J Life Sci Res 2014; 12: 9-14
Ⓒ 2014 Osaka Prefecture University

Journal of Life Science Research
Online ISSN 2186-5809

Original

Neural impulse amplitude of taste nerve fiber branches depends on taste receptor sites
in bullfrogs
Ken-ichi KASAHARA１, Yoshitaka OHTUBO１, Takashi KUMAZAWA２ and Kiyonori YOSHII†１
Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Hibikino 2-4, Kitakyushu-shi 808-0196, Japan１

２Graduate School of Engineering, Saitama Institute of Technology, Fukaya 369-0293, Japan.

Received 26 June 2014; accepted 17 July 2014

The branches of each single taste nerve fiber selectively innervate the same taste receptor site on different
taste receptor cells. Here we show that the impulse amplitude and firing pattern of nerve branches depends ontaste receptor sites in bullfrogs. L-Threonine, L-serine, and galactose elicited larger impulses in amplitude thanquinine HCl. L-Leucine, L-phenylalanine, and papaverine HCl elicited both impulses, where the train of thesmall impulses preceded that of the large ones. The large impulses were suppressed in the presence of salts,
but the small impulses were not. Washing subjects’ tongues with an alkaline solution facilitated the firing of
the large impulses. The firing of the small impulses stopped shortly, but that of the large impulses kept firing
during taste stimulation. The present results showed that taste receptor sites examined could be classified into
salt-sensitive and salt-insensitive sites, and that the nerve branches generating the large impulses innervatedsalt-sensitive sites while the nerve branches generating the small impulses innervated salt-insensitive sites.
These results suggest that the excitability of taste nerve branches contributes to the selective innervation. We
discuss the firing of the large and small impulses.
Key words : taste nerve responses; suction electrodes; impulse amplitude; antidromic impulses; alkaline treatment

1 Introduction
Single taste nerve fibers of mammals, frogs, and fishes
more or less selectively respond to taste substances.1−8
Notwithstanding that single taste nerve fibers ramify andinnervate different taste receptor cells, each single taste
nerve fiber comprising the coherent response of its branches
showed selective responsiveness. These results showed thatthe branches of each single taste nerve fiber innervate thesame taste receptor site on different taste receptor cells, andsuggest that these branches differentiate for selective innervation. The differentiation may affect the electrophysiological properties of the taste nerve fiber branches, and theneural impulses of branches may reflect the differentiation.
Frog taste receptors are taste disks innervated by ~10
myelinated fibers and a few unmyelinated fibers on the tip
of fungiform papillae.9, 10 They are homologous with mammalian taste buds, though they are much bigger than taste
buds. In bullfrogs, the branch of each single taste nerve fiber
innervates 6.6 taste disks.6 Therefore, action potentials oftaste nerve fibers generated in response to taste substances
are not only transmitted to the brain but also antidromicallyCorresponding author, Email: yoshii@brain.kyutech.ac.jp†

transmitted to neighboring taste disks on different fungiform papillae. These antidromic action potentials have beenrecorded extracellularly by sucking the fungiform papillae
with suction electrodes.11−14 The amplitude of antidromic
neural impulses thus recorded depends on the amplitude ofaction currents, and hence shows the electrophysiologicalproperties of the taste nerve fiber branches.
Our previous study showed that although the instantaneous component (phasic response) of integrated taste nerve
responses of bullfrogs to hydrophobic amino acids,
L-leucine, L-phenylalanine, etc. was insensitive to salts, thesubsequent long-lasting component (tonic response) was
suppressed in the presence of salts.15 Bullfrog taste nerve
responses to galactose, a hydrophilic substance, were suppressed in the presence of salts.16 These results showed thatthese taste receptor sites could be classified as salt-sensitiveor salt-insensitive.
In the present study, we took advantage of bullfrog
taste receptors and investigated the neural impulses of taste
nerve fiber branches innervating the salt-sensitive and saltinsensitive sites by recording these antidromic impulses
with suction electrodes. In addition to amino acids andgalactose, we chose papaverine HCl and quinine HCl as10hydrophobic substances, in order to examine the relations
among the hydrophobicity/hydrophilicity of taste substances, the salt-sensitivity of taste receptor sites for them,and the height of neural impulses they elicited. The present
results showed that taste nerve branches innervating thesalt-sensitive sites generated larger impulses in amplitude
than taste nerve branches innervating the salt-insensitivesites. Also we showed that the small impulses formed thephasic responses and the large impulses formed the tonic
responses. These results suggest that taste nerve fiber
branches differentiate or adjust to selective innervation, andthat the differentiation or adjustment affects the impulse
amplitude. We discuss the firing of these impulses.
2 Materials and Methods
All experimental protocols were conducted in compliance with the Guiding Principles for the Care and Use of
Animals in the Field of Physiological Sciences approved by
the council of the Physiological Society of Japan, and werepermitted by the Animal Institutional Review Board of
Saitama Institute of Technology in accordance with theguidelines of the U.S. National Institutes of Health.
2.1 Recording of antidromic impulses
We purchased adult bullfrogs, Rana catesbeiana froma local commercial source, and anesthetized them as described in a previous paper.17 In brief, we anesthetized bullfrogs of 180~250 g with intraperitoneal injection ofurethane (0.2 g/100 g body weight), recorded the taste responses from the branch of glossopharyngeal nerves innervating fungiform papillae with suction electrodes as thetrain of antidromic neural impulses as described in previous
papers.11−14 The impulses were amplified, band-pass filtered(300-3000 Hz) with homemade devices, and stored in acomputer. A small amount of urethane was added to maintain anesthetization. These animals were killed by decapitation under anesthetization after the experiments.
Since each fungiform papilla contained ~10 myelinated nerve fibers and a few unmyelinated fibers,9, 10 suction
electrodes recorded antidromic impulses from these myelinated fibers. Also suction electrodes might record the antidromic impulses of unmyelinated fibers, if they weredetectable.
2.2 Stimulation
All taste substances were dissolved in deionized wateror salt solutions, and applied to the whole ventral surfaceof tongues to simulate whole fungiform papillae acclimatedto their respective vehicles through a silicon tube. Althoughbullfrog taste disks respond to deionized water,18−20 we irrigated taste disks with deionized water until the water response in the firing frequency became negligible. All
chemicals used were of analytical grade purchased from

Wako Pure Chemical Industries, Ltd, and all experiments
were carried out at room temperature.
3 Results
3.1 Alkaline treatment
The application of taste substances to the tongue
elicited antidromic impulses (Figure 1). We previously
showed that a wash of the tongue with an alkaline solution(2.5 mM NaHCO3-Na2CO3 buffer, pH 10.0) for ~5 min enhanced the tonic responses to amino acids.15 In the present
study, the alkaline treatment increased the frequency of thelarge antidromic impulses generated by the application of
50 mM L-leucine. Similar results were found in response to
the other amino acids, papaverine HCl, and galactose. In
the following experiments we performed alkaline treatment
before all tongue examinations.
3.2 Current amplitude
Several hydrophilic amino acids, L-threonine, L-serine,and glycine and galactose primarily generated higher-amplitude antidromic impulses, whereas quinine HCl primarily
generated smaller antidromic impulses (Figure 2). We classified these impulses obtained from the same fungiform
papillae into two groups by their amplitude. When the amplitude of given impulses was larger than one-third of the
maximum impulse in amplitude, we referred to them aslarge impulses. Impulses smaller than the criteria but 1.5times larger than the fluctuation amplitude of the current
traces in the absence of stimulation were referred to as small
impulses. The other impulses were neglected as noises. We
stopped the recording when the amplitude of the maximum
impulses was changed by 20%.
Hydrophobic amino acids such as L-leucine, L-phenylalanine, and L-tryptophan generated both large and small
impulses (Figure 2A). Hydrophilic amino acids such as
L-threonine, L-serine, and glycine typically generated large
impulses (Figure 2B). Galactose generated large impulses,

Fig. 1 Antidromic impulses in response to 50-mM L-leucine before (upper traces) and after alkaline treatment (lower
traces) obtained from the same fungiform papilla.
L-Leucine dissolved in deionized water (left traces) or 10 mM

NaCl (right traces) was applied to the whole tongue surface acclimated to respective vehicles before and after an alkaline treatment.
Arrowheads show the onset of the stimulation in this and following figures.

11

Fig. 3 The number of large (open circles) and small (closed circles) impulses in response to L-leucine in the first 7 s after
the onset of stimulation as a function of L-leucine concentration.
L-Leucine was dissolved in deionized water and applied to thewhole tongue surface. Plotted are means and SDs (3 fungiform
papillae of different frogs).

Table 1

Fig. 2

Antidromic impulses in response to hydrophobic aminoacids, L-leucine, L-phenylalanine, and L-tryptophan (A),hydrophilic amino acids, L-threonine, L-serine, andglycine (B), and other taste substances (C) in the absence(left traces) and presence of 10 mM NaCl (right traces).

Taste substances were dissolved in deionized water (left traces) or
10 mM NaCl (right traces). We recorded from the same fungiform
papilla to compare the effect of NaCl. All stimuli were applied to
the whole tongue surface acclimated to respective vehicles after
alkaline treatment in this and all subsequent figures.

papaverine HCl generated large and small impulses, andquinine HCl generated small impulses (Figure 2C).
The concentration-response curves for L-leucine
showed that the threshold concentration of the large impulses was between 3 mM and 10 mM (Figure 3). The
threshold concentration of the small impulses was in thesame range. The number of large and small impulses firedin the first 7 s after the onset of stimulation was similar at
10 mM.
3.3 Salt-sensitivity
The addition of 10 mM NaCl to acclimating solutions
and taste-stimulating solutions suppressed the firing of thelarge impulses (Figure 2). The responses to galactose wereremarkably suppressed, and the responses to quinine HClremained unchanged. The quantitative comparison showedthat the addition of 10 mM NaCl significantly decreased thenumber of large impulses generated in response to 50 mM
L-threonine and 1 M galactose, and that the number of thesmall impulses generated remained unchanged (Table 1).

Salt-sensitivity of neural impulses in response to aminoacids and galactose

The numerals are means and SDs (6 fungiform papillae of different frogs) of summated impulse number over 7 s after the onsetof the stimulation with the indicated taste substances. There wereno significant differences between the number of small impulses
in the absence and presence of 10 mM NaCl (p > 0.05, two-tailed
t-test).

Similar results were obtained from the tongue untreatedwith the alkaline solution (Figure 1), showing that the saltsensitivity did not result from the alkaline treatment.
The number of large impulses in response to 50 mM
L-leucine was decreased with increasing NaCl concentration(Figure 4A). The addition of Na4Fe(CN)6 and K2SO4 also
decreased the number of large impulses with increasing
concentration, and no large impulses appeared at 10 mM(Figure 4B).
The large impulses elicited in response to 50 mM
L-leucine disappeared in the presence of 1 mM K2SO4 and10 mM Na4Fe(CN)6 and NaCl (Figure 4B). Although thenumber of the large impulses might decrease with increasing salt concentration, the large SDs prevented further
analyses.
3.4 Other differences of the large and small impulses
The train of small impulses always preceded that of
the large impulses by ~1 s in response to hydrophobic
amino acids, L-leucine, L-phenylalanine, and L-tryptophan,

12Fig. 5 Accumulated relative number of impulses as a functionof time after the onset of stimulation with 50 mM
L-leucine.
We accumulated the number of the large impulses every 0.5 s from2 s before to 8.5 s after the onset of stimulation, divided them withthe total number of the large impulses elicited during the stimulation period of 8.5 s, and plotted this value as a function of stimulation time. The small impulse number was similar normalized.
Plotted are the means and SDs (10 fungiform papillae of different
frogs) of the relative number of small (closed circles) and large
impulses (open circles). Stimulating solutions were applied to thewhole tongue surface.
Fig. 4

Salt-dependent suppression of antidromic impulses in response to 50 mM L-leucine.

A, antidromic impulses in different NaCl concentrations.
B, relative number of large impulses in the frist 7 s after the onsetof stimulation as a function of salt concentrations added to the
L-leucine solution. Plotted are the means and SDs for NaCl andNa4Fe(CN)6 (3 fungiform papillae of different frogs) and means
for K2SO4 (2 fungiform papillae of different frogs). The total large
impulse number in the 7 s was calculated relative to the total impulse number elicited in the same duration in the presence of 10−6
M respective salts. We applied stimulating solutions to the whole
tongue surface.

and papaverine HCl (Figs. 1, 2 and 4). The firing of thesmall impulses generated by 50 mM L-leucine stopped
shortly in ~4 s after the onset of firing, but the large ones
continued firing during stimulation (Figure 5). Similar inactivation in the firing of the small impulses was found in
response to the other hydrophobic amino acids, quinine
HCl, and papaverine HCl (Figure 2).
4 Discussion
In the present study, we classified the antidromic impulses of taste nerve fiber branches into large and small impulses. It is likely that thicker branches generate the large
impulses and thinner branches generate the small impulses,
if both branches are equal in membrane properties including
voltage-gated channel molecules and their densities.21 In
brief, impulse amplitude depends on the local circuit current
magnitude of taste nerve fiber branches. The current mag-nitude is proportional to the axon radius, the second derivative of the membrane potential with respect to time, and isinversely proportional to the square of the conduction velocity and the axoplasm resistivity. The axoplasm resistivity
is similar among axons. When the kinetics and the densityof voltage-gated channels are the same between the
branches, the second derivative depends on the branch radius. The conduction velocity would also depend on the
branch radius. Therefore, the branch radius primarily decides the impulse amplitude under these assumptions.
The firing of the small impulses stopped shortly, butthat of the large impulses was long-lasting. These results
suggest that taste receptor cells that respond to hydrophobic
substances inactivate easily. It is also possible that taste
nerve fibers that conduct the response to hydrophobic substances by firing small impulses inactivate easily. We previously investigated integrated taste nerve responses tohydrophobic amino acids recorded from the trunk of theglossopharyngeal nerve, and showed that salt-insensitivesites yielded phasic responses and salt-sensitive sites
yielded tonic responses.15 The present results not only
agreed with the previous study but also showed that thesmall impulses yielded the phasic responses and the large
impulses yielded the tonic responses. The transient firingof the small impulses suggest that salt-insensitive taste receptor cells or the small impulse-generating taste nerve
fibers easily inactivate.

13

The train of the small impulses in response to hydrophobic amino acids and papaverine HCl always precededthat of the large impulses by ~1 s. Since the distance
between stimulated and recorded fungiform papillae was a
few centimeters, even the slowest, unmyelinated nerves
conduct impulses between them in 10 ms. Therefore, thedifference in the conduction velocity of taste nerves neverproduces this lag time. We assume that the small impulses
are fired before the large impulses. In other words, taste receptor cells expressing salt-insensitive sites may more rapidly trigger the neurotransmitter releases than thoseexpressing salt-sensitive sites.
The application of quinine HCl, a hydrophobic substance, only fired the small impulses. Although hydrophobic amino acids and papaverine HCl fired the large
responses, the small impulses always preceded the large impulses. Since many hydrophobic substances are bitter andtoxic,22 the small impulses may stop swallowing toxic
hydrophobic substances. By contrast, the large impulses
may urge or allow swallowing. Therefore, it is likely thattaste nerve fibers firing the small impulses and the large
impulses may have different roles.
The firing of large and small impulses showed thathydrophilic amino acids and galactose primarily stimulatedsalt-sensitive sites; hydrophobic amino acids and papaverine HCl stimulated both the salt-sensitive and salt-insensitive sites; and quinine HCl primarily stimulated thesalt-insensitive sites. Mammalian taste receptors express
two taste receptor molecule families, T1Rs and T2Rs.23−25
T1Rs consist of three subtypes1−3. The complex of T1R1
and T1R3 forms taste receptor sites for amino acids, andthat of T1R2 and T1R3 for sweet substances.26, 27 T2Rs are
taste receptor sites for bitter substances,24 and are expressedin different taste receptor cells from those expressing
T1Rs.23 So far, a few reports have detected genes for T2Rs
but not T1Rs in frogs.28, 29 However, we assume that bullfrog
taste receptors express their functional counterparts, andsuggest that the counterparts of frog T1Rs are salt-sensitiveand frog T2Rs are salt-insensitive. Since hydrophobic
amino acids are bitter substances, it is likely that they stimulate both the counterparts of frog T1R1/T1R3 complex andsome frog T2Rs.
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