Uptake and Release of NeurotransmitterCandidates, [3H]serotonin, [3H]glutamate,

and [3H]g-Aminobutyric Acid, in TasteBuds of the Mudpuppy, Necturus maculosus

TAKATOSHI NAGAI,1,2 RONA J. DELAY,2 JOAN WELTON,3

AND STEPHEN D. ROPER2,4*1Department of Physiology, Teikyo University School of Medicine, Tokyo 173, Japan

2Rocky Mountain Taste and Smell Center, University of Colorado Health Sciences Center,Denver, Colorado 80262

3Department of Anatomy and Neurobiology, Colorado State University,Ft. Collins, Colorado 80523

4Department of Physiology and Biophysics, University of Miami School of Medicine,Miami, Florida 33101

ABSTRACTNeurotransmitters in vertebrate taste buds have not yet been identified with confidence.

Serotonin, glutamate, and g-aminobutyric acid (GABA) have been postulated, but theevidence is incomplete. We undertook an autoradiographic study of [3H]serotonin, [3H]gluta-mate, and [3H]GABA uptake in lingual epithelium from the amphibian, Necturus maculosus,to determine whether taste bud cells would accumulate and release these substances. Lingualepithelium containing taste buds was incubated in low concentrations (0.4–6 µM) of thesetritiated transmitter candidates and the tissue was processed for light microscopic autoradiog-raphy. Merkel-like basal taste cells accumulated [3H]serotonin. When the tissue was treatedwith 40 mM K1 after incubating the tissue in [3H]serotonin, cells released the radiolabelledtransmitter. Furthermore, depolarization (KCl)-induced release of [3H]serotonin was Ca-dependent: if Ca21 was reduced to 0.4 mM and 20 mM Mg21 added to the high K1 bathingsolution, Merkel-like basal cells did not release [3H]serotonin. In contrast, [3H]glutamate wastaken up by several cell types, including non-sensory epithelial cells, Schwann cells, and sometaste bud cells. [3H]glutamate was not released by depolarizing the tissue with 40 mM K1.[3H]GABA uptake was also widespread, but did not occur in taste bud cells. [3H]GABAaccumulated in non-sensory epithelial cells and Schwann cells.

These data support the hypothesis that serotonin is a neurotransmitter or neuromodula-tor released by Merkel-like basal cells in Necturus taste buds. The data do not support (norrule out) a neurotransmitter role for glutamate or GABA in taste buds. J. Comp. Neurol.392:199–208, 1998. r 1998 Wiley-Liss, Inc.

Indexing terms: synapses; gustatory; monoamines; excitatory amino acids; Merkel cells

Vertebrate sensory end organs in taste consist of 50–100cells arranged in tight clusters called taste buds. Fordecades, it is has been believed that the taste receptor cellsin these sensory organs represent a passive interfacebetween the external environment and sensory afferentaxons. Taste receptor cells were thought merely to trans-duce chemical stimuli in the external environment intoelectrical signals that were transmitted to sensory neu-rons and relayed to higher centers in the brain for decod-ing and interpretation. However, evidence is now accumu-lating that a certain degree of information processing

takes place in the peripheral end organs of taste before thesignals are transmitted to the central nervous system.That is, lateral synaptic interactions, both electrical andchemical, have been reported in taste buds (reviewed in

Grant sponsor: NIH/NIDCD; Grant numbers: DC00374 and DC00244.*Correspondence to: Dr. Stephen D. Roper, Department of Physiology and

Biophysics, University of Miami School of Medicine, P.O. Box 016430,Miami, FL 33101-6430. E-mail: sroper@newssun.med.miami.edu

Received 26 February 1997; Revised 6 October 1997;Accepted 8 October 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 392:199–208 (1998)

r 1998 WILEY-LISS, INC.

Roper, 1992). Evidence for these lateral interactions in-cludes morphological descriptions of synapses betweentaste bud cells and electrophysiological recordings fromtaste bud cells and from sensory axons that innervate tastebuds.

Receptor cells in taste buds form synaptic connectionswith sensory afferent fibers. However, taste cells are alsobelieved to synapse with other cells within the taste bud,at least in the amphibian (reviewed in Roper, 1992).Presently, one of the major gaps in our understanding ofsynaptic interactions in taste buds is the identification ofneurotransmitters. This has slowed physiological and phar-macological analyses of taste bud function and investiga-tions of signal processing in taste end organs. The stron-gest evidence to date for a transmitter in vertebrate tastebuds is for serotonin. This monoamine is found in a specificsubset of taste bud cells. In fish and amphibia, serotonin islocalized to Merkel-like basal cells that lie near the base oftaste buds. Merkel-like basal cells are club-shaped cellsand do not have processes that extend to the taste pore.Consequently, these cells are not believed to participatedirectly in taste reception. Instead, they may act asinterneurons or neuromodulators (Reutter, 1971, 1978;Delay and Roper, 1988; Ewald and Roper, 1994a). Inmammals, serotonin is found in elongate cells that moreclosely resemble receptor cells. Serotonergic taste bud cellsin mammals extend processes from the base of the tastebud into the taste pore. In mammals, serotonergic tastecells may be identical to what have been identified as TypeIII taste bud cells, although their specific morphology (e.g.,Type III) remains to be more firmly established (Murray,1986; Fujimoto et al., 1987; Kim and Roper, 1995). Seroto-nin is believed to modulate chemosensory responses ofreceptor cells, although it may have other functions as wellfor taste buds and other tissues (Seuwen and Pouyssegur,1990; Lauder, 1993; Toyoshima, 1994). Other neurotrans-mitters have been proposed, including norepinephrine,acetylcholine, glutamate, g-aminobutyric acid (GABA),and certain peptides (Landgren et al., 1954; Morimoto andSato, 1982; Nagahama and Kurihara, 1985; Jain andRoper, 1991; Ewald and Roper, 1994b; Nagai et al., 1996).However, the evidence for any one of these other transmit-ter candidates is less than compelling.

Morphological approaches can help identify neurotrans-mitters in many neural systems. These techniques includeimmunocytochemistry for specific transmitter candidatesand uptake studies utilizing [3H]-labelled transmittercandidates. Autoradiographic analyses of radiolabelledtransmitter uptake are predicated on the ability of presyn-aptic terminals to transport synaptically-released transmit-ter. Transmitter reuptake helps terminate synaptic trans-mission and prevent the accumulation of neuroactivesubstances in the synaptic cleft. Autoradiographical analy-ses of transmitter uptake have proven quite powerful inidentifying neurotransmitters in the vertebrate retina,where acetylcholine, glutamate, GABA, and other transmit-ters have been localized to specific cell types (e.g., Marcand Lam, 1981; O’Malley and Masland, 1989; Muller andMarc, 1990; Mize et al., 1992). This has led to a fairlycomplete understanding of the elaborate synaptic interac-tions in the retina. In the taste bud, certain neurotransmit-ter candidates, including serotonin, glutamate, and GABA,have also been localized to specific cellular elements byusing immunocytochemistry. Namely, Merkel-like basalcells in taste buds from fish and amphibia and putative

Type III taste cells in mammalian vertebrates are in-tensely immunopositive for serotonin. Furthermore, nervefibers that innervate taste buds in the salamander, Nectu-rus maculosus, are immunostained with antibodies toGABA and to glutamate (Jain and Roper, 1991). Jain andRoper speculated that these amino acids may be involvedin synaptic interactions in the taste bud, although thelocalization to axons, not taste cells, was enigmatic (Jainand Roper, 1991).

The present study was undertaken to extend the immu-nocytochemical studies of neurotransmitters in taste endorgans by investigating which cellular elements in Nectu-rus taste buds, if any, take up, store, and upon depolariza-tion, release serotonin, glutamate, or GABA. We selectedthe amphibian, Necturus, to study transmitter uptake andrelease because at least one of the transmitter candidates,serotonin, has been identified in a subset of taste bud cellsin this species (see above). Furthermore, taste buds andtaste cells in this species are very large and the outlines ofindividual taste cells are readily visible in microscopicsections (see Farbman and Yonkers, 1971). This greatlyfacilitated the quantitative autoradiography. The resultsfor serotonin are consistent with previous immunocyto-chemical findings and greatly strengthen the argument forthis monoamine being a neurotransmitter or neuromodula-tor in taste buds, at least in Necturus. The data forglutamate and GABA are less convincing. These findingshave been presented in abstract form (Welton and Roper,1992; Nagai et al., 1994).

MATERIALS AND METHODS

Mudpuppies (Necturus maculosus) were obtained fromcommercial vendors. Animals were maintained in 300gallon polyethylene tanks filled with 5% artificial seawater and kept at 10°C. Animals were fed minnows weeklyad libitum. For the series of experiments on serotoninuptake, Necturus received a single injection of pargyline(50 mg/ml in 0.9% NaCl), a blocker of monoamine oxidase,at a dose of 100 mg/kg the day prior to dissecting thelingual tissues (below).

Dissection and incubation

Mudpuppies were deeply anesthetized with ice-cooledwater, pithed and quickly decapitated. All procedures werereviewed and approved by the institution’s Animal Careand Use Committee. Pieces of lingual epithelium thatcontained taste buds were removed from the tongues withblunt dissection. Samples were placed in 1 ml Eppendorftest tubes and incubated in oxygenated amphibian physi-ological saline (APS; 112 mM NaCl, 2 mM KCl, 8 mMCaCl2, and 3 mM HEPES buffer, pH 7.2) that contained[3H]labeled serotonin, glutamate or GABA for 15 minutes.Ascorbic acid (0.1%) was included in the APS to reduce thedegradation of serotonin. Elevated CaCl2 helped to pre-serve the integrity of the tissue. Following incubation inAPS that contained radiolabeled transmitters, lingualtissues were rinsed for 2 minutes with fresh APS andimmediately fixed with 2% glutaraldehyde in 0.05 Msodium cacodylate buffer (pH 7.2). Preservation with glu-taraldehyde covalently linked the radioligands to cytosolicproteins and fixed them in place (Peters and Ashley, 1967).

To study whether radiolabeled transmitters were re-leased when cells were depolarized, a second series ofsamples was incubated in radioactive transmitters as

200 T. NAGAI ET AL.

described above, but subsequently rinsed for 2 minuteswith APS that contained 40 mM KCl (replaced for anequivalent amount of NaCl). To determine whether anydepolarization-induced release was Ca-dependent, afterradioligand incubation, a third series of samples wasrinsed with APS that contained 40 mM KCl plus 20 mMMgCl2 and 0.4 mM CaCl2.

Radioligands

Radiolabeled transmitters were tritiated ligands: seroto-nin; 5-hydroxy tryptamine creatine sulfate ([3H]5HT, Am-ersham Life Science, Inc., Arlington Heights, IL); gluta-mate; glutamic acid ([3H]Glu, DuPont NENt ResearchProducts, Boston, MA) and GABA; g-aminobutyric acid([3H]GABA, DuPont NENt Research Products). Two con-centrations of each transmitter were tested in the incuba-tion steps. Aliquots of radioligand were added to theincubation solutions (total volume; 400–1,000 µl) to pro-duce the following final concentrations: 0.4 µM and 2.2 µM[3H]5-HT (10-20 Ci/mmol); 0.5 µM and 5 µM [3H]Glu (57.4Ci/mmol); 0.6 µM and 6 µM [3H]GABA (33.6 Ci/mmol).These low concentrations of neurotransmitters were usedin the incubation steps to elicit transport through high-affinity mechanisms, as opposed to low-affinity uptakethat operates in the mM range (Logan and Snyder, 1971;Johnston, 1981; Dall’Asta et al., 1983).

Autoradiography

Fixed tissues were embedded in Spurr’s plastic, sec-tioned at 1.5–2.5 µm thickness, and mounted onto glassslides. Slides were coated with nuclear track emulsion(Kodak NTB-2, Eastman Kodak, Rochester, NY), air-driedovernight, and kept in a light-tight box for 3 to 12 days at4°C. Slides were developed in Kodak D-19 and fixed in 25%sodium thiosulfate. After air drying, slides were cover-slipped in glycerine.

Samples were evaluated with bright-field, phase-con-trast, Nomarski differential interference contrast or dark-field optics with a Zeiss Axiophot microscope (Carl Zeiss,Inc., Thornwood, NY). To quantify the results, we selectedtaste buds that were sectioned longitudinally through theregion of the taste pore. Cell areas and numbers of grainswere measured with imaging software (NIH Image, NIH,Betheda, MD). For tissues incubated with [3H]Glu and[3H]GABA, a circular template of fixed size (10 µm diam-eter, 80 square µm) was projected over five differentregions in the lingual tissue, and the numbers of silvergrains within this circular area were counted. Theseregions were: (1) taste bud cells; (2) stratum basale cellsadjacent to taste buds; (3) lamina propria subjacent totaste buds, but excluding glial cells; (4) cells in thesuperficial epithelial layer; and (5) mucus-secreting gobletcells in the non-sensory epithelium. Five separate measure-ments were made from each of these regions in everysection inspected. The average grain densities for each ofthe regions was computed for that section. Data fromseveral different sections (reported below as N sections)were collected and pooled to arrive at population means forthe different experimental treatments. This approach wasdictated by the widespread uptake of [3H]Glu and[3H]GABA. This was in marked contrast to the morelimited and focal uptake of [3H]5HT into Merkel-like basalcells of taste buds (see Results, below). In the case oftissues labeled with [3H]5HT where the uptake was local-ized to a single cell type, it was possible to count the

number of silver grains over each Merkel-like basal cell,determine the surface area of the Merkel-like basal cell,and directly calculate the number of silver grains persquare micron surface area. We calculated the statisticalsignificance of differences between means among the sam-ple sets with the Student’s t-test.

RESULTS

When lingual samples from the amphibian, Necturus,were incubated in low concentrations of [3H]labelled sero-tonin ([3H]5HT), glutamate ([3H]Glu) or g-aminobutyricacid ([3H]GABA), cells accumulated the radiolabelled com-pounds and could subsequently be visualized in the fixedtissues with autoradiographic techniques, as described inMethods. The localization of radioligand was most focal for[3H]5HT, and widespread for [3H]Glu and [3H]GABA.

Uptake and release of serotonin

Certain cells in taste buds intensely and consistentlyaccumulated [3H]5HT when the tissue was bathed in lowconcentrations of the radiolabelled monoamine. The tastebud cells that were labelled with [3H]5HT were club-shaped cells situated at the lateral base of taste buds.Accumulation of radiolabel was evident when the lingualsamples had been incubated even with 0.4 µM [3H]5HT.When samples were incubated in a higher concentration of[3H]5HT (2.2 µM), the basal cells were more heavilylabeled, although non-specific background labelling alsoincreased (Fig. 1). Cells that transported [3H]5HT wereidentically situated and similar in shape to the serotoner-gic Merkel-like basal cells that have been described previ-ously by us and others (Reutter, 1971, 1978; Toyoshima etal. 1984; Toyoshima and Shimamura, 1987; Delay et al.,1993; Kim and Roper, 1995).

Uptake of [3H]5HT into other cells or structures inlingual epithelium was not as evident except for thepresence of radiolabel in occasional non-taste epithelialcells. The radiolabelled cells outside taste buds resembledMerkel-like basal taste bud cells. Merkel cells are found inlingual epithelium (Toyoshima et al., 1987; Toyoshima,1989; Nagai and Koyama, 1994) and cutaneous Merkelcells are known to contain 5HT (Hartschuh and Weihe,1988; Garcia-Caballero et al., 1989). We interpret our datato indicate that serotonergic Merkel cells, whether insidetaste buds or in the surrounding, non-taste epithelium,accumulated [3H]5HT. However, since the main point ofthis report is on transmitter candidates in taste buds, wefocussed our attention on the presence of [3H]5HT ingustatory end organs.

When lingual samples were incubated with [3H]5HT inthe presence of imipramine, a selective blocker of high-affinity 5HT uptake, the density of silver grains overMerkel-like basal cells was greatly reduced. For example,when we examined tissues that had been incubated in 0.4µM [3H]5HT, no detectable label was found over any tastebud cells when 1–10 µM imipramine had been included inthe incubation medium (Fig. 2). These data suggest thatimipramine had completely inhibited the accumulation of[3H]5HT, consistent with the role of this substance inblocking serotonin uptake in other tissues.

To study whether 5HT is released when Merkel-like cellsare depolarized, as would be expected if the monoaminewas a neurotransmitter in taste buds, lingual sampleswere incubated in 0.4 µM [3H]5HT, as before, followed byincubation in amphibian physiological saline (APS) that

NEUROTRANSMITTERS IN NECTURUS TASTE BUDS 201

contained 40 mM KCl. This concentration of K would beexpected to depolarize cells from a normal resting poten-tial of about -90 mV to -15 mV. This is assuming that [K]i <80 mM (Kinnamon and Roper, 1987, 1988) and that the

resting potential is predominantly due to an electrochemi-cal gradient for K1. When cells were depolarized, radiola-bel over Merkel-like basal cells was significantly less (54%;P 5 0.013) compared to samples that were rinsed withstandard APS (Fig. 3). These data suggest that Merkel-likebasal cells release [3H]5HT when they are depolarized.Furthermore, depolarization-evoked release of [3H]5HTfrom Merkel-like basal cells was calcium-dependent: afterincubating lingual tissues with 0.4 µM [3H]5HT, thesamples were rinsed with APS in which KCl had beenelevated to 40 mM but CaCl2 lowered to 0.4 mM and 20mM MgCl2 added. With this treatment there was no loss ofradiolabel over Merkel-like basal cells (Fig. 3). That is,there was no statistical difference between the density ofsilver grains over Merkel-like basal cells in the presenceand absence of 40 mM KCl if Ca21 was lowered and Mg21

elevated.Collectively, these data strongly suggest that serotonin

is a neurotransmitter in Merkel-like basal cells. [3H]5HTis accumulated via a high-affinity uptake system, and it isreleased in a Ca-dependent fashion upon depolarization.

Uptake and release of glutamate

When lingual tissues were incubated in 0.5 µM or 5.0µM [3H]Glu, the pattern of accumulation was much differ-ent than was observed for the accumulation of [3H]5HT.Generally speaking, there was much more backgroundlabelling. This might be expected for such a ubiquitousmetabolite as glutamic acid. Autoradiographic labellingoccurred over non-sensory cells as well as over a subset oftaste bud cells. The most dense radiolabelling was foundover cells in the connective tissue (lamina propria) through-out the samples (Fig. 4A,B). The disposition of these cells

Fig. 1. Merkel-like basal cells in Necturus taste buds take up[3H]serotonin via high affinity transport mechanisms. This sectionshows two labelled Merkel-like basal cells at the periphery of the tastebud (arrows). The tissue had been incubated for 15 minutes in 2.2 µM[3H]serotonin (10-20 Ci/mmol). This photo micrograph is a double-exposure with dark-field and phase-contrast optics. Scale bar 5 50 µm.

Fig. 2. The uptake of [3H]5HT by Merkel-like basal cells inNecturus taste buds is blocked by imipramine. Lingual tissues wereincubated in 0.4 µM [3H]5HT in the presence and absence of imipra-mine (1 and 10 µM). Bars show the mean (6S.E.M.) number of silvergrains per square micron over Merkel-like basal cells for these threeconditions. N, Number of cells.

Fig. 3. Merkel-like basal cells release [3H]5HT in a Ca-dependentmanner when the cells are depolarized. Lingual tissues were incu-bated in 0.4 µM [3H]5HT and then rinsed either in APS; in APScontaining 40 mM KCl (‘‘40 mM K-APS’’); or in APS containing 40 mMKCl, 0.4 mM CaCl2 and 20 mM MgCl2 (‘‘Low Ca-APS’’) and processedfor autoradiography. Bars show the mean (6S.E.M.) number of silvergrains per square micron over Merkel-like basal cells for these threeconditions. N, Number of cells; asterisk, data were significantlydifferent from those collected in APS (P 5 0.013) and in Low Ca-APS(P 5 0.008).

202 T. NAGAI ET AL.

and close association with nerve fibers suggests that thesewere Schwann cells, but without specific immunocyto-chemical staining or electron microscopy, we could notverify this identification. Silver grains were conspicuouslymissing over nerve fibers (Fig. 4E,F). There was alsoconsistent and relatively uniform radiolabel in most cellsin the stratum basale of the non-sensory epithelium (Fig.4A,B). Large circular-shaped gaps in the radiolabellingover non-sensory epithelium were identified at highermagnification as mucus-secreting goblet cells. There was agradient in [3H]Glu accumulation from basal to superficialstrata in the lingual epithelium. The more superficial cellsin the epithelium were only lightly radiolabelled, if at all.Lastly, certain elongated cells in taste buds accumulated[3H]Glu (Fig. 4A–D). These cells resembled receptor cells.Processes of radiolabelled taste cells extended from thetaste pore to the base of the taste bud. The density of silvergrains over taste bud cells appeared to be identical to thatover adjacent cells in the non-sensory stratum basale. Onaverage, 13% of taste bud cells accumulated [3H]Glu,based on the proportion of labeled cells to total cells in eachsection of taste bud that was examined (n 5 9 sections).This value is an overestimate of the actual percentage oftaste bud cells that accumulate glutamate because we didnot include sections that did not possess at least oneradiolabelled cell.

We attempted to quantify the radiolabelling in thedifferent regions of the lingual epithelium to assess therelative accumulation of [3H]Glu in the different cell typesand to investigate whether depolarizing the cells with 40mM KCl elicited a release of glutamate. Samples wereincubated with 5 µM [3H]Glu and conditions for autoradi-ography were standardized to 8 day exposures. We mea-sured the number of silver grains in a circular area thathad a fixed diameter of 10 µm, about the diameter of theperikarya of Necturus taste cells. The circular templatewas moved to different regions on a sample and the densityof silver grains was counted. This worked well for allregions except for the putative Schwann cells, which weretoo heavily labelled to distinguish and count separatesilver grains in this fashion. The quantitative resultsverified the above qualitative description (Fig. 5). Thedensity of silver grains over taste cells and non-sensorystratum basale cells was approximately 10-fold higherthan that over goblet cells and cells in the superficialregion of the epithelium.

To test whether glutamate is released when taste cellsare depolarized, lingual samples were incubated in 5 µM[3H]Glu, followed by rinsing in APS that contained 40 mMKCl. When samples were inspected that had been pro-cessed identically (incubation in 5 µM [3H]Glu, autoradio-graphic exposure for 8 days) except for the presence orabsence of 40 mM KCl in the rinse solution, there were nosignificant differences between the grain densities overcells in taste buds or in the non-sensory stratum basale(Fig. 5). Furthermore, rinsing tissues with APS in whichKCl had been elevated to 40 mM but CaCl2 lowered to 0.4mM and 20 mM MgCl2 added also did not consistentlyalter the grain densities (Fig. 5). Depolarizing the tissuewith high K1 did not appear to elicit [3H]Glu release fromtaste cells or stratum basale cells.

Uptake and release of g-aminobutyric acid

[3H]GABA was accumulated by cells in regions similar tothose above for [3H]Glu, but with one notable exception.

No [3H]GABA labelled taste bud cells were observed.Presumptive Schwann cells and cells in the stratum basaleof non-sensory epithelium were labelled (Fig. 6). Silvergrains were absent from taste buds and from goblet cells.Samples were rinsed with APS that contained 40 mM KClto depolarize the tissue, as was done above, to test whetherthis treatment affected the distribution of silver grains.Qualitatively, no obvious differences were observed in theaccumulation of [3H]GABA whether the tissues had beenrinsed with APS; APS with 40 mM KCl; or APS with 40 mMKCl, 20 mM MgCl2, and 0.0.4 mM CaCl2. Given the factthat [3H]GABA did not accumulate in taste bud cells andthat our focus was on putative transmitters in taste cells,we did not measure grain densities and quantify the datafor GABA uptake.

DISCUSSION

Histochemical, immunocytochemical, and physiologicaldata have implicated 5HT, glutamate, and GABA ascandidates for taste bud neurotransmitters (reviewed inNagai et al., 1996). The present investigation utilizesanother methodology and reports findings that extend,amplify, and clarify the role of these transmitter candi-dates in amphibian taste buds. Specifically, high affinityuptake of tritiated labelled compounds was examined totest whether taste cells transport these transmitter candi-dates in this amphibian species. The principal finding isthat [3H]5HT is selectively taken up by and released upondepolarization in a Ca-dependent fashion from a selectsubset of taste bud cells, the Merkel-like basal cells.[3H]glutamate is taken up by a different subset of taste budcells, ones that more closely resemble taste receptor cells.However, there is no evidence for depolarization-depen-dent release of glutamate from these cells. Lastly,[3H]GABA is not taken up by taste bud cells, but accumu-lates in other cells in lingual tissue. Given these results,the evidence most strongly supports 5HT as being aneurotransmitter or neuromodulator in Necturus tastebuds. The evidence from uptake studies is weaker forglutamate, and non-existent for GABA, as being neuro-transmitters.

Serotonin as a neurotransmitteror neuromodulator in taste buds

Serotonin is found in high concentrations in specifictaste bud cells, and has long attracted attention as apotential neurotransmitter in peripheral taste organs.Early studies using histofluorescent techniques for aminessuggested that certain cells in taste end organs in fish,amphibia, and mammals contained monoamines (Reutter,1971, 1978; Dehan and Graziadei, 1973; Savushkina et al.,1974; Goossens and Vandenberghe, 1974; Hirata andNada, 1975; Nada and Hirata, 1975; Toyoshima et al.,1984; Toyoshima and Shimamura, 1987). These findingswere later refined using specific antibodies to serotoninand immunocytochemical methodology. There is now strongevidence that Merkel-like basal cells in taste buds fromnon-mammalian vertebrates, and a subset of receptor cellsin mammalian taste buds contain serotonin (Uchida, 1985;Fujimoto et al., 1987; Delay et al., 1993; Kim and Roper,1995). Studies utilizing intracellular and patch microelec-trode recordings have shown that serotonin producespostsynaptic responses when focally applied to taste cellsand modulates receptor cell properties in Necturus and inrats (Ewald and Roper, 1994a; Delay et al., 1997; Chen et

NEUROTRANSMITTERS IN NECTURUS TASTE BUDS 203

Figure 4

204 T. NAGAI ET AL.

al., 1996). Lastly, injecting serotonin into the blood streamnear taste buds in amphibia modifies taste responsesrecorded from the glossopharyngeal nerve (Morimoto andSato, 1977; Esakov et al., 1983; Nagahama and Kurihara,1985). Taken together with the findings reported in thepresent study, the data suggest that serotonin, releasedfrom specific cells in amphibian (and perhaps mammalian)taste buds, modulates taste transduction in the end or-gans. This might explain, for example, certain taste abnor-malities that are side effects of tricyclic antidepressants,such as imipramine, in clinical medicine (reviewed inNagai et al., 1996). These antidepressants are powerfulblockers of serotonin uptake and may exert peripheraleffects on the end organs of taste as well as act in the CNSto control depression. Clearly, what remains unanswerednow is what is the stimulus that provokes serotoninrelease from Merkel-like basal cells in taste buds fromamphibia, and from a subset of receptor cells in mamma-lian taste buds. Efferent input from the central nervoussystem is one possibility. Cholinergic parasympatheticinput from lingual ganglia is another speculation. Lastly,excitation from primary taste receptor cells directly ontoserotonergic taste buds cells may elicit release of themonoamine (Ewald and Roper, 1994a).

Parenthetically, the function of serotonergic Merkel-likebasal cells in amphibian taste buds remains unclear.These cells are so named because of their morphologicalresemblence with cutaneous Merkel cells. CutaneousMerkel cells have been associated with mechanoreception(e.g., Ogawa, 1996). However, this role has been chal-lenged and it has been suggested that cutaneous Merkelcells function in the growth, development, and mainte-nance of epidermis and its nerve supply (Diamond et al.,1988; Tachibana, 1995). Toyoshima (1994) speculated thatin taste buds, Merkel-like basal cells exert a neurotropicinfluence on the ingrowth of sensory nerve fibers duringearly development and provide a continuing trophic influ-ence over the survival and maintenance of taste buds inthe adult. Our findings do not resolve the function ofMerkel-like basal taste buds cells, but suggest that what-ever is their final role, serotonin will be a player.

Glutamate as a neurotransmitterin taste buds

Jain and Roper (1991) reported that nerve fibers inner-vating taste buds in Necturus were immunoreactive toantibodies raised against glutamate. On the basis of thesefindings, they suggested that glutamate may be involvedin neurotransmission in taste, although the axonal localiza-tion, rather than in taste bud cells, was enigmatic. Jainand Roper (1991) discussed the possibility that glutamatewas an efferent transmitter, or alternatively that thepresence of glutamate in peripheral nerve endings in tastebuds may simply reflect the presence of glutamatergicneurotransmission at central endings of the sensory affer-ent neurons. The immunocytochemical localization of glu-tamate in nerve fibers does not at all match the uptake of[3H]glutamate in lingual structures in Necturus found inthe present study. This discrepancy is not readily ex-plained. [3H]glutamate accumulated in certain taste budcells and also in non-taste epithelial cells in the surround-ing stratum basale. However, the most intense accumula-tion of [3H]glutamate was in glial cells in the laminapropria of the lingual mucosa. It is possible that radiola-

Fig. 4. Necturus taste bud cells, epithelial cells, and glial cells inthe lamina take up [3H]glutamate via high affinity transport mecha-nisms. A: This photomicrograph shows a section that contains tworadiolabelled cells in the taste bud (curved arrows), radiolabelled cellsin the surrounding non-taste stratum basale (asterisks), and stronglylabelled glial cells in the lamina propria (arrowheads). The section wasphotographed with bright field illumination. B: Same section as in A,photographed with phase contrast optics. C: Another taste bud,photographed with dark-field illumination. Stratum basale cells (aster-isks), but not mucus-secreting goblet cells (double arrowheads) trans-port [3H]glutamate. This taste bud contains at least 2 radiolabelledtaste cells (white arrows). D: Same section as in C, but showingdouble-exposures that combine dark-field illumination with phase-contrast optics. E: Another experimental tissue, showing intenselylabeled glial cells (arrowheads) that accompany nerve fibers innervat-ing the taste organ. This montage of 3 micrographs was photographedwith bright field illumination. The original micrographs were scannedinto a Macintosh Quandra 800 (Apple, Cupertino, CA) using a UMAXPS2400X scanner (UMAX Technologies, Fremont, CA). The gray levelswere balanced and cut lines removed using Adobe Photoshop v.3(Mountain View, CA). The final output was produced on a Kodak8650DS printer (Eastman Kodak, Rochester, NY). F: Same section asin E, showing double-exposures that combine dark-field illuminationwith phase-contrast optics. Myelinated axons travelling in the nervebundle are visible (arrow). The tissues in A–F had been incubated for15 minutes in 5 µM [3H]glutamate (57.4 Ci/mmol). Scale bars 5 50 µmfor A and B, 50 µm for C–F.

Fig. 5. Epithelial stratum basale cells and a subset of taste budcells preferentially take up [3H]glutamate. Other cells in lingual tissueshow only marginal accumulation of [3H]glutamate. Furthermore,[3H]glutamate in taste cells and stratum basale cells is not released bydepolarizing the tissue. Lingual tissues were incubated in 5 µM[3H]glu and then rinsed either in APS; in APS containing 40 mM KCl(‘‘40 mM K-APS’’); or in APS containing 40 mM KCl , 0.4 mM CaCl2 and20 mM MgCl2 (‘‘Low Ca-APS’’) and processed for autoradiography.Bars show the mean (6S.E.M.) number of silver grains in a 10 µmdiameter circle (ca. 80 square microns) placed over five differentregions (taste cells, stratum basale cells, lamina propria excludingSchwann cells, goblet cells, and superficial epithelial cells) for thesethree conditions. The numbers over the bars show the number of cellssampled (or in the case of lamina propria, the number of areassampled); asterisk, the data for goblet cells were significantly differentfrom those collected in APS and in low Ca-APS (P 5 0.05). No othersample means were significantly different between APS, 40 mMK-APS or low Ca-APS.

NEUROTRANSMITTERS IN NECTURUS TASTE BUDS 205

belled fine axons would not be resolved in thin (1.5–2.5 µm)sections.

Thus, the question of whether glutamate is a neurotrans-mitter at amphibian taste bud synapses cannot be an-swered on the basis of the present findings. The accumula-tion of [3H]glutamate in lingual tissue does not readily fitthe pattern for a neurotransmitter at taste bud synapses.In the brain of mammals and retinae of fish and reptiles,[3H]glutamate uptake has been successfully used to iden-tify sites of glutamatergic synaptic transmission (Marcand Lam, 1981; Balcar and Li, 1992; Sherry and Ulshafer,1992). Based on those reports, the absence of glutamateuptake and release we report here suggests that thisamino acid is not a transmitter in Necturus taste buds. Onthe other hand, glutamate is likely to be the neurotransmit-ter at afferent synapses in the cochlea (e.g., Li et al., 1994).Yet, cochlear hair cells do not take up [3H]glutamate

(Gulley et al., 1979). Instead, glutamine, the precursor forglutamate at excitatory amino acid synapses, is trans-ported into cochlear hair cells in the guinea-pig and gerbil(Eybalin and Pujol, 1983; Ryan and Schwartz, 1984).Indeed, Gulley et al (1979) concluded that ‘‘it appearsunlikely that high affinity uptake can be useful for identi-fying the neurotransmitter at the hair cell to afferentsynapse’’. Thus, based on the reports in the mammaliancochlea, the present negative results in taste buds forglutamate as a neurotransmitter may not rule out theexistence of glutamatergic synapses.

Uptake of glutamate into lingual epithelial cells inNecturus may reflect the widespread use of glutamic acidin a variety of cellular metabolic processes not directlyrelated to neurotransmission, including its participationas an energy source in the tricarboxylic acid cycle, in ureaand glutathione synthesis, and in protein synthesis (Meis-

Fig. 6. Necturus lingual epithelial cells and glial cells in the laminapropria take up [3H]GABA via high affinity transport mechanisms.Two different taste buds are shown. A: Treated with 0.6 µM [3H]GABA;B: with 6 µM [3H]GABA. C: Higher magnification of the box shown inB. Taste bud cells do not take up [3H]GABA at either concentrationtested. Glial cells (arrowheads) in the lamina propria are labelled.

Cells in the stratum basale (asterisks) of the non-taste epithelium arealso labelled, especially when the tissue was treated with the higherconcentration of GABA. Sections were photographed with doubleexposures that combine dark field illumination with phase contrastoptics. Scale bars 5 50 µm for A, B, 20 µm for C.

206 T. NAGAI ET AL.

ter, 1979). Indeed, non-neuronal cells in a variety of othertissues, including hepatocytes, kidney epithelium, andfibroblasts transport and utilize glutamate for metabolicpurposes (Sacktor et al., 1981; Haussinger and Gerok,1983; Balcar et al., 1994).

Other roles for glutamate transport include the establish-ment of blood/tissue barriers. For example, retinal pig-ment epithelium in rats has a powerful glutamate uptakemechanism to assist in creating a blood-retina barrier andmaintain a low interstitial concentration of glutamic acidin the synaptic spaces within the retina (Salceda andSaldana, 1993), where glutamate is a neurotransmitter(Cervetto and MacNichol, 1972; Murakami et al., 1972;Sugawara and Negishi, 1973). Similarly, insect epidermalcells transport glutamate, perhaps also functioning tomaintain a low blood plasma concentration and to pre-serve the integrity of synaptic transmission where gluta-mate is a transmitter at neuromuscular junctions (McLeanand Caveney, 1993). However, it is unlikely that theglutamate uptake observed in lingual epithelium andSchwann cells produces a blood/taste bud barrier. There isno ultrastructural barrier surrounding taste buds and thelamina propria immediately subjacent to taste buds isrichly vascularized, including a bed of fenestrated capillar-ies and sinuses (Graiziadei and Monti Graziadei, 1978).Substances injected into the blood stream readily pen-etrate into and stimulate taste buds, a phenomenon termedintravenous taste (Bradley, 1973).

Lastly, the mere accumulation of [3H]glutamate in Nec-turus lingual cells may not necessarily represent netuptake, but simply exchange of glutamate between intra-cellular and extracellular compartments. More detailedexperiments would need to be conducted in lingual tissuesto ascertain whether the appearance of [3H]glutamate incertain cells was net accumulation or merely transmem-brane exchange.

There are other reasons to doubt whether glutamate is aneurotransmitter in taste buds. The plasma concentrationof glutamate is relatively high (e.g., in humans, up to 250µM; Table 18-2 in Henry et al., 1974). This is inconsistentwith glutamic acid being a neurotransmitter at sites, suchas taste buds, that are not protected by a blood/tissuebarrier. For example, in the retina where glutamic acid is aneurotransmitter, there is an efficient blood/retina barrier.The interstitial concentration of glutamate is about 20 µM(i.e., aqueous humor, Table 18-3 in Henry et al., 1974), onlya fraction that of the plasma. Similarly, glutamate is one ofthe most ubiquitous excitatory neurotransmitters in thebrain, where the blood/brain barrier maintains a lowconcentration of 1–2 µM (i.e., in cerebrospinal fluid, Table18-3 in Henry et al., 1974). Although glutamate does havean effect on taste buds, at least in mammals, it is in theform of a taste stimulus (e.g., monosodium glutamate).The taste of monosodium glutamate is presumably trans-duced by specific receptors at the apical chemosensory tipsof taste cells (Chaudhari et al., 1996).

GABA as a neurotransmitter in taste buds

[3H]GABA uptake into neurons is an accurate andreliable sign of GABAergic transmission in several neuralsystems where it has been investigated, including the cellbodies and nerve fibers in the retina, CNS, gut, andnematode ganglia (O’Malley and Masland, 1989; Mullerand Marc, 1990; Guastella and Stretton, 1991; Krantis andClark, 1991). We found no evidence for GABA transport

into taste cells or nerve fibers in Necturus. The failure toobserve [3H]GABA uptake in Necturus taste buds is notdue to any access barriers since [3H]glutamate and [3H]se-rotonin readily penetrated to and were taken up by tastebud cells. Furthermore, cells in the immediate proximity oftaste buds strongly accumulated [3H]GABA. Althoughattention has been focussed on the neurotransmitter rolefor GABA, there are numerous non-neural tissues thattake up and accumulate this amino acid, including kidneyepithelium, hepatocytes, platelets, spermatozoa and oth-ers (see Table 4 in Erdo, 1992). GABA clearly has functionsapart from its role as a neurotransmitter, perhaps viatransamination to glutamate and the metabolism of gluta-mate. However, we have no specific explanation for thehigh affinity uptake of GABA into non-sensory epithelialcells and glial cells.

ACKNOWLEDGMENTS

We are indebted to Drs. S. Hayashi, M. Ichikawa, TokyoMetropolitan Institute for Neurosciences, and Dr. Y. Oka,University of Tokyo, for technical assistance. We acknowledgeMs. E. Gilfoyle and J. Voss, D.V.M., for authorship for J.W.

LITERATURE CITED

Balcar, V.J., J. Shien, S. Bao, and N.J.C. King (1994) Na-dependent highaffinity uptake of L-glutamate in primary cultures of human fibroblastsisolated from three different types of tissue. FEBS Lett. 339:50–54.

Balcar, V.J., and Y. Li (1992) Heterogeneity of high affinity uptake ofl-glutamate and l-aspartate in the mammalian central nervous system.Life Sci. 51:1467–1478.

Bradley, R.M. (1973) Electrophysiological investigations of intravasculartaste using perfused rat tongue. Am. J. Physiol. 224:300–304.

Cervetto, L., and E.F. MacNichol (1972) Inactivation of horizontal cells inturtle retina by glutamate and aspartate. Science 178:767–768.

Chaudhari, N., H. Yang, C. Lamp, E. Delay, C. Cartford, and S.D. Roper(1996) The taste of monosodium glutamate: Membrane receptors intaste buds. J. Neurosci. 16:3817-–826.

Chen, Y., X.-D. Sun, and M.S. Herness (1996) Serotonin inhibits calcium-activated potassium currents in rat taste receptor cells. Soc. Neurosci.Abstracts 22:1827.

Dall’Asta, V., G.C. Gazzola, R. Franchi-Gazzola, O. Bussoloti, N. Longo, andG.G. Guidotti (1983) Pathways of L-glutamic acid transport in culturedhuman fibroblasts. J. Biol. Chem. 258:6371–6379.

Dehan, R.S., and P.P.C. Graziadei (1973) The innervation of frog’s tasteorgan. Life Sci. 13:1435–1449.

Delay, R.J., S.C. Kinnamon, and S.D. Roper (1997) Serotonin modulatesvoltage-dependent calcium current in Necturus taste cells. J. Neuro-physiol.77:2515–2524.

Delay, R. J., and S. D. Roper (1988) Ultrastructure of taste cells andsynapses in the mudpuppy. Necturus maculosus. J. Comp. Neurol.277:268–280.

Delay, R.J., R. Taylor, and S.D. Roper (1993) Merkel-like basal cells inNecturus taste buds contain serotonin. J. Comp. Neurol. 335:606–613.

Diamond J., L.R. Mills, and K.M. Mearow (1988) Evidence that the Merkelcell is not the transducer in the mechanosensory Merkel cell-neuritecomplex. Prog. Brain Res. 74:51–56.

Erdo, S.L. (1992) Non-neuronal GABA systems: An overview. In S.L. Erdo(ed): GABA Outside the CNS. Berlin: Springer-Verlag, pp. 97–110.

Esakov, A.I., K.V. Golubtsov, and N.A. Solov’eva (1983) The role of serotoninin taste reception in the frog Rana temporaria. J. Evol. Biochem.Physiol. 19:56–61.

Ewald, D.A., and S.D. Roper (1994a) Bidirectional synaptic transmission inNecturus taste buds. J. Neurosci. 14:3791–3804.

Ewald, D.A., and S.D. Roper (1994b) Cholinergic responses of taste cells inNecturus taste buds. Soc. Neurosci. Abstracts. 20:980.

Eybalin M., and R. Pujol (1983) A radioautographic study of [3H]L-glutamate and [3H]L-glutamine uptake in the guinea-pig cochlea.Neuroscience 9:863–871.

Farbman, A.I., and J.D. Yonkers (1971) Fine structure of the taste bud inthe mud puppy, Necturus maculosus. Amer. J. Anat. 131:353–369.

NEUROTRANSMITTERS IN NECTURUS TASTE BUDS 207

Fujimoto, S., H. Ueda, and H. Kagawa (1987) Immunocytochemistry on thelocalization of 5-hydroxytryptamine in monkey and rabbit taste buds.Acta Anat. 128:80–83.

Garcia-Caballero, T., R. Callego, E. Rosen, D. Basanti, and G. Morel (1989)Localization of serotonin-like immunoreactivity in the Merkel cells ofpig snout skin. Anat. Rec. 225:267–271.

Graiziadei, P.P.C., and G.A. Monti Graziadei (1978) Observations on theultrastructure of ganglion cells in the circumvallate papilla of rat andmouse. Acta Anat. 100:289–305.

Goossens, N., and M.-P. Vandenberghe (1974) The basal cells in the papillaefungiformes of the tongue of the common frog, Rana temporaria. Arch.Histol. Jap. 36:173–179.

Guastella, J., and A.O.W. Stretton (1991) Distribution of [3H]GABA uptakesites in the nematode Ascaris. J. Comp. Neurol. 307:598–608.

Gulley, R.L., J. Fex, and R.J. Wenthold (1979) Uptake of putative neurotrans-mitters in the organ of Corti. Acta Otolaryngol. 88:177–182.

Hartschuh, W., and E. Weihe (1988) Multiple messenger candidates andmarker substances in the mammalian Merkel cell-axon complex: A lightand electron microscopic immunohistochemical study. Prog. Brain Res.74:181–187.

Haussinger, D., and W. Gerok (1983) Hepatocyte heterogeneity in gluta-mate uptake by isolated perfused rat liver. Eur. J. Biochem. 136:421–425.

Henry, R.J., D.C. Cannon, and J.W. Winkelman (1974) Clinical Chemistry,2nd Edition, New York: Harper and Row.

Hirata, K., and O. Nada (1975) A monoamine in the gustatory cell of thefrog’s taste organ. Cell Tissue Res. 159:101–108.

Jain, S.B., and S.D. Roper (1991) Immunocytochemistry of g-aminobutyricacid, glutamate, serotonin and histamine in Necturus taste buds. J.Comp. Neurol. 307:675–682.

Johnston, G.A.R. (1981) Glutamate uptake and its possible role in neuro-transmitter inactivation. In P.J. Roberts, J. Storm-Mathisen, andG.A.R. Johnston (eds): Glutamate: Transmitter in the Central NervousSystem. New York: Wiley, pp. 77–87.

Kim, D.-J., and S.D. Roper (1995) Localization of serotonin in taste buds: Acomparative study in four vertebrates. J. Comp. Neurol. 353:364–370.

Kinnamon, S.C., and S. Roper (1987) Passive and active membraneproperties of mudpuppy taste receptor cells. J. Physiol. 383:601–614.

Kinnamon, S.C., and S. Roper (1988) Evidence for a role of voltage-sensitiveapical K1 channels in sour and salt taste transduction. Chem. Senses13:115–121.

Krantis, A., and D. Clark (1991) High-affinity uptake of [3H]GABA bysubmucous ganglion cells, nerve fibres and peri- and para-vascularfibres in guinea-pig and rat intestine. J. Auton. Nerv. Syst. 32:251–258.

Landgren, S., G. Liljestrand, and Y. Zotterman (1954) Chemical transmis-sion in taste fibre endings. Acta Physiol. Scand. 30:105–114.

Lauder, J.M. (1993) Neurotransmitters as growth regulatory signals.Trends Neurosci. 16:233–240.

Li, H-S., A.S. Niedzielski, K.W. Beisel, H. Hiel, R.J. Wenthold, and B.J.Morley (1994) Identification of a glutamate/aspartate transporter in therat cochlea. Hearing Res. 78:235–242.

Logan, W.J., and S.H. Snyder (1971) Unique high affinity uptake systemsfor glycine, glutamic and aspartic acids in central nervous tissue of therat. Nature (Lond.) 234:297–299.

Marc, R.E., and D.M.K. Lam (1981) Uptake of aspartic and glutamic acid byphotoreceptors in goldfish retina. Proc. Natl. Acad. Sci. USA 78:7185–7189.

McLean, H., and S. Caveney (1993) Na1-dependent medium-affinity uptakeof L-glutamate in the insect epidermis. J. Comp. Physiol. B 163:297–306.

Meister, A. (1979) Biochemistry of glutamate: glutamine and glutathione.In Filer, L.J., Kare, M.R., Garattini, S., and Reynolds, W.A. (eds):Glutamic Acid: Advances in Biochemistry and Physiology. New York:Raven, pp. 69–84.

Mize, R.R., R.E. Marc, and A.M. Sillito (1992) GABA in the Retina andCentral Visual System. (Prog. Brain Res. Volume 90), Amsterdam:Elsevier.

Morimoto, K., and M. Sato (1977) Is serotonin a chemical transmitter in thefrog taste organ? Life Sci. 21:1685–1696.

Morimoto, K., and M. Sato (1982) Role of monoamines in afferent synaptictransmission in frog taste organ Jpn. J. Physiol. 32:855–871.

Muller, J.F., and R.E. Marc (1990) GABA-ergic and glycinergic pathways inthe inner plexiform layer of the goldfish retina. J. Comp. Neurol.291:281–304.

Murakami, M., K. Ohtsu, and T. Ohtsuka (1972) Effects of chemicals onreceptors and horizontal cells in the retina. J. Physiol. (Lond.) 227:899–913.

Murray, R.G. (1986) The mammalian taste bud type III cell: A criticalanalysis. J. Ultrastructural Mol. Res. 95:175–188.

Nada, O., and K. Hirata (1975) The occurrence of the cell type containing aspecific monoamine in the taste bud of the rabbit’s foliate papilla.Histochemistry 43:237–240.

Nagahama, S., and K. Kurihara (1985) Norepinephrine as a possibletransmitter involved in synaptic transmission in frog taste organs andCa dependence of its release. J. Gen. Physiol. 85:431–442.

Nagai, T., R.J. Delay, and S.D. Roper (1994) In vitro uptake of [3H]gluta-mate and [3H]GABA in Necturus taste buds. Neurosci. Abstr. 20:980.

Nagai, T., D.-J. Kim, R.J. Delay, and S.D. Roper (1996) Neuromodulation oftransduction and signal processing in the end organs of taste. Chem.Senses 21:353–365.

Nagai, T., and H. Koyama (1994) Ultrastructure of Merkel- like cells labeledwith carbocyanine dye in the non-taste lingual epithelium of the axolotl.Neurosci. Lett. 178:1–4.

Ogawa, H . (1996) The Merkel cell as a possible mechanoreceptor cell. Prog.Neurobiol. 49:317–334.

O’Malley, D.M., and R.H. Masland (1989) Co-release of acetylcholine andgamma-aminobutyric acid by a retinal neuron. Proc. Natl. Acad. Sci.USA 86:3414–3418.

Peters, T., Jr., and C.A. Ashley (1967) An artefact in radioautography due tobinding of free amino acids to tissues by fixatives. J. Cell Biol. 33:53–60.

Reutter, K. (1971) Die Geschmacksknospen des Zwergwelses Amiurusnebulosus (Lesueur): Morphologische und histochemische Untersuchun-gen. Z. Zellforsch. 120:280–308.

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

Roper, S.D. (1992) The microphysiology of peripheral taste organs. J.Neurosci. 12:1127–1134.

Ryan, A.F., and I.R. Schwartz (1984) Preferential glutamine uptake bycochlear hair cells: Implications for the afferent cochlear transmitter.Brain Res. 290:376–379.

Sacktor, B., I.L. Rosenbloom, C.T. Liang, and L. Cheng (1981) Sodiumgradient- and sodium plus potassium gradient-dependent L-glutamateuptake in renal basolateral membrane vesicles. J. Membr. Biol. 60:63–71.

Salceda, R., and M.R. Saldana (1993) Glutamate and taurine uptake byretinal pigment epithelium during rat development. Comp. Biochem.Physiol. 104C:311–316.

Savushkina, M.A., E.M. Krokhina, and A.I. Esakov (1974) Examination ofthe taste buds of the frog tongue by fluorescent microscopy. Byull. eks.biol. med. 78:118–121.

Seuwen, K., and J. Pouyssegur (1990) Serotonin as a growth factor.Biochem. Pharmacol. 39:985–990.

Sherry, D.M., and R.J. Ulshafer (1992) Neurotransmitter-specific identifica-tion and characterization of neurons in the all-cone retina of Anoliscarolinensis. II: Glutamate and aspartate. Vis. Neurosci. 9:313–323.

Sugawara, K., and K. Negishi (1973) Effects of some amino acids on thehorizontal cell membrane potential in the isolated carp retina. VisionRes. 13:2479–2489.

Tachibana, T. (1995) The Merkel cell: Recent findings and unresolvedproblems. Arch. Histol. Cytol. 58:379–396.

Toyoshima, K. (1989) Chemoreceptive and mechanoreceptive paraneuronsin the tongue. Arch. Histol. Cytol. 52 (Suppl.):383–388.

Toyoshima, K. (1994) Role of Merkel cells in the taste organ morphogenesisof the frog. In K. Kurihara, N. Suzuki, and H. Ogawa (eds): Olfactionand Taste XI. Tokyo: Springer-Verlag, pp. 13–15.

Toyoshima, K., K. Miyamoto, A. Itoh, and A. Shimamura (1987) Merkel-neurite complexes in the fungiform papillae of two species of monkeys.Cell Tissue Res. 250:237–239.

Toyoshima, K., O. Nada, and A. Shimamura (1984) Fine structure ofmonoamine-containing basal cells in the taste buds on the barbels ofthree species of teleosts. Cell Tissue Res. 235:479–484.

Toyoshima, K., and A. Shimamura (1987) Monoamine-containing basalcells in the taste buds of the newt, Triturus pyrrhogaster. Archs. OralBiol. 32:619–621.

Uchida, T. (1985) Serotonin-like immunoreactivity in the taste bud of themouse circumvallate papilla. Jpn. J. Oral Biol. 27:132–139.

Welton, J., and S. Roper (1992) In vitro uptake of 3H serotonin in taste budsof the mudpuppy, Necturus maculosus. Chem. Senses 17:719.

208 T. NAGAI ET AL.