and physiology. In Northcutt RG and Davis RE, editors. Fish neurobiology: vol. 1. Brain stem and sense organs. Ann Arbor: The University of Michigan Press. p 311-350.

Wisden Wand Farrant M. 2002. Insights into GABAA receptor complexity from the study of cerebellar granule cells. In Egebjerg J, Schousboe A, and Krogsgaard­Larsen P, editors. Glutamate and GABA receptors and transporters. Structure, function and pharmacology. New York: Taylor & Francis. p 189-201.

Yamamoto T, Nagai T, Shimura T, and Yasoshima Y. 1998. Roles of chemical mediators in the taste system. Jpn J Pharmacol 76:325-348.

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

rabbit antibodies (with final dilution): anti-L-glutamate (GLU, 1 :32000 dilution), anti-y­

aminobutyric acid (GABA, 1:32000 dilution), anti-taurine (TAU, 1:16000 dilution), anti­

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

numerous taste receptor cells (Fig. 2.1B). Dark, granular pigment cells were noted

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

fibers innervate taste buds (Lu and Roper, 1993), non-NMDA glutamate receptor activity

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.

References

Atema J. 1971. Structures and functions of the sense of taste in the catfish, Ictalurus natalis. Brain Behav EvoI4:273-294.

Bardach JE, Todd JH, and Crickmer R. 1967. Orientation by taste in fish of the genus Ictalurus. Science 155:1276-1278.

Bensouilah M and Denizot J-P. 1991. Taste buds and neuromasts of Astyanaxjordani: Distribution and immunochemical demonstration of co-localized substance P and enkephalins. Eur J Neurosci 3:407-414.

Boudriot F and Reutter K. 2001. Ultrastructure of the taste buds in the blind cave fish Astyanax jordani ("Anoptichthys") and the sighted river fish Astyanax mexicanus (Teleostei, Characidae). J Comp Neurol 434:428-444.

Bradley RM and Grabauskas G. 1998. Neural circuits for taste - Excitation, inhibition, and synaptic plasticity in the rostral gustatory zone of the nucleus of the solitary tract. Ann NY Acad Sci 855:467-474.

Caicedo A, Kim KN, and Roper SD. 2000. Glutamate-induced cobalt uptake reveals non­NMDA receptors in rat taste cells. J Comp NeuroI417:315-324.

Caprio J, Brand JG, Teeter JH, Valentincic T, Kalinoski DL, Kohbara J, Kumazawa T, and Wegert S. 1993. The taste system of the channel catfish: From biophysics to behavior. Trends Neurosci 16:192-197.

Crisp M, Lowe GA, and Laverack MS. 1975. On the ultrastructure and permeability of taste buds of the marine teleost Ciliata mustela. Tissue Cell 7: 191-202.

Delay RJ, Taylor R, and Roper SD. 1993. Merkel-like basal cells in Necturus taste buds contain serotonin. J Comp Neurol 335:606-613.

Eram M and Michel WC. 2001a. Amino acid immunoreactivity of channel catfish peripheral taste system. Chern Sens 26(8), 1125.

Eram M and Michel WC. 2001 b. Comparison of the amino acid composition of vagal and facial nerve innervated taste buds in the channel catfish. 31 st Annual Soc for Neurosci 287.10, 148.

48

Farbman AI. 1965. Fine structure of the taste bud. J Ultrastruct Res 12:328-350.

Fujimoto S and Yamamoto K. 1980. Electron microscopy of tenninal buds on the barbels of the silurid fish, Corydoras paleatus. The Anatomical Record 197:133-141.

Ganchrow JR. 2000. Taste cell function: Structural and biochemical implications. Physiology and Behavior 69:29-40.

Hamasaki K, Seta Y, Yamada K, and Toyoshima K. 1998. Possible role of serotonin in Merkel-like basal cells of the taste buds of the frog, Rana nigromaculata. J Anat 193:599-610.

Hansen A, Reutter K, and Zeiske E. 2002. Taste bud development in the zebrafish, Danio rerio. Dev Dyn 223:483-496.

Hirata Y. 1966. Fine structure of the terminal buds on the barbels of some fishes. Arch Histol Jpn 26:507-523.

Huang YJ and Lu KS. 1996. Immunohistochemical studies on protein gene product 9.5, serotonin and neuropeptides in vallate taste buds and related nerves of the guinea pig. Arch Histol Cytol 59:433-441.

Ito M. 1986. The innermost cell layer of the outer root sheath in anagen hair follicle: light and electron microscopic study. Arch Dermatol Res 279:112-119.

Ito M, Tazawa T, Shimizu N, Ito K, Katsuumi K, Sato Y, and Hashimoto K. 1986. Cell differentiation in human anagen hair and hair follicles studied with anti-hair keratin monoclonal antibodies. J Invest Dermatol 86:563-569.

Jain S and Roper SD. 1991. Immunocytochemistry of gamma-aminobutyric acid, glutamate, serotonin, and histamine in Necturus taste buds. J Comp Neurol 307:675-682.

Jakubowski M. 1983. New details of the ultrastructure (TEM, SEM) of taste buds in fishes. Z Mikrosk Anat Forsch-62.

Jakubowski M and Whitear M. 1990. Comparative morphology and cytology of taste buds in teleosts. Z Mikrosk Anat Forsch 104:529-560.

Johnson EW, Eller PM, Jafek BW, and Norman AW. 1992. Calbindin-like immunoreacti vity in two peripheral chemosensory tissues of the rat: taste buds and the vomeronasal organ. Brain Res 572:319-324.

Joyce EC and Chapman GB. 1978. Fine structure of the nasal barbel of the channel catfish, Ictalurus punctatus. J MorphoI158:109-153.

Kalloniatis M and Fletcher EL. 1993. Immunocytochemical localization of the amino acid neurotransmitters in the chicken retina. J.Comp Neurol. 336, 174-193.

Kanwal JS and Caprio J. 1983. An electrophysiological investigation of the oro­pharyngeal (IX-X) taste system in the channel catfish, Ictalurus punctatus. J Comp Physiol A 150:345-357.

Kanwal JS, Hidaka I, and Caprio J. 1987. Taste responses to amino acids from facial nerve branches innervating oral and extra-oral taste buds in the channel catfish, Ictalurus punctatus. Brain Res 406: 105-112.

Kim D-J and Roper SD. 1995. Localization of serotonin in taste buds: A comparative study in four vertebrates. J Comp Neurol 353:364-370.

49

Lu K-S and Roper SD. 1993. Electron microscopic immunocytochemistry of glutamate­containing nerve fibers in the taste bud of mudpuppy (Necturus maculosus). Microsc Res Tech 26:225-230.

Marc RE and Liu W. 2000. Fundamental GABAergic amacrine cell circuitries in the retina: nested feedback, concatenated inhibition, and axosomatic synapses. J Comp NeuroI425:560-582.

Marc RE, Liu WL, Scholz K, and Muller JF. 1988. Serotonergic and serotonin­accumulating neurons in the goldfish retina. J Neurosci 8:3427-3450.

Marc RE, Murry RF, and Basinger SF. 1995. Pattern recognition of amino acid signatures in retinal neurons. J Neurosci 15:5106-5129.

Marc RE, Wei-Ley SL, Kalloniatis M, Raiguel SF, and Van Haesendonck E. 1990. Patterns of glutamate immunoreactivity in the goldfish retina. J Neurosci 10:4006-4034.

Marui T and Caprio J. 1992. Teleost gustation. In Hara TJ, editor. Chemoreception in fishes. Amsterdam: Elsevier. p 171-198.

Medler KF, Margolskee RF, and Kinnamon SC. 2003. Electrophysiological characterization of voltage-gated currents in defined taste cell types of mice. J Neurosci 23:2608-2617.

Miyawaki Y, Morisaki I, Tabata MJ, Kurisu K, and Wakisaka S. 1996. Calbindin D28k­like immunoreactivity in the gustatory epithelium in the rat. Neurosci Let 214:29-32.

Miyawaki Y, Morisaki I, Tabata MJ, Maeda T, Kurisu K, and Wakisaka S. 1998. Calbindin D28k-like immunoreactivity in the developing and regenerating circumvallate papilla of the rat. Cell Tissue Res 291 :81-90.

Montmayeur JP and Matsunami H. 2002. Receptors for bitter and sweet taste. Curr Opin Neurobiol 12:366-371.

50

Murray RO. 1971. Ultrastructure of taste receptors. In Beidler LM, editor. Handbook of sensory physiology. Volume IV. Chemical senses. Part 2. Berlin: Springer­Verlag. p 31-50.

Murray RO. 1973. The ultrasturcture of taste buds. In Friedman I., editor. The ultrastructure of sensory organs. Amsterdam: North-Holand Publishing C. p3-37.

Nagai T, Delay RJ, Welton J, and Roper SD. 1998. Uptake and release of neurotransmitter candidates, [3H]serotonin, [3H]glutamate, and [3H]gamma­aminobutyric acid, in taste buds of the mudpuppy, Necturus maculosus. J Comp Neurol 392: 199-208.

Nelson OM and Finger TE. 1993. Inlmunolocalization of different fonns of neural cell adhesion molecule (NCAM) in rat taste buds. J Comp Neurol 336:507-516.

Obata H, Shimada K, Sakai N, and Saito N. 1997. OABAergic neurotransmission in rat taste buds: immunocytochemical study for OABA and GABA transporter SUbtypes. Brain Res Mol Brain Res 49:29-36.

Orwin DF. 1971. Cell differentiation in the lower outer sheath of the Romney wool follicle: a companion cell layer. Aust J BioI Sci 24:989-999.

Reutter K. 1971. The taste-buds of Amiurus nebulosus (Leseur) Morphological and histochemical investigations. Z Zellforsch 120:280-308.

Reutter K. 1973. Morphological and neurohistochenlical investigations on Xiphophorus helleri Heckel (Poeciliidae, Cyprinodontifonnes, Teleostei). Z Zellforsch 143:409-423.

Reutter K. 1978. Taste organ in the bullhead (Teleostei). Adv Anat Embryol Cell BioI 55:1-98.

Reutter K. 1986. Chemoreceptors. In J.Bereiter-Hahn, A.O.Matoltsy, and K.S.Richards, editors. Biology of the integument: Vol. 2 Vertebrates. New York: Springer­Verlag. p 586-604.

Royer SM and Kinnamon JC. 1996. Comparison of high-pressure freezing/freeze substitution and chemical fixation of catfish barbel taste buds. Microsc Res Tech 35:385-412.

Smeraski CA, Bottger B, and Finger TE. 2001. Kainate-activated cobalt uptake in the primary gustatory nucleus in goldfish: visualization of the morphology and distribution of cells expressing AMPAlkainate receptors in the vagal lobe. J Comp NeuroI431:59-74.

Smith DV, Akeson RA, and Shipley MT. 1993. NCAM expression by subsets of taste cells is dependent upon innervation. J Comp Neurol 336:493-506.

51

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.

Takeda M, Suzuki Y, Obara N, and Breipohl W. 1999. Expression of the neural cell adhesion molecule in mouse taste buds after denervation. J Electron Microsc (Tokyo) 48:39-45.

Toyoshima K. 1989. Ultrastructure and immunohistochemistry of the basal cells in the taste buds of the loach, Misgumus anguillicaudatus. J Submicrosc Cytol Pathol 21:771-774.

Van Bel AJ, Ehlers K, and Knoblauch M. 2002. Sieve elements caught in the act. Trends Plant Sci 7: 126-132.

Welton J, Taylor R, Porter AJ, and Roper SD. 1992. Immunocytochemical survey of putative neurotransmitters in taste buds from Necturus maculosus. J Comp Neurol 324:509-521.

Yang RB, Tabata S, Crowley HH, Margolskee RF, and Kinnamon JC. 2000. Ultrastructural localization of gustducin immunoreactivity in microvilli of type II taste cells in the rat. J Comp NeuroI425:139-151.

Yee CL, Yang R, Bottger B, Finger TE, and Kinnamon JC. 2001. "Type III" cells of rat taste buds: immunohistochemical and ultrastructural studies of neuron-specific enolase, protein gene product 9.5, and serotonin. J Comp NeuroI440:97-108.

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.

Facial (7th cranial), glossopharyngeal (9th cranial) and vagal (10th cranial) nerves

innervate taste buds in the specific regions of oropharyngeal cavity. In catfish, a highly

54

elaborated facial taste system sensitive to amino acid stimuli (Kanwal and Caprio, 1983;

1987; Caprio et aI., 1993) innervates taste buds located in the anterior mouth and over the

entire body, and is important for food search behavior, while oropharyngeal taste buds

innervated by the glossopharyngeal or vagal nerves are critical for the final acceptance or

rejection of food (Atema, 1971). We recently reported that while facial nerve innervated

taste buds (FITBs) had more taste cells and a larger nerve plexus (personal

communication, Eram and Michel, in review), vagal nerve innervated taste buds (VITBs)

contained significantly more GABA positive elements and may thus be subject to greater

inhibitory regulation.

Physiological studies have identified alanine-best, arginine-best and arginine­

proline nerve fibers (Kohbara et aI., 1992) with subsets of these fibers variably sensitive

to quinine (Ogawa et aI., 1997). Such diversity in the response of individual afferent

nerve fibers presumably requires receptor elements with similar diversity. Several

transduction mechanisms are known to occur in catfish taste cells. For example, alanine

and several other amino acids engage G-protein coupled receptors (Kalinoski et aI., 1987;

1992), while arginine and proline acti vate ionotropic receptors (Teeter et aI., 1990; 1992).

Taste cells in fish have been classified as dark, light or intermediate cells based on

ultrastructural criteria (Crisp et aI., 1975; Kapoor et aI., 1975; Grover-Johnson and

Farbman, 1976; Reutter, 1978; Tucker, 1983; Reutter, 1986; Caprio, 1988; Reutter and

Witt, 1993). Three cell types are insufficient to account for the range of specificities

described above indicating that one or more of these cell groups must have additional

heterogenei ty.

In the present investigation of FITBs and VITBs of the channel catfish, we have

classified taste cells based on levels of immunoreactivity to y-aminobutyric acid

(GABA), glutamate, aspartate, taurine, alanine and glutathione. This

immunocytochemically-based profiling technique has previously been used to classify

retinal neurons (Marc et aI., 1995). While cells with similar metabolite profiles occur in

both types of taste buds, the relative distribution of cell classes varies considerably.

Materials and Methods

Animal Care

55

Six juvenile catfish, Ictalurus punctatus, (5-12 grams, 7-10 cm total length) were

held up to three days 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 in accord with

experimental procedures approved by the Institutional Animal Care and Use Committee

of the University of Utah.

Tissue Preparation

Following decapitation of the animals, maxillary barbels and the 2nd through 5th

gill arches were rapidly dissected 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 immediately

transferred to a cold fixative (4°C) containing 1 % paraformaldehyde, 2.5%

glutaraldehyde, 3% sucrose, 0.01 % CaCh in 0.1 M phosphate buffer (PB), pH 7.4,

overnight. Fixed tissue was dehydrated through graded methanol (75%, 85%, 95%, and

100%) and 100% acetone for 20 minutes each, agitated in 50% acetone and 50% Eponate

56

plastic (Ted Pella Inc., Redding, CA, USA) overnight, and taken through two 1 hour

changes of fresh 100% Eponate before curing overnight at 65°C. U"ltra-thin (50-70 nm)

horizontal serial sections of taste buds were obtained using a Leica Ultracut T microtome

(Leica, Vienna, Austria) and diamond knife (Delaware Diamond Knife, Inc., Wilmington,

DE, USA) and collected in individual wells of Teflon-coated spot slides (Erie Scientific

Co., Portsmouth, NH, USA). To insure that sections were obtained from comparable

regions of different taste buds, each bud was sectioned from base to apex with a series of

consecutive 50-70 nm thick sections collected for immunostaining at 10 J.Lm intervals.

For each taste bud, only the series of sections collected at the widest part of the bud were

analyzed.

Immunocytochemistry

Postembedding immunocytochemical procedures employed techniques described

by Marc et al. (1990, 1995). In brief, sections were deplasticized with 25% sodium

ethoxide for 7 minutes, rinsed in 100% methanol (3 x 2 minutes), rinsed in ultra-pure

water for 5 minutes, and dried. Individual sections were then incubated overnight at

room temperature in a humidified chamber with one of the following rabbit polyclonal

primary antibodies conjugated with bovine serum albumin (BSA) by glutaraldehyde

(Marc et at, 1988; 1990): anti-L-glutamate (GLU, 1:32000 dilution), anti-y-aminobutyric

acid (GABA, 1:32000 dilution), anti-taurine (TAU, 1:16000 dilution), anti-glutathione

(GSH, 1:4000 dilution), anti-L-alanine (ALA, 1:8000 dilution), or anti-L-aspartate (ASP,

1:2000 dilution) (Signature Immunologics, Salt Lake City, UT, USA). The primary

antibodies were diluted in 0.1 M PB containing 1 % goat serum and 0.05% thiomerosal

57

(pH 7.4). After rinsing with 0.1 M PB, sections were incubated for 1 hour at room

temperature in nanogold-conjugated goat anti-rabbit secondary antibody (1 nm, 1:50

dilution; Amersham Corp., Arlington Heights, IL, USA), washed with 0.1 M PB for 1

hour, and silver intensified using 0.14% silver nitrate in a hydroquinone (43 mM)/citrate

buffer (64 mM, citric acid; 141 mM sodium citrate) solution for 3 minutes at 32°C

(Kalloniatis and Fletcher, 1993). The slides were coversliped using cover glass and

Eponate and then cured at 65°C overnight. Incubation of the tissue without the primary

antibodies and only secondary antibody eliminated immunoreactivity (IR).

Image Acquisition, Registration and Analysis

Taste buds, viewed at high magnification (100x) with a Zeiss Axioplan2

microscope, were captured as 8-bit gray scale digital images (1300 x 1030 pixels) using a

Zeiss Axiocam CCD camera and Axiovision imaging software 3.0 (Thornwood, NY,

USA). Identical illumination, microscope and camera settings were used in a single

session for capturing images of each metabolite across the six facial and six vagal­

innervated taste buds examined. Details of the imaging technique have been described by

Marc et al. (1995). Each of the immunostained images of a taste bud was aligned using

image analysis software (Geomatica software 8.0, PCI Remote Sensing, Richmond Hill,

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

66

1.0 A.GABA

1.0 B.TAU

1.0 C.GSH

0.8 O.S 0.8

0.6 0.6 0.6

..... 0.4 0.4 0.4

§ 0 0.2 0.2 0.2 U aJ 0.0 0.0 0.0 ~ 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 c: ] D.ALA E.GLU F.ASP

1 1.0 1.0 1.0

0.8 0.8 0.8

~ 0.6 0.6 0.6

0.4 0.4 0.4

0.2 0.2 0.2

0.0 0.0 0 50 100 150 200 250 0 50 100 150 200 250 50 100 150 200

Relative Concentration

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.

C 240 160 80 II

240 >-. 160

."'::: 80 ~ O ~ ...............

..s 160

GABA

GSH ~ 240 ~ ....... 80 ... ~ 0 +-............ --... l,.I, L,-I, ..... -... .... -..

i:5: 240 Q) 1M 00 80

E! 0 Q)

3: 2411 ....... 160

80

160 80

240 1 O+-.......... -....-..J\I,... .............. -...IIIIIII-.

CJ C2 C3 C4 C5

Cluster # C6

74

75

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.

77

240

160

80

0

240

160

80

---~ ~ 0

'-'

.0 .~

240 r:J'J

5 160 ...... ~ 80 'il li< 0 j£ (L)

~ as

240

> 160 < 80

0

240

160

80

0

240

160

80

0

CI C2 C3 C4 C5 C6 C7 C8 C9 CIO CII Cl2 C13 C14 CI5

Cluster #

78

Cluster 9. The intermediate TAU IR of Clusters 7 and 11 were significantly lower than

Clusters 8, 12 and 15. Cells in Cluster 7 could be distinguished from cells in Cluster 11

by their higher GSH and GLU IR. High GLU and ASP levels separated Cluster 8 from

Clusters 12 and 15. Cells in Clusters 12 and 15 were separated on the basis of ASP and

GSH IR. Results of the pairwise comparison for each metabolite between every pair of

clusters are shown in Table 3.1. In each cluster there were significantly different API

values for the metabolites in comparison to other clusters. For example, with respect to

GABA, Cluster 1 had a significantly different API value from Clusters 2 and Clusters 4-

15. The GABA API values of clusters 1 and 3 were not significantly different.

The only cluster that contained cells found exclusively from either FITBs or

VITBs was Cluster 3 with two FITB cells. However, differences in the relative numbers

of FITB and VITB cells were noted in other clusters (Table 3.2). Since different numbers

of cells were sampled from FITBs and VITBs the percentages of total FITB or VITB

cells rather than absolute numbers of cells is a more appropriate comparison. The

clusters containing the GABA high cells contained of 15.2% of the total VITB cells, but

only 5.6% of the total FITB cells. The greatest disparities were noted in Clusters 1 and 2

with more than 4-fold more VITB than FITB cells. In contrast, Cluster 4 contained

similar numbers of VITB and FITB cells. Cluster 5 of GABA intermediate cells was 3

times more common in VITBs, while cells in Cluster 6 were 4 times more common in

FITBs. Overall there were similar numbers of cells in the GABA low clusters but there

was considerable variability in the indi vidual clusters. Clusters 9-11 contained from 3 to

7.8 times more FITB cells. In contrast, Cluster 15 contained approximately 8.5 times

more VITB cells.

Table 3.1. Significant differences among clusters based on pairwise comparisons (p > 0.05)

79

OABA TAU OSH

Cluster 1 2,4-15 2,4-15 4-8,10-15

2 1,4-15 1,3-10,12-15 4-12,14,15

3 5-15 2,4-12,15 4,5,7,8,10-12,14

4 1,2,5-15 1-3,5-15 1-3,5-15

5 1-4,6-15 1-4,6,7,9-15 1-4,6-11,13-15

6 1-5,7-15 1-5,7-15 1-5,7-13,15

7 1-6,10-15 1-6,8-15 1-6,8-15

8 1-6,10-15 1-4,6,7,9-15 1-7,9-11,13-15

9 1-6,10-15 1-8,10-15 2,4-8,10-15

10 1-9,12-15 1-9,11-15 1-9,11-15

11 1-9,12-15 1,3-10,12-15 1-10,12,13,15

12 1-11,15 1-11,13,14 1-4,6,7,9-11,13-15

13 1-11,14,15 1,2,4-12,15 1,4-12

14 1-11,13,15 1,2,4-12,15 1-5,7-10,12,13,15

15 1-14 1-11,13-15 1,2,4-12,14

ALA OLU

2-15 2-4,6-15

1,3-15 1,4-15

1,2,4-12,15 1,5,6,8,10,12,14

1-3,5-15 1-3,5-10,13-15

1-4,7,9,12-15 2-4,6-11,13-15

1-4, 7,11-15 1,2,5,7-10,13-15

1-6,8-11,13-15 1,2,4-6,8-15

1-4,7,12-15 1-7,9-15

1-5,7,10-15 1,2,4-8,10-15

1-4,7,9,11-15 1-9,11-13,15

1-4,6,7,9,10,12-15 1,2,5,7-10,13-15

1-6,8-11,13-15 1-3,7-11,13-15

1,2,4-12,15 1,2,4-12,14

1,2,4-12,15 1-9,11-13,15

1-14 1,2,4-12,14

ASP

3,6-15

1,3-14

1,2,4-7,9-15

2,3,5-15

2-4,6-11,13-15

1-5,7-15

1-6,8-15

1,2,4-7,9-15

1-8,10,13-15

1-9,11-15

1-8,10,13-15

1-3,6-10,13-15

1-12,14,15

1-13,15

1,3-14

00 o

Table 3.2. The distJibution of FITB and VITB cells based on their GABA immunoreactivity pattern.

81

NTota1= 2118 Npacial=1303 NVagal=815

Cluster NTotal %Total NTotal %Total NTotal %Total

1 52 2.5 14 1.1 38 4.7 GABA 2 57 2.7 8 0.6 49 6.0 high 3 2 0.1 2 0.2 0 0.0

4 86 4.1 49 3.8 37 4.5

Total 197 9.4 73 5.7 124 15.2

GABA 5 171 8.1 46 3.5 125 15.3 int. 6 192 9.1 168 12.9 24 2.9

Total 363 17.1 214 16.4 149 18.2

7 124 5.9 90 6.9 34 4.2 8 27 1.3 23 1.8 4 0.5 9 430 20.3 357 27.4 73 9.0

GABA 10 159 7.5 148 11.4 11 1.4 low 11 147 6.9 119 9.1 28 3.4

12 184 8.7 138 10.6 46 5.6 13 101 4.8 65 5.0 36 4.4 14 22 1.0 19 1.5 3 0.4 15 364 17.2 57 4.4 307 37.7

Total 1558 73.6 1016 78.0 542 66.5

82

Radial Distribution of the Cells

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.

en ......t ......t

a ~ o ~ (I.)

~ z

120 1001 Class 1

80 60 40 20

FITBs _VITBs

01 , .i-.'~~ I I

0.0 0.2 0.4 0.6 0.8 1.0 1.2

120 100~ Class 6

80 60 40 20 o 1c=J' II " II 11 II II 11 " I

0.0 0:2 0:4 0~6 0:8 1 :0 1 :2

120 100 t Class 10

80 60 40 20 o 1==/ /I II /I II I~I II " II I

0.0 0:2 0~4 oJi 0:8 1 :0 1 :2

120 1001 Class 14

80 60 40 20 o I r=1 r=1 .............. ,....,-

0.0 0:2 0:4 0:6 0:8 1:0 1 :2

120 100 ~ Class 2

80 60 40 20

01 ,--, __ ,!I!I,IIII., i

0.0 0.2 0.4 0.6 0.8 1.0 1.2

1201 Class 7 100

80

120 100 ~ Class 4

80 60 40 20 o 1 1-M,p!II!~~O~"',C1 0.0 0.2 0.4 0.6 0.8 1.0 1.2

1201 Class 8 100

80

601 60 40 40

2~ =, -==.nnU[b, 2~ I , , 1'""'=/ I=,= , 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2

120 Class 11 120, Class 12 100 100

80 80 60 60 40 40 20 20 o 0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2

120 1001 Class 15

80 60 40 20

0.2 0.4 0.6 0.8 1.0 1.2

Distance from the Center of the Bud

120 100 ~ Class 5

80 60 40 20 o , t==1 -'"'' II 1_ 0.0 0:2 0:4 <16 0:8 1 :0 1 :2

120 1001 Class 9

80 60 40 20

01 ...... ' " II " ., U II 11 11 !

0.0 0:2 0:4 0:6 0:8 1 :0 1 :2

120 1001 Class 13

80 60 40 20 0, "'C:"=if " '0' " Ir--I

0.0 0:2 0:4 0:6 0:8 1:0 1 :2

00 ~

85

tissues (Huxtable, 1992; 1996; Eram and Michel, 2001 a; Schuller-Levis and Park, 2003);

it reportedly functions as a neuromodulator or a tissue protective osmolite. As a major

endogenous nonprotein thiol, glutathione is involved in protecting cells from reactive

oxygen species (Dickinson and Forman, 2002). The presence of each of these

metabolites in particular subtypes of taste cells is considered to represent a measure of the

functional diversity of these cells.

Our approach to classification using low molecular weight markers is compatible

with electron microscopy, not restricted to two to three markers, and comparable across

species. We used a battery of five amino acids and the peptide GSH as markers in the

current study but could have tested more had other useful markers been identified.

Classification by metabolite profiling provides some information about cellular function,

for example the cell's neurotransmitter profile, but understanding specific functions is

likely to require information about macromolecular expression, for example, taste

receptor expression.

Using patterns of codistribution, we identified 15 clusters of channel catfish taste

cells, each with a distinct metabolite profile. The present approach to taste cell

classification was organized around GABA, since GABA IR profiles have the most

diverse pattern of expression of the five amino acids under consideration and since

GABA is widely acknowledged as a potent inhibitory neurotransmitter and/or

neuromodulator, which could significantly influence gustatory transduction in taste cells.

The k-means classification identified four GABA rich clusters comprising 9.4%, two

GABA intermediate clusters comprising 17.1 % and nine GABA low clusters comprising

73.6% of the cells sampled. Cells from each of these clusters were found in both FITBs

86

and VITBs, but differences in their relative abundance were noted and may contribute to

differences in the response properties noted in afferent nerve recordings (Kanwal and

Caprio, 1983; 1987; Kanwal et aI., 1987).

The potential role of GABA as a modulator of transductive events within taste

buds is an intriguing possibility. Although GABA was first reported in Necturus (Jain

and Roper, 1991) and later in mouse (Obata et aI., 1997) taste buds, its function was not

determined. In catfish, the majority of GABA rich and GABA intermediate cells appear

to correspond to dark cells (personal communication, Eram and Michel, in review),

whose thin glial like processes wrap around and thereby separate the light cells from one

another. Several studies (Hirata, 1966; Graziadei, 1969; Fujimoto and Yamamoto, 1980;

Toyoshima et aI., 1984; Jakubowski and Whitear, 1990) have suggested that dark cells

serve in a supporting capacity; however, given their high GABA levels and close

apposition to the light cells, a more active functional role seems probable. If GABA

serves as an inhibitory modulator of peripheral gustatory activity, the higher number of

GABA rich cells (~ 211bud) in VITBs compared to FITBs (~ 121bud) may explain the

higher vagal and glossopharyngeal, compared to the facial, nerve thresholds for the

amino acid stimuli in catfish (Kanwal and Caprio, 1983; 1987; Kanwal, Hidaka, and

Caprio, 1987). Further study is warranted to determine if GABA is involved in these

differences in gustatory sensitivity.

Two to three taste cell types have been reported in fish (Kapoor, Evans, and

Pevzner, 1975; Tucker, 1983; Reutter, 1986; Caprio, 1988; Boudriot and Reutter, 2001;

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.

References

Adler E, Hoon MA, Mueller KL, Chandrashekar J, Ryba NJP, and Zuker CS. 2000. A novel family of mammalian taste receptors. Cell 100:693-702.

89

Aiuchi T, Kamo N, Kurihara K, and Kobatake Y. 1976. Physicochemical studies of taste reception VI: Interpretation of anion influences on taste response. Chern Senses Flav 2:107-119.

Atema J. 1971. Structures and functions of the sense of taste in the catfish, Ictalurus natalis. Brain Behav EvoI4:273-294.

Boudriot F and Reutter K. 2001. Ultrastructure of the taste buds in the blind cave fish Astyanaxjordani ("Anoptichthys") and the sighted river fish Astyanax mexican us (Teleostei, Characidae). J Comp Neurol 434:428-444.

Caprio J. 1988. Peripheral filters and chemoreceptor cells in fishes. In Atema J, Fay RR, Popper AN, and Tavolga WN, editors. Sensory biology of aquatic animals. NY: Springer-Verlag. p 313-338.

Caprio J, Brand JG, Teeter JH, Valentincic T, Kalinoski DL, Kohbara J, Kumazawa T, and Wegert S. 1993. The taste system of the channel catfish: From biophysics to behavior. Trends Neurosci 16:192-197.

Chandrashekar J, Mueller KL, Hoon MA, Adler E, Feng LX, Guo W, Zuker CS, and Ryba NJP. 2000. T2Rs function as bitter taste receptors. Cell 100:703-711.

Crisp M, Lowe GA, and Laverack MS. 1975. On the ultrastructure and permeability of taste buds of the marine teleost Ciliata mustela. Tissue Cell 7:191-202.

Dickinson DA and Forman HJ. 2002. Glutathione in defense and signaling: lessons from a small thiol. Ann N Y Acad Sci 973:488-504.

Eram M and Michel WC. 2001a. Amino acid immunoreactivity of channel catfish peripheral taste system. Chern Sens 26(8), 1125.

Farbman AI. 1965. Fine structure of the taste bud. J Ultrastruct Res 12:328-350.

Finger TE and Simon CM. 2000. Cell biology of taste epithelium. In Finger TE, Silver WL, and Restrepo D, editors. The neurobiology of taste and smell. New York: Wiley-Liss. p 287-314.

Fujimoto S and Yamamoto K. 1980. Electron microscopy of terminal buds on the barbels of the silurid fish, Corydoras paleatus. The Anatomical Record 197:133-141.

Graziadei PPC. 1969. The ultrastructure of vertebrate taste buds. In Pfaffmann C, editor. Olfaction & taste III. New York: Rockefeller Univ. Press. p 315-330.

90

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.

Hansen A, Reutter K, and Zeiske E. 2002. Taste bud development in the zebrafish, Danio rerio. Dev Dyn 223:483-496.

Hirata Y. 1966. Fine structure of the terminal buds on the barbels of some fishes. Arch Histol Jpn 26:507-523.

Hoon MA, Adler E, Lindemeier J, Battey JF, Ryba NJ, and Zuker CS. 1999. Putative mammalian taste receptors: a class of taste-specific GPCRs with distinct topographic selectivity. Cell 96:541-551.

Huxtable RJ. 1992. Physiological actions of taurine. Physiol Rev 72:101-163.

Huxtable RJ. 1996. Taurine. Past, present, and future. Adv Exp Med BioI 403:641-650.

Jain S and Roper SD. 1991. Immunocytochemistry of gamma-aminobutyric acid, glutamate, serotonin, and histamine in Necturus taste buds. J Comp Neurol 307:675-682.

Jakubowski M and Whitear M. 1990. Comparative morphology and cytology of taste buds in teleosts. Z Mikrosk Anat Forsch 104:529-560.

Kalinoski DL, Bruch RC, and Brand JG. 1987. Differential interaction of lectins with chemosensory receptors. Brain Res 418:34-40.

Kalinoski DL, Johnson LC, Bryant BP, and Brand JG. 1992. Selective interactions of lectins with amino acid taste receptor sites in the channel catfish. Chern Sens 17:381-390.

Kalloniatis M and Fletcher E. 1993. Immunocytochemical localization of the amino acid neurotransmitters in the chicken retina. J Comp Neurol 336:174-193.

Kanwal J and Caprio J. 1987. Central projections of the glossopharyngeal and vagal nerves in the channel catfish, lctalurus punctatus: clues to differential processing of visceral inputs. J Comp Neurol 264:216-230.

Kanwal JS and Caprio J. 1983. An electrophysiological investigation of the oro­pharyngeal (IX-X) taste system in the channel catfish, lctalurus punctatus. J Comp Physiol A 150:345-357.

Kanwal JS, Hidaka I, and Caprio J. 1987. Taste responses to amino acids from facial nerve branches innervating oral and extra-oral taste buds in the channel catfish, lctalurus punctatus. Brain Res 406: 105-112.

Kapoor BG, Evans HE, and Pevzner RA. 1975. The gustatory system in fish. Adv Mar BioI 13:53-108.

91

Kinnamon JC, Taylor BJ, Delay RJ, and Roper SD. 1985. Ultrastructure of mouse vallate taste buds. I. Taste cells and their associated synapses. J Comp Neurol 235:48-60.

Kitagawa M, Kusakabe Y, Miura H, Ninomiya Y, and Hino A. 2001. Molecular genetic identification of a candidate receptor gene for sweet taste. Biochem Biophys Res Commun 283:236-242.

Kohbara J, Michel W, and Caprio J. 1992. Responses of single facial taste fibers in the channel catfish, lctalurus punctatus, to amino acids. J Neurophysiol 68: 1012-1026.

Marc RE, Liu WL, Scholz K, and Muller JF. 1988. Serotonergic and serotonin­accumulating neurons in the goldfish retina. J Neurosci 8:3427-3450.

Marc RE, Murry RF, and Basinger SF. 1995. Pattern recognition of amino acid signatures in retinal neurons. J Neurosci 15:5106-5129.

Marc RE, Wei-Ley SL, Kalloniatis M, Raiguel SF, and Van Haesendonck E. 1990. Patterns of glutamate immunoreactivity in the goldfish retina. J Neurosci 10:4006-4034.

Matsunami H, Montmayeur JP, and Buck LB. 2000. A family of candidate taste receptors in human and mouse. Nature 404:601-602.

Montmayeur JP and Matsunami H. 2002. Receptors for bitter and sweet taste. Curr Opin NeurobiolI2:366-371.

Murray RG. 1969. Cell types in rabbit taste buds. In Pfaffmann C, editor. Olfactory and taste, Vol III. New York: Rockeffeler UP. p 331-344.

Murray R. 1973. The ultrasturcture of taste buds. In Friedman I, editor. The ultrastructure of sensory organs. Amsterdam: North-Holland Publishing Co. p 3-37.

Obata H, Shimada K, Sakai N, and Saito N. 1997. GABAergic neurotransmission in rat taste buds: immunocytochemical study for GABA and GABA transporter SUbtypes. Brain Res Mol Brain Res 49:29-36.

Ogawa K, Marui T, and Caprio J. 1997. Quinine suppression of single facial taste fiber responses in the channel catfish. Brain Res 769:263-272.

Reutter K. 1978. Taste organ in the bullhead (Teleostei). Adv Anat Embryol Cell BioI 55:1-98.

Reutter K. 1986. Chemoreceptors. In Bereiter-Hahn J, Matoltsy AG, and Richards KS, editors. Biology of the Integument: Vol. 2 Vertebrates. New York: Springer­Verlag. p 586-604.

Reutter K and Witt M. 1993. Morphology of vertebrate taste organs and their nerve supply. In Simon SA and Roper SD, editors. Mechanisms of taste transduction. Boca Raton: CRC. p 29-82.

92

Royer SM and Kinnamon JC. 1996. Comparison of high-pressure freezing/freeze substitution and chemical fixation of catfish barbel taste buds. Microsc Res Tech 35:385-412.

Schousboe A, Sonnewald U, and Waagepetersen HS. 2003. Differential roles of alanine in GABAergic and glutamatergic neurons. Neurochem Int 43:311-315.

Schuller-Levis GB and Park E. 2003. Taurine: new implications for an old amino acid. FEMS Microbiol Lett 226: 195-202.

Teeter JH, Brand JG, and Kumazawa T. 1990. A stimulus-activated conductance in isolated taste epithelial membranes. Biophys J 58:253-259.

Teeter JH, Kumazawa T, Brand JG, Kalinoski DL, Honda E, and Smutzer G. 1992. Amino acid receptor channels in taste cells. Soc Gen Physiol Ser 47:291-306.

Toyoshima K, Nada 0, and Shimamura A. 1984. Fine structure of monoamine-containing basal cells in the taste buds on the barbels of three species of teleosts. Cell Tissue Res 235:479-484.

Tucker D. 1983. Fish chemoreception: Peripheral anatomy and physiology. In Northcutt RG and Davis RE, editors. Fish neurobiology: Vol. 1. Brain stem and sense organs. Ann Arbor: The University of Michigan Press. p 311-350.

Wong GT, Gannon KS, and Margolskee RF. 1996. Transduction of bitter and sweet taste by gustducin. Nature 381:796-800.

Yang RB, Tabata S, Crowley HH, Margolskee RF, and Kinnamon JC. 2000. Ultrastructural localization of gustducin immunoreactivity in microvilli of type II taste cells in the rat. J Comp NeuroI425:139-151.

Yee CL, Yang R, Bottger B, Finger TE, and Kinnamon JC. 2001. "Type III" cells of rat taste buds: immunohistochemical and ultrastructural studies of neuron-specific enolase, protein gene product 9.5, and serotonin. J Comp NeuroI440:97-108.

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

fixative containing 1 % parafonnaldehyde, 2.5% glutaraldehyde, 3% sucrose, 0.01 %

CaCh in 0.1 M phosphate buffer (PB), pH 7.4, overnight at 4°C. For cryostat sections, a

cold fixative consisting of 4% parafonnaldehyde, 3% sucrose, 0.02% CaCh, in 0.1 M PB

solution (pH 7.4) was used for 1 hour at room temperature.

Plastic Sections

Postembedding immunocytochemical procedures have been described (Marc et al.

1995). Briefly, the fixed tissues were dehydrated through graded methanol (50%, 75%,

85%, 95% and 100%), or ethanol for electron microscopy, followed by 100% acetone (2

x 20 minutes). The tissue was then agitated in 50% acetone and 50% Eponate (Ted Pella

Inc., Redding, CA, USA) overnight, transferred through two changes of fresh 100%

Eponate (1 hour), oriented in embedding molds, and cured overnight at 65°C. Ultra-thin

(50-70 nm) serial sections were cut 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 collected in wells of Teflon-coated spot

slides (Erie Scientific Co., Portsmouth, NH, USA) or placed on fonnvar-coated gold slot

grids for electron microscopy. Sections for light microscopy were deplasticized with

2S% sodium ethoxide (saturated sodium hydroxide in absolute ethanol) in absolute

ethanol (7 minutes), dehydrated in pure methanol (3 x 2 minutes each), rinsed in ultra­

pure water (S minutes), and then dried.

99

Individual sections were incubated overnight at room temperature in a humidified

chamber with polyclonal rabbit anti-GABA (1:32000) or rabbit anti-taurine (1:16000)

antibodies (Signature Immunologies, Salt Lake City, UT, USA) diluted in 0.1 M PB

containing 1 % goat serum and O.OS% thiomerosal, pH 7.4 (Marc et aI., 1990). Following

a rinse with 0.1 M PB, sections were incubated in nanogold-conjugated goat anti-rabbit

secondary antibody (1 nm, 1 :SO dilution; Amersham Corp., Arlington Heights, IL, USA)

for 1 hour at room temperature, rinsed with 0.1 M PB (1 hour), and silver intensified (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). The

tissue was coverslipped using Eponate plastic and cover glass cured overnight at 6SoC.

Elimination of each of the primary antibodies and only secondary antibody resulted in

lackofIR.

Cryostat Sections

The tissue was cryo-protected in graded sucrose (10%, 20% and 30%) in 0.1 M

PB and held in 30% sucrose solution overnight at 4°C. After transfer to cryo-embedding

compound (Microm International GmbH, Walldorf, Germany), the tissue was frozen and

kept at -20°C. Twelve J.lm sections were cut using a cryostat microtome (HM SOSE,

Microm International GmbH, Walldorf, Germany), collected on gelatin-coated slides, and

stored at -20°C. Before immunostaining, the sections were treated with S% goat serum

100

and 0.2% Triton X-100 in filtered 0.1 M PB (40 minutes) to block nonspecific antibody

binding. Following three 10-minute rinses in 0.1 M PB, the sections were double labeled

with an affinity-purified monoclonal mouse anti-rat GAD65 (1:400) antibody (Alpha

Diagnostics Int., San Antonio, TX, USA) diluted in 0.2% Triton X-100 and filtered 0.1 M

PB and one of the following primary antibodies: affinity-purified rabbit polyclonal anti­

rat GAD67 (1: 100, Alpha Diagnostics Int., San Antonio, TX, USA), affinity-purified

rabbit polyclonal anti-GAT-1 (1:50), anti-GAT-2 (1:100), or anti-GAT-3 (1:100)

(Chemicon International, Temecula, CA, USA) and affinity-purified rabbit polyclonal

anti-GABAAR a1 (1:100) and a3 (1:300) subunits (Alomone Labs Ltd., Jerusalem,

Israel) diluted in 0.2% Triton X-100 and filtered 0.1 M PB. Sections were kept in a

humidified chamber overnight at 4°C. The sections were rinsed in 0.1 M PB (3 x 10

minutes), treated with secondary affinity purified goat anti-mouse Alexa Fluoro 555 and

goat anti-rabbit Alexa Fluoro 488 IgGs (1:400) (Molecular Probes, Eugene, OR, USA)

diluted in 0.2% Triton X-100 and 0.1 M PB for 1 hour at room temperature, rinsed in 0.1

M PB (3 x 10 minutes), coverslipped with Vectashield mounting medium (Vector

Laboratories, Burlingame, CA, USA), and viewed with a laser scanning confocal

microscope (Zeiss LSM510, Zeiss Inc, Thornwood, NY, USA). Elimination of primary

antibody resulted in no staining.

Electron Microscopy

The aldehyde fixed tissues were not osmicated prior to plastic embedding to

minimize the loss of metabolite immunoreactivity. Ultrathin serial sections were stained

with 3% uranyl acetate in distilled water (45 minutes) followed by Reynold's lead citrate

101

(20 minutes) and examined with a Hitachi H-7100 electron microscope (Hitachi, San

Jose, CA, USA). The samples were 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 (Microtek, Carson, CA, USA) and Adobe

Photoshop 5.5 software (Adobe Systems Inc., San Jose, CA, USA).

Immunoassay (Western Blot)

Following decapitation, the maxillary barbels of six catfish were removed and

transferred immediately into ice-cold wash buffer (WB) containing 0.05 M Tris-HCI, 0.5

mM CaCh and 0.1 M NaCI (pH 7.8). The barbel taste epithelium was removed by

holding one end of the barbel with a toothed forceps and scraping the epithelium with a

sharp scalpel blade under ice-cold WB. The tissue fragments were collected with a glass

pipette into two or three chilled 1.5 ml Eppendorf tubes, and gently pelleted by

centrifugation at 1000g for 2 minutes at 4°C. The supernatant was removed and the

tissue was resuspended in approximately nine volumes of ice-cold homogenization buffer

(HB) containing 0.05 M Tris-HCI (pH 7.8), 1 mM ethylene diamine tetraacetic acid

(EDTA), 1 % Triton X-100 and freshly added protease inhibitor cocktail (Sigma, St.

Louis, MO, USA). The tissue was homogenized by hand on ice using a disposable nylon

homogenizer and centrifuged at 14,000g for 20 minutes at 4°C. An aliquot of the

supernatant was taken for protein assay, and the reminder was mixed with an equal

volume of 2 x SDS sample buffer, heated for 3 minutes at 85°C for 3 minutes, and stored

at -20°C. Equal volumes (20 JlL) of the sample were subjected to 4-15% gradient SDS­

PAGE. The separated proteins were electrophoretically transferred to polyvinylidine

102

difluoride membranes (Invitrogen, Carlsbad, CA, USA) according to the method of

Laemmli (1970). Nonspecific binding sites were blocked by incubation of the membrane

in 5% (w/v) nonfat dry milk on a shaker overnight at 4°C. The membranes were then

incubated with one of the following primary antibodies: affinity-purified mouse

monoclonal anti-rat GAD65 (1: 1000, Alpha Diagnostics Int., San Antonio, TX, USA),

affinity-purified rabbit polyclonal anti-rat GAD67 (1 :500, Alpha Diagnostics Int., San

Antonio, TX, USA), affinity-purified rabbit polyclonal anti-GAT-1 (1:200), rabbit anti­

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

expression (Fig. 4.5A, arrow) but VITB basal cells apparently lack GAT-1 expression

(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.

References

AdlerE, Hoon MA, Mueller KL, Chandrashekar J, Ryba NJP, and Zuker CS. 2000. A novel family of mammalian taste receptors. Cell 100:693-702.

Atema J. 1971. Structures and functions of the sense of taste in the catfish, letalurus natalis. Brain Behav Evol 4:273-294.

Borden LA. 1996. GABA transporter heterogeneity: phannacology and cellular localization. Neurochem Int 29:335-356.

122

Borden LA, Smith KE, Gustafson EL, Branchek T A, and Weinshank RL. 1995. Cloning and expression of a betaine/GABA transporter from human brain. J Neurochem 64:977-984.

Borden LA, Smith KE, Hartig PR, Branchek T A, and Weinshank RL. 1992. Molecular heterogeneity of the gamma-aminobutyric acid (GABA) transport system. Cloning of two novel high affinity GABA transporters fronl rat brain. J BioI Chern 267:21098-21104.

Bu DF, Erlander MG, Hitz BC, Tillakaratne NJ, Kaufman DL, Wagner-McPherson CB, Evans GA, and Tobin AJ. 1992. Two human glutamate decarboxylases, 65-kDa GAD and 67-kDa GAD, are each encoded by a single gene. Proc Natl Acad Sci U SA 89:2115-2119.

Caicedo A, Jafri MS, and Roper SD. 2000a. In situ Ca2+ imaging reveals neurotransmitter

receptors for glutamate in taste receptor cells. J Neurosci 20:7978-7985.

Caicedo A, Kim KN, and Roper SD. 2oo0b. Glutamate-induced cobalt uptake reveals non-NMDA receptors in rat taste cells. J Comp NeuroI417:315-324.

Caprio J, Brand JG, Teeter JH, Valentincic T, Kalinoski DL, Kohbara J, Kumazawa T, and Wegert S. 1993. The taste system of the channel catfish: from biophysics to behavior. Trends Neurosci 16: 192-197.

Chaudhari N, Yang H, Lamp C, Delay E, Cartford C, Than T, and Roper S. 1996. The taste of monosodium glutamate: membrane receptors in taste buds. J Neurosci 16:3817-3826.

Crisp M, Lowe GA, and Laverack MS. 1975. On the ultrastructure and permeability of taste buds of the marine teleost Ciliata mustela. Tissue Cell 7: 191-202.

Delay RJ and Roper SD. 1988. Ultrastructure of taste cells and synapses in the mudpuppy Necturus maculosus. J Comp NeuroI277:268-280.

Delay RJ, Taylor R, and Roper SD. 1993. Merkel-like basal cells in Necturus taste buds contain serotonin. J Comp NeuroI335:606-613.

Durkin MM, Smith KE, Borden LA, Weinshank RL, Branchek T A, and Gustafson EL. 1995. Localization of messenger RNAs encoding three GABA transporters in rat brain: an in situ hybridization study. Brain Res Mol Brain Res 33:7-21.

Eram M and Michel WC. 2001a. Amino acid immunoreactivity of channel catfish peripheral taste system. Chern Sens 26(8), 1125.

Eram M and Michel WC. 2003. Immunohistochemical characerization of GABA and GAD in facial and vagal nerve innervated taste buds of channel catfish. Chern Sens 28(6), 561.

Erlander MG, Tillakaratne NJ, Feldblum S, Patel N, and Tobin AJ. 1991. Two genes encode distinct glutamate decarboxylases. Neuron 7:91-100.

Erlander MG and Tobin AJ. 1991. The structural and functional heterogeneity of glutamic acid decarboxylase: a review. Neurochem Res 16:215-226.

Esclapez M, Tillakaratne NJ, Kaufman DL, Tobin AJ, and Houser CR. 1994.

123

Comparative localization of two forms of glutamic acid decarboxylase and their mRNAs in rat brain supports the concept of functional differences between the forms. J Neurosci 14:1834-1855.

Ewald DA and Roper SD. 1994. Bidirectional synaptic transmission in Necturus taste buds. J Neurosci 14:3791-3804.

Grover-Johnson N and Farbman AI. 1976. Fine structure of taste buds in the barbel of the catfish, Ictalurus punctatus. Cell Tissue Res 169:395-403.

Guastella J, Nelson N, Nelson H, Czyzyk L, Keynan S, Miedel MC, Davidson N, Lester HA, and Kanner BI. 1990. Cloning and expression of a rat brain GAB A transporter. Science 249: 1303-1306.

Hemess S and Chen YS. 1997. Serotonin inhibits calcium-activated K+ current in rat taste receptor cells. Neuroreport 8:3257-3261.

Hemess S, Zhao FL, Kaya N, Lu SG, Shen T, and Sun XD. 2002a. Adrenergic signalling between rat taste receptor cells. J Physiol 543:601-614.

Hemess S, Zhao FL, Lu SG, Kaya N, and Shen T. 2002b. Expression and physiological actions of cholecystokinin in rat taste receptor cells. J Neurosci 22: 10018-10029.

Herrick CJ. 1901. The cranial nerves and cutaneous sense organs of the north american siluroid fishes. J Comp Neurol 11(3): 178-249.

Herrick CJ. 1904. The organ and sense of taste in fishes. Bull US Fish Commission 22:237 -272.

Itouji A, Sakai N, Tanaka C, and Saito N. 1996. Neuronal and glial localization of two GABA transporters (GAT1 and GAT3) in the rat cerebellum. Brain Res Mol Brain Res 37:309-316.

Jain S and Roper SD. 1991. Immunocytochemistry of gamma-aminobutyric acid, glutamate, serotonin, and histamine in Necturus taste buds. J Comp Neurol 307:675-682.

124

Kalloniatis M and Fletcher E. 1993. Immunocytochemical localization of the amino acid neurotransmitters in the chicken retina. J Comp NeuroI336:174-193.

Kanner BI and Schuldiner S. 1987. Mechanism of transport and storage of neurotransmitters. CRC Crit Rev Biochem 22: 1-38.

Kanwal JS and Caprio J. 1983. An electrophysiological investigation of the oro­pharyngeal (IX-X) taste system in the channel catfish, Ictalurus punctatus. J Comp Physiol A 150:345-357.

Kanwal JS, Hidaka I, and Caprio J. 1987. Taste responses to amino acids from facial nerve branches innervating oral and extra-oral taste buds in the channel catfish, Ictalurus punctatus. Brain Res 406:105-112.

Kapoor AS and Ojha PP. 1973. The olfactory apparatus in the flatfish Cynoglossus oligolepis. Trans Amer Micros Soc 92:298-304.

Kapoor BG, Evans HE, and Pevzner RA. 1975. The gustatory system in fish. Adv Mar BioI 13:53-108.

Kaufman DL, Houser CR, and Tobin AJ. 1991. Two forms of the gamma-aminobutyric acid synthetic enzyme glutamate decarboxylase have distinct intraneuronal distributions and cofactor interactions. J Neurochem 56:720-723.

Kim D-J and Roper SD. 1995. Localization of serotonin in taste buds: a comparative study in four vertebrates. J Comp Neurol 353:364-370.

Krogsgaard-Larsen P, Falch E, Larsson OM, and Schousboe A. 1987. GABA uptake inhibitors: relevance to anti epileptic drug research. Epilepsy Res 1:77-93.

Laemmli UK. 1970. Cleavage of structural proteins during assembly of the head of the bacteriophage T4. Nature 277:680-685.

Lam DM, Lasater EM, and Naka KI. 1978. gamma-Aminobutyric acid: a neurotransmitter candidate for cone horizontal cells of the catfish retina. Proc Natl Acad Sci USA 75:6310-6313.

Liu QR, Lopez-Corcuera B, Mandiyan S, Nelson H, and Nelson N. 1993. Molecular characterization of four pharmacologically distinct gamma-aminobutyric acid transporters in mouse brain [corrected]. J BioI Chern 268:2106-2112.

Lopez-Corcuera B, Liu QR, Mandiyan S, Nelson H, and Nelson N. 1992. Expression of a mouse brain cDNA encoding novel gamma-aminobutyric acid transporter. J BioI Chern 267:17491-17493.

Macdonald RL and Olsen RW. 1994. GABAA receptor channels. Annu Rev Neurosci 17:569-602.

Marc RE and Liu W. 2000. Fundamental GABAergic amacrine cell circuitries in the retina: nested feedback, concatenated inhibition, and axosomatic synapses. J Comp Neurol 425:560-582.

125

Marc RE, Murry RF, and Basinger SF. 1995. Pattern recognition of amino acid signatures in retinal neurons. J Neurosci 15:5106-5129.

Marc RE, Wei-Ley SL, Kalloniatis M, Raiguel SF, and Van Haesendonck E. 1990. Patterns of glutamate immunoreactivity in the goldfish retina. J Neurosci 10:4006-4034.

Montmayeur JP and Matsunami H. 2002. Receptors for bitter and sweet taste. Curr Opin Neurobiol 12:366-371.

Mroz EA and Sewell WF. 1989. Pharmacological alterations of the activity of afferent fibers innervating hair cells. Hear Res 38: 141-162.

Murakami M, Otsu K, and Otsuka T. 1972. Effects of chemicals on receptors and horizontal cells in the retina. J PhysioI227:899-913.

Nagai T, Delay RJ, Welton J, and Ror,er SD. 1998. Uptake and release of neurotransmitter candidates, [ H]serotonin, eH]glutamate, and eH]gamma­aminobutytic acid, in taste buds of the mudpuppy, Necturus maculosus. J Comp NeuroI392:199-208.

Obata H, Shimada K, Sakai N, and Saito N. 1997. GABAergic neurotransmission in rat taste buds: immunocytochemical study for GABA and GABA transporter SUbtypes. Brain Res Mol Brain Res 49:29-36.

Ogawa K, Marui T, and Caprio J. 1997. Quinine suppression of single facial taste fiber responses in the channel catfish. Brain Res 769:263-272.

Ogura T. 2002. Acetylcholine increases intracellular Ca2+ in taste cells via activation of

muscarinic receptors. J Neurophysiol 87:2643-2649.

Pietrini G, Suh YJ, Edelnlann L, Rudnick G, and Caplan MJ. 1994. The axonal gamma­aminobutyric acid transporter GAT -1 is sorted to the apical membranes of polarized epithelial cells. J BioI Chern 269:4668-4674.

Pirker S, Schwarzer C, Wieselthaler A, Sieghart W, and Sperk G. 2000. GABA(A) receptors: immunocytochemical distribution of 13 subunits in the adult rat brain. Neurosci 101:815-850.

Radian R, Ottersen OP, Storm-Mathisen J, Castel M, and Kanner BI. 1990. Immunocytochemical localization of the GABA transporter in rat brain. J Neurosci 10:1319-1330.

Ren Y, Shimada K, Shirai Y, Fujimiya M, and Saito N. 1999. Immunocytochemical localization of serotonin and serotonin transporter (SET) in taste buds of rat. Brain Res Mol Brain Res 74:221-224.

126

Reutter K. 1978. Taste organ in the bullhead (Teleostei). Adv Anat Embryol Cell BioI 55:1-98.

Reutter K. 1986. Chemoreceptors. In Bereiter-Hahn I, Matoltsy AG, and Richards KS, editors. Biology of the integument: Vol. 2 Vertebrates. New York: Springer­Verlag. p 586-604.

Reutter K and Witt M. 1993. Morphology of vertebrate taste organs and their nerve supply. In Sin10n SA and Roper SD, editors. Mechanisms of taste transduction. Boca Raton: CRC. p 29-82.

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.

Royer SM and Kinnamon IC. 1996. Comparison of high-pressure freezing/freeze substitution and chemical fixation of catfish barbel taste buds. Microsc Res Tech 35:385-412.

Schousboe A and Kanner B. 2002. GABA transporters: functional and pharmacological properties. In Egebjerg I, Schouboe A., and Krogsgaard-Larsen P., editors. Glutamate and GABA receptors and transporters. Structure, function, and pharmacology. New York: Taylor and Francis. p 337-349.

Schousboe A. 1981. Transport and metabolism of glutamate and GABA in neurons are glial cells. Int Rev Neurobiol 22: 1-45.

Sieghart W. 1995. Structure and pharmacology of gamma-aminobutyric acid A receptor SUbtypes. Pharmacol Rev 47:181-234.

Tucker D. 1983. Fish chemoreception: Peripheral anatomy and physiology. In Northcutt RG and Davis RE, editors. Fish neurobiology: Vol. 1. Brain stem and sense organs. Ann Arbor: The University of Michigan Press. p 311-350.

Welton I, Taylor R, Porter AI, and Roper SD. 1992. Immunocytochemical survey of putative neurotransmitters in taste buds from Necturus maculosus. I Comp Neurol 324:509-521.

Wisden Wand Farrant M. 2002. Insights into GABAA receptor complexity from the study of cerebellar granule cells. In Egebjerg I, Schousboe A, and Krogsgaard­Larsen P, editors. Glutamate and GABA receptors and transporters. Structure, function and pharmacology. New York: Taylor & Francis. p 189-201.

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;

Murray, 1969; 1973; Reutter, 1971; Grover-Johnson and Farbman, 1976; Reutter, 1978).

Awareness of heterogeneity within taste cell populations has grown with recent

histochemical, genetic and physiological assessments (Medler et aI., 1998; Finger and

Simon, 2000), and we no longer limit our descriptions to the two to three cell types

originally reported (Farbman, 1965; Hirata, 1966; Murray, 1969; 1973; Reutter, 1971;

131

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

relatively low extracellular GABA concentrations favors reverse transport. Thus, GABA

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.

References

Atema J. 1971. Structures and functions of the sense of taste in the catfish, Ictalurus natalis. Brain Behav Evol 4:273-294.

Caprio J, Brand JG, Teeter JR, Valentincic T, Kalinoski DL, Kohbara J, Kumazawa T, and Wegert S. 1993. The taste system of the channel catfish: from biophysics to behavior. Trends Neurosci 16:192-197.

Chandrashekar J, Mueller KL, Roon MA, Adler E, Feng LX, Guo W, Zuker CS, and Ryba NJP. 2000. T2Rs function as bitter taste receptors. Cell 100:703-711.

137

Edwards JG and Michel WC. 2002. Odor-stimulated glutamatergic neurotransmission in the zebrafish olfactory bulb. J Comp Neurol 454:294-309.

Farbman AI. 1965. Fine structure of the taste bud. J Ultrastruct Res 12:328-350.

Finger TE and Simon CM. 2000. Cell biology of taste epithelium. In Finger TE, Silver WL, and Restrepo D, editors. The neurobiology of taste and smell. New York: Wiley-Liss. p 287-314.

Gilbertson TA, Damak S, and Margolskee RF. 2000. The molecular physiology of taste transduction. CUff Opin Neurobiol1O:519-527.

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.

Herness MS and Gilbertson T A. 1999. Cellular mechanisms of taste transduction. Ann Rev Physiol 61 :873-900.

Herrick CJ. 1901. The cranial nerves and cutaneous sense organs of the North American siluroid fishes. J Comp Neurol11(3):178-249.

Hirata Y. 1966. Fine structure of the terminal buds on the barbels of some fishes. Arch Histol Jpn 26:507-523.

Jain S and Roper SD. 1991. Immunocytochemistry of gamma-aminobutyric acid, glutamate, serotonin, and histamine in Necturus taste buds. J Comp Neurol 307:675-682.

Kalinoski DL, Bryant BP, Shaulsky G, Brand JG, and Harpaz S. 1989. Specific L­arginine taste receptor sites in the catfish, lctalurus punctatus: Biochemical and neurophysiological characterization. Brain Res 488: 163-173.

Kanwal JS and Caprio J. 1983. An electrophysiological investigation of the oro­pharyngeal (IX-X) taste system in the channel catfish, lctalurus punctatus. J Comp Physiol A 150:345-357.

Kanwal JS, Hidaka I, and Caprio J. 1987. Taste responses to amino acids from facial nerve branches innervating oral and extra-oral taste buds in the channel catfish, lctalurus punctatus. Brain Res 406:105-112.

Kohbara J, Michel W, and Caprio J. 1992. Responses of single facial taste fibers in the channel catfish, lctalurus punctatus, to amino acids. J Neurophysiol 68: 1012-1026.

Kumazawa T, Brand JG, and Teeter JH. 1998. Amino acid-activated channels in the catfish taste system. Biophys J 75:2757-2766.

138

Marc RE. 1999a. Kainate activation of horizontal, bipolar, amacrine, and ganglion cells in the rabbit retina. J Comp Neurol 407:65-76.

Marc RE. 1999b. Mapping glutamatergic drive in the vertebrate retina with a channel­permeant organic cation. J Comp NeuroI407:47-64.

Marc RE, Murry RF, and Basinger SF. 1995. Pattern recognition of amino acid signatures in retinal neurons. J Neurosci 15:5106-5129.

Medler KF, Hansen A, and Bruch RC. 1998. Odorant receptor gene expression in catfish taste tissue. Neuroreport 9:4103-4107.

Michel Wand Caprio J. 1988. Responses of single facial taste units to amino acids in the sea catfish, Arius felis. Chern Sens 13, 718.

Michel Wand Caprio J. 1989. Organization of amino acid taste information of single facial taste fibers in the marine catfish, Ariusfelis. Chern Sensl4, 728-729.

Michel Wand Caprio J. 1991. Responses of single facial taste fibers in the sea catfish, Ariusfelis, to amino acids. J Neurophysiol 66:247-260.

Murray RG. 1969. Cell types in rabbit taste buds. In Pfaffmann C, editor. Olfactory and taste, Vol III. New York: Rockeffeler UP. p 331-344.

Murray R. 1973. The ultrasturcture of taste buds. In Friedman I, editor. The ultrastructure of sensory organs. Amsterdam: North-Holland Publishing Co. p 3-37.

Nagai T, Delay RJ, Welton J, and Ro~er SD. 1998. Uptake and release of neurotransmitter candidates, [ H]serotonin, eH]glutamate, and eH]gamma­aminobutyric acid, in taste buds of the mudpuppy, Necturus maculosus. J Comp Neurol 392: 199-208.

Obata H, Shimada K, Sakai N, and Saito N. 1997. GABAergic neurotransmission in rat taste buds: immunocytochemical study for GABA and GABA transporter subtypes. Brain Res Mol Brain Res 49:29-36.

Ogawa K, Marui T, and Caprio J. 1997. Quinine suppression of single facial taste fiber responses in the channel catfish. Brain Res 769:263-272.

Pirker S, Schwarzer C, Wieselthaler A, Sieghart W, and Sperk G. 2000. GABA(A) receptors: immunocytochemical distribution of 13 subunits in the adult rat brain. Neurosci 101:815-850.

Pumplin DW, Yu CS, and Smith DV. 1997. Light and dark cells of rat vallate taste buds are morphologically distinct cell types. J Comp Neurol 378:389-410.

Reutter K. 1971. The taste-buds of Amiurus nebulosus (Leseur) Morphological and histochemical investigations. Z Zellforsch 120:280-308.

Reutter K. 1978. Taste organ in the bullhead (Teleostei). Adv Anat Embryol Cell BioI 55:1-98.

139

Richerson GB and Wu Y. 2003. Dynamic equilibrium of neurotransmitter transporters: not just for reuptake anymore. J Neurophysiol 90: 1363-1374.

Roper SD. 1993. Synaptic interaction in taste buds. In Simon SA and Roper SD, editors. Mechanisms of Taste Transduction. Boca Raton: CRe. p 275-293.

Saxena NC and Macdonald RL. 1996. Properties of putative cerebellar gamma­aminobutyric acid A receptor isoforms. Mol PharmacoI49:567-579.

Takeda M, Suzuki Y, Obara N, and Nagai Y. 1992. Neural cell adhesion molecule of taste buds. J Electron Microsc (Tokyo) 41:375-380.

Wong GT, Gannon KS, and Margolskee RF. 1996. Transduction of bitter and sweet taste by gustducin. Nature 381:796-800.

Yamamoto T, Nagai T, Shimura T, and Yasoshima Y. 1998. Roles of chemical mediators in the taste system. Jpn J Pharmacol 76:325-348.

Yee CL, Yang R, Bottger B, Finger TE, and Kinnamon JC. 2001. "Type III" cells of rat taste buds: immunohistochemical and ultrastructural studies of neuron-specific enolase, protein gene product 9.5, and serotonin. J Comp NeuroI440:97-108.