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The Journal of Neuroscience, January 15, 2002, 22(2):493-504
Postnatal Development of Membrane Excitability in Taste Cells of
the Mouse Vallate Papilla
Albertino
Bigiani1,
Rosella
Cristiani2,
Francesca
Fieni1,
Valeria
Ghiaroni1,
Paola
Bagnoli2, and
Pierangelo
Pietra1
1 Dipartimento di Scienze Biomediche, Sezione di
Fisiologia, Università di Modena e Reggio Emilia, 41100 Modena,
Italy, and 2 Dipartimento di Fisiologia e Biochimica "G.
Moruzzi," Università di Pisa, 56127 Pisa, Italy
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ABSTRACT |
The mammalian peripheral taste system undergoes functional changes
during postnatal development. These changes could reflect age-dependent
alterations in the membrane properties of taste cells, which use a vast
array of ion channels for transduction mechanisms. Yet, scarce
information is available on the membrane events in developing taste
cells. We have addressed this issue by studying voltage-dependent
Na+, K+, and
Cl
currents
(INa,
IK, and
ICl, respectively) in a subset of
taste cells (the so-called "Na/OUT" cells, which are electrically
excitable and thought to be sensory) from mouse vallate papilla.
Voltage-dependent currents play a key role during taste transduction,
especially in the generation of action potentials. Patch-clamp
recordings revealed that INa,
IK, and
ICl were expressed early in postnatal development. However, only IK and
ICl densities increased significantly in
developing Na/OUT cells. Consistent with the rise of
IK density, we found that action potential
waveform changed markedly, with an increased speed of repolarization
that was accompanied by an enhanced capability of repetitive firing. In
addition to membrane excitability changes in putative sensory cells, we
observed a concomitant increase in the occurrence of glia-like taste
cells (the so called "leaky" cells) among patched cells. Leaky
cells are likely involved in dissipating the increase of extracellular K+ during action potential discharge in chemosensory
cells. Thus, developing taste cells of the mouse vallate papilla
undergo a significant electrophysiological maturation and
diversification. These functional changes may have a profound impact on
the transduction capabilities of taste buds during development.
Key words:
development; taste cells; membrane excitability; gustatory; patch clamp; vallate papilla
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INTRODUCTION |
Electrophysiological recordings from
gustatory nerves have shown that in many mammalian species the
peripheral taste system undergoes functional changes during postnatal
development. For example, neural responses to substances like sugars
and sodium ions mature over several weeks after birth (Mistretta and
Hill, 1995
; Stewart et al., 1997). In addition to morphogenetic events, it is likely that these functional modifications may arise from age-dependent changes in the expression and regulation of transduction mechanisms at the level of taste cells. Taste cells use a vast array of
ion channels and receptors to transduce chemical signals into afferent
nerve discharge (Lindemann, 1996); changes in the expression and
regulation of these membrane components would have obvious
repercussions on the functionality of the taste system during development.
Voltage-gated ion channels play a key role in taste transduction,
especially in the events downstream of the early interaction with
chemical stimuli (Herness and Gilbertson, 1999). Taste cells in adult
mammals are electrophysiologically heterogeneous, and many of them are
electrically excitable by possessing voltage-gated Na+ and K+
channels, which, as in neurons, underlie action potential generation (Roper, 1983; Kinnamon and Roper, 1987; Chen et al., 1996). Action potential firing appears to be one important step in the
transduction and signaling of sensory information in taste buds. For
example, the frequency of spike discharge is related to the
concentration of certain stimuli (Gilbertson et al., 1992; Cummings et
al., 1993). In addition, membrane depolarization during action
potential seems to be necessary to activate
Ca2+ channels for neurotransmitter release
underlying signal transfer to afferent nerves (Béhé et al.,
1990; Furue and Yoshii, 1997). Finally, action potentials may be used
for intercellular communications through gap junctions inside the taste
buds (Bigiani and Roper, 1995).
The pattern and time course of the expression of voltage-gated
Na+ and K+
channels underlying membrane excitability is known to change during
development in neurons and sensory cells (Gao and Ziskind-Conhaim, 1998; Kros et al., 1998). However, in taste cells of the rat fungiform papillae, Kossel et al. (1997) established that voltage-gated Na+ and K+
currents do not change during postnatal development. In this paper, we
have reexamined the issue of membrane excitability in developing taste
cells by studying with the patch-clamp technique the
electrophysiological properties of a subset of taste cells from the
mouse vallate papilla (the so called "Na/OUT" cells, which are
thought to be sensory). Unlike findings in rat, our data show that the
density of voltage-gated K+ and
Cl currents increases significantly in
these cells with the age of the animals. Moreover, we found that the
electrophysiological heterogeneity of taste cells in adult animals is
set during postnatal development. Thus, our data indicate that
postnatal changes in ionic currents of rat and mouse taste sensory
cells are very different. As a whole, our findings suggest that in the
mouse the functional maturation of the peripheral taste system might
rely, at least in part, on alterations of membrane excitability of
taste cells.
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MATERIALS AND METHODS |
Tissue preparation. C57BL/6J mice were used. Vallate
taste buds were isolated with an enzymatic-mechanical procedure
(Bigiani, 2001). Mice were deeply anesthetized with
CO2, followed by dislocation of cervical
vertebrae. Tongues were rapidly removed and placed in Tyrode solution
containing (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES,
10 glucose, 10 Na pyruvate, pH 7.4 with NaOH. Two milligrams of
elastase (Worthington Biochemical Corporation, Freehold, NJ), and 2 mg
of dispase (grade II; Boehringer Mannheim, Mannheim, Germany) in 1.0 ml
of Tyrode solution were injected (0.2-0.4 ml per tongue) between the
lingual epithelium and muscle layer. Tongues were incubated in
Ca2+-free Tyrode solution at 30°C for
~15-70 min, depending on the age of the animal (longer incubation
times were required for juveniles) (Kossel et al., 1997). After
incubation, the lingual epithelium was peeled free from the underlying
tissue, pinned serosal side up in a Sylgard-lined Petri dish, and
incubated in Ca2+-free Tyrode solution
for ~ 5-20 min. Vallate taste buds were removed by gentle
suction with a fire-polished pipette and plated onto glass slides
coated with Cell-Tak (Collaborative Research, Bedford, MA). Drugs were
dissolved in modified Tyrode solutions to maintain osmolarity. All
chemicals were from Sigma (St. Louis, MO), except tetrodotoxin (TTX;
Alomone, Jerusalem, Israel).
Recording techniques. Membrane currents of single cells in
isolated taste buds were studied by whole-cell patch clamp (Hamill et
al., 1981) with use of an Axopatch 1D amplifier (Axon Instruments, Foster City, CA). Signals were recorded and analyzed using a 486-based computer equipped with Digidata 1200 data acquisition system and pClamp6 software (Axon Instruments).
Patch pipettes were made from soda lime glass capillaries (Baxter
Scientific Products, McGaw Park, IL) on a vertical puller (model PB-7,
Narishige, Tokyo, Japan). For voltage-clamp recordings, the pipette
solution contained (in mM): 120 KCl, 1 CaCl2, 2 MgCl2, 10 HEPES,
11 EGTA, 2 ATP, 0.4 GTP, pH 7.3 with KOH. For current-clamp recordings,
KCl was replaced by an equal concentration of potassium gluconate.
Pipette resistances were 3-5 M
when filled with intracellular solution. Leakage and capacitive currents were not subtracted from
currents under voltage clamp, and all voltages have been corrected for
liquid junction potential (LJP) (~4 mV for KCl pipette solution and
~10 mV for potassium gluconate pipette solution) (Neher, 1992). Input
resistance and cell membrane capacitance were measured as described
previously (Bigiani, 2001).
Immunohistochemical procedures. As a morphological marker
for the functional maturation of taste buds, we used
immunocytochemistry to determine the expression of 
-gustducin, a
taste-specific G-protein (Wong et al., 1996; Ming et al., 1999). Whole
mounts of the vallate papilla, obtained after enzymatic treatment and
peeling (Kim and Roper, 1995), were fixed with 4% paraformaldehyde in
0.1 M phosphate buffer (PB) for 3 hr at room
temperature. Then, they were incubated for 3 d at 40°C with a
primary antibody (Ggust, 1:100; Santa Cruz Biotechnology,
Santa Cruz, CA) in 0.1 M PB containing 5% normal
goat serum and 0.5% Triton X-100. After they were rinsed in 0.1 M PB, whole-mount preparations were immersed in a
secondary antibody, a fluorescein isothiocyanate (FITC)-conjugated
anti-rabbit IgG (1:100; Vector Laboratories, Burlingame, CA) in 0.1 M PB for 12 hr at 40°C. Specificity of the
immune reaction was assessed according to previous studies (Boughter et
al., 1997; Cho et al., 1998). Immunofluorescence was examined using a
Nikon TE 800 fluorescence microscope and also with a Bio-Rad Laser
Scanning Microscope Radiance Plus (Bio-Rad, Hemstead Herts, UK).
Electronic images from the confocal microscope were processed using
Adobe Photoshop 5.0 (Adobe Systems, Mountain View, CA).
Immunocytochemical data were analyzed according to previous studies
(Boughter et al., 1997; Cho et al., 1998).
Statistical analysis. Results are presented as means ± SEM. Data comparisons were made with a two-tailed independent
t test, and significance level was taken as
p < 0.05. Distribution of ion current magnitude was
displayed in the form of a box plot. In this plot, boxes show the
middle half of the data (approximately the 25th and 75th percentiles)
and the horizontal line marks the median, whereas the "whiskers"
extending from the top and bottom of the boxes show the main body of
the data. Outliers or extreme values are plotted individually with circles.
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RESULTS |
Isolated taste buds during postnatal development
Taste bud formation and taste cell differentiation occur
postnatally in the mouse (Nolte and Martini, 1992; Cooper and Oakley, 1998). After isolation, taste cells typically remained aggregated at
their apical poles, resembling a taste bud (Fig.
1A-D).
However, partially disaggregated taste buds and sometimes isolated
taste cells (Fig. 1E,F)
could be obtained during the dissociation procedure (Béhé
et al., 1990). Isolated taste cells were more commonly found when taste
tissue was dissociated from mouse pups (Fig. 1E,F). The presence of
solitary taste cells during the first week of postnatal life has been
documented recently in the rat vallate papilla (Sbarbati et al., 1999).
Taste buds could be isolated reliably at postnatal day 4 (PD 4),
although the size was smaller as compared with buds obtained from older
animals (Fig. 1). This was consistent with earlier histological studies
on vallate taste buds of the rat, indicating that taste bud size
increases during postnatal development (Hosley and Oakley, 1987). With
mice younger than PD4, identification of isolated taste
buds proved to be difficult, and the preparation yield was poor.
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Figure 1.
Development of mouse vallate taste buds.
A-D, Differential interference contrast
photomicrographs depicting taste buds isolated from the mouse vallate
papilla at different postnatal ages. Taste buds increase in size during
development, which is most likely attributable to an increase in the
number of cells per bud. E, F, Isolated
taste cells from juvenile vallate papilla. Note the cellular processes.
PD, Postnatal day. Arrowhead indicates
apical pole. Scale bar, 25 µm.
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Using the patch-clamp technique, we were able to study the
electrophysiological properties of single cells in taste buds isolated from mouse vallate papilla as early as PD 4. Stable recordings were
obtained from a total of 470 cells, and data were referred to the
following age groups: PD 4-7; PD 8-14; PD 15-21; PD 22-28; and
PD > 28 (adults).
Functional populations of taste cells during development
Taste cells in adult mammals are functionally heterogeneous as to
the expression of voltage-dependent ion currents (Akabas et al., 1990;
Béhé et al., 1990; Chen et al., 1996; Kossel et al., 1997;
Bigiani, 2001). Accordingly, in a first series of experiments we
studied the membrane currents elicited in mouse taste cells by
depolarizing voltage steps from a reference holding potential of 
84
mV (LJP corrected). In this experimental condition we were able to
identify three main functional groups of taste cells in the vallate
papilla of the adult mouse.
Most of the recorded cells (~57%; 82 of 145 cells) were
characterized by the presence of voltage-gated, TTX-sensitive
Na+ currents
(INa) and voltage-dependent outward
currents (Iout) (Fig.
2A). These cells were
named Na/OUT cells, according to Bigiani (2001). Pharmacological
dissection revealed that Iout was
mediated by potassium and chloride ions. However, as indicated by data reported in Figure 3, the relative
proportion of K+ currents and
Cl currents to
Iout was highly variable. In some
cells, tetraethylammonium (TEA), a potassium channel blocker (Rudy,
1988; Castle et al., 1989), totally abolished the outward currents
(Fig. 3A), whereas in other cells it affected these currents
only partially (Fig. 3B). Conversely,
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a known
Cl conductance blocker in taste cells
(Taylor and Roper, 1994; Herness and Sun, 1999), did not affect the
outward currents in some cells (data not shown) or partially affected
them in others (Fig. 3C).
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Figure 2.
Membrane currents in three different populations
of taste cells from vallate papilla of adult mice. Cells were held at
84 mV and stepped in 10 mV increments from 74 to +56 mV.
A, Na/OUT cells were characterized by voltage-dependent
inward Na+ currents
(INa) and outward currents
(Iout) carried by
K+ and Cl. As shown by the
current-voltage (I-V)
relationships on the right,
INa and Iout
typically activated at approximately 50 mV and approximately 20 mV,
respectively. Im, Membrane currents.
B, OUT cells possessed only
Iout currents, which were carried
predominantly by K+. Outward currents activated at a
voltage similar to the one observed in Na/OUT cells. C,
Leaky cells were characterized by the presence of a conspicuous leakage
current carried by K+
(IK) that was almost negligible in
the other two groups of cells. In all
I-V plots, outward currents were
measured at the end of the 30 msec voltage pulses.
Vm, Membrane potential.
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Figure 3.
Contribution of K+ currents and
Cl currents to the voltage-dependent outward
currents in Na/OUT cells from adult mice. Membrane currents were
elicited by a series of 30 msec depolarizing pulses between 74 mV and
+76 mV, in 10 mV increments, from a holding potential of 84 mV.
A, In some cells, outward currents were carried almost
exclusively by K+ ions, as indicated by their high
sensitivity to TEA. In this example, outward currents recorded in
regular Tyrode solution (control) were totally
abolished by 20 mM TEA. Note that TEA did not affect
voltage-gated Na+ currents (downward deflections in
the current records). B, In other cells, on the
contrary, 20 mM TEA blocked the outward currents only
partially, indicating that TEA-insensitive currents occurred in taste
cell membranes. C, Consistent with the partial block by
TEA was the finding that DIDS affected outward currents. In this cell,
outward currents recorded in regular Tyrode solution
(control) were partially abolished by 0.5 mM DIDS. The effect was reversible, as indicated by the
recovery of the currents during washout (wash). In
B and C, recordings were obtained in the
presence of 1 µM TTX to block voltage-gated
Na+ currents.
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A second group of taste cells (~12%; 17 of 145 cells) was
characterized by the presence of voltage-dependent outward currents only (Fig. 2B). For this reason, they were named OUT
cells (Bigiani, 2001). Outward currents in these cells were
predominantly carried by potassium ions (data not shown).
Finally, ~32% (46 of 145 cells) of taste cells in the vallate taste
buds of adult animals displayed strong leakage currents (Fig.
2C). Accordingly, they were named leaky cells. As
demonstrated recently (Bigiani, 2001), leaky cells are endowed with a
conspicuous resting potassium conductance (responsible for the leakage
currents), whereas voltage-dependent conductances are negligible.
After establishing the functional subsets of taste cells in the adults,
we addressed the following issue: do these subsets occur also in
developing taste buds? To determine this, we recorded membrane currents
from taste cells as early as PD 4. Electrophysiologically identified
cells were assigned to the functional subsets described above and
pooled according to five age groups (PD 4-7; PD 8-14; PD 15-21; PD
22-28; PD > 28). Patch-clamp recordings demonstrated that Na/OUT
cells, OUT cells, and leaky cells could be identified at all ages (Fig.
4). However, when we evaluated the
relative percentage of each cell type, we found that their occurrence
changed significantly during postnatal development. In particular,
leaky cells were almost absent during the first 2 weeks; Na/OUT cells and OUT cells were, as a whole, the most frequent in this period (Fig.
4). At the third postnatal week, that is, 1 week before weaning in the
mouse, the percentage of leaky cells increased considerably,
approaching the value found in the adult vallate papilla (Fig. 4).
Thus, there was a rearrangement in the relative occurrence of the
functional subsets of taste cells in the vallate taste buds during
postnatal development.
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Figure 4.
Electrophysiological subsets of taste cells in the
mouse vallate papilla during postnatal development. Histograms
represent the percentage of Na/OUT cells, OUT cells, and leaky cells
for five age groups (PD, postnatal day). The occurrence
of leaky cells changed significantly with the age of the animals. Total
number of tested cells: 85 (PD
4-7); 61 (PD
8-14); 49 (PD
15-21); 40 (PD 22-28);
145 (PD > 28).
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Mammalian taste cells are structurally heterogeneous, and different
cell morphotypes have been identified, such as elongated cells
(subdivided in type I, type II, and type III cells) and round basal
cells (Roper, 1989; Lindemann, 1996). At the moment, we do not have
data on the structural features of the taste cells belonging to the
functional subsets identified in mouse vallate papilla (that is, Na/OUT
cells, OUT cells, and leaky cells). Further studies involving
correlation of electrophysiological recordings with cell identification
at the electron microscopic level are required to establish whether the
functional subsets correspond to any of the known morphotypes.
Development of membrane properties in Na/OUT cells
Several observations suggest that taste cells capable of firing
action potentials are involved in chemotransduction. Action potential
discharges are regularly recorded from taste cells during chemostimulation in mammals (Béhé et al., 1990; Avenet and
Lindemann, 1991; Gilbertson et al., 1992; Cummings et al., 1993; Furue
and Yoshii, 1997; Ohtubo et al., 2001). Given the presence of
voltage-gated Na+ currents, Na/OUT cells
are able to fire action potentials (see below). Thus, it is conceivable
that Na/OUT cells, or at least part of them, represent sensory cells.
For this reason, we focused our attention on the electrophysiological
properties of these cells by addressing the following question: do the
membrane properties of Na/OUT cells change during postnatal
development? The low occurrence of OUT cells throughout development
(Fig. 4) prevented us from performing a detailed analysis of their
electrophysiological properties. A similar argument holds also for
leaky cells, which display a very low incidence during early
development (Fig. 4).
Development of passive membrane properties
In the whole-cell patch-clamp configuration, two parameters are
commonly used to characterize the passive membrane properties of
excitable cells: the zero-current potential
(V0, an estimation of the cell resting
potential) and the input resistance
(Rin, an estimation of the cell
membrane resistance) (Barry and Lynch, 1991; Bigiani et al., 1996). In
Na/OUT cells, these electrophysiological parameters changed during
postnatal development (Fig. 5). Taste cells from young animals displayed a significantly less negative V0 than adults (Fig. 5,
top). Estimation of the resting potential with the
patch-clamp technique might require significant corrections because of
shunt to ground by the seal resistance (Barry and Lynch, 1991).
Age-related variations of seal resistances could have affected V0 measurements. However, we did not
detect any significant variations in seal resistance when patch
electrodes were applied to taste cells of different age groups (data
not shown). Although the actual resting potential of Na/OUT cells might
be more negative than that measured in our experimental conditions, the
change in V0 during development was
indicative of variations in the "resting" properties of these
cells.
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Figure 5.
Zero-current potential
(V0), input resistance
(Rin), and membrane capacitance
(Cm) of Na/OUT cells during postnatal
development. Cells were grouped into five age classes, and measurements
of each electrophysiological parameter were averaged within each group.
V0 changed significantly during development,
becoming more negative (n = 40/43/20/20/35; error
bars represent SEM). Rin decreased in older
animals, but changes were not significant (n = 41/43/19/18/38). Finally, there was not a significant change in
Cm and, therefore, in membrane surface area
of single vallate taste cells during development (n = 41/44/20/17/37). Asterisks indicate significant
differences. PD, Postnatal day.
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Rin was evaluated as the slope
resistance of the linear current-voltage relationship at a membrane
potential of 84 mV (corresponding to our standard holding potential
corrected for LJP). Concomitantly with
V0 changes, we observed also a
decrease in Rin during development (Fig. 5, middle), although the differences between age
groups were not statistically significant.
Finally, we also measured the cell membrane capacitance
(Cm) of Na/OUT cells.
Cm is directly related to the cell
surface area and can give an estimation of the extension of the cell
membrane in single as well as in coupled taste cells (Bigiani and
Roper, 1993). As shown in Figure 5 (bottom),
Cm did not differ significantly among
the various age groups. This suggests that Na/OUT cells reach their
mature surface size early in development. A similar finding was
obtained also by Kossel et al. (1997) for taste cells of rat fungiform papillae.
Development of voltage-dependent currents
Electrophysiological recordings demonstrated that Na/OUT cells
were already present in the vallate papilla from early developmental stages (Fig. 4). As reported above, these cells are endowed with voltage-gated Na+ currents
(INa) and voltage-dependent outward
currents (Iout) mediated by
K+ and Cl
(Figs. 2, 3). In other excitable cells, such as neurons and sensory cells, these voltage-dependent currents are known to undergo
significant changes during development. In particular, variations in
current amplitude, which affects profoundly membrane excitability, are well documented (Baraban and Lothman, 1994; Gao and Ziskind-Conhaim, 1998; Kros et al., 1998). Thus, we addressed the following issue: does
the amplitude of voltage-dependent currents change in Na/OUT cells
during postnatal development? First we analyzed
INa. Figure 6A shows the
I-V plots for
INa evaluated in taste cells belonging to different age groups (PD 4-7, PD 15-21, PD > 28). In the
adults (PD > 28), INa activated
at approximately 50 mV and peaked at approximately 20 mV. There was
not a significant change of these biophysical characteristics during
development (Fig. 6A). Moreover, the average peak
value of INa did not show any
significant pattern of variation during development (PD 4-7:
609 ± 136 pA, n = 45; PD 8-14: 762 ± 144, n = 42; PD 15-21: 558 ± 144, n = 22; PD 22-28: 765 ± 235, n = 18; PD > 28: 745 ± 113, n = 49). These results were in agreement with those reported for taste cells of rat
fungiform papillae (Kossel et al., 1997).
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Figure 6.
Voltage-gated Na+ currents
(INa) in Na/OUT cells during
postnatal development. A, Current-voltage
relationships. Cells were pooled into three age groups
(PD 4-7, PD
15-21, and PD > 28), and INa values for each
membrane potential (Vm) were averaged
within each group (n = 44/23/44).
INa activated at approximately 50 mV and
peaked at approximately 20 mV in all age groups. There was no
significant pattern of variation for the average maximum value of
INa during development. The holding
potential was 84 mV. PD, Postnatal day.
B, Peak value distribution of
INa displayed in the form of a box
plot (see Materials and Methods for details) for three
age groups (PD 4-7, PD
15-21, and PD > 28). Note that the median (the horizontal
line in the middle of each box)
increases during development, whereas the data range does not.
n = 61 (PD
4-7), 36 (PD
15-21), 67 (PD > 28).
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In our experiments, Na+ currents were not
studied in isolation. However, it is unlikely that the kinetics of
outward currents could influence the activation threshold and peak
magnitude of INa. As shown by Figure
7B, outward currents activate
at approximately 20 mV and do not reach a sizeable magnitude until
~0 mV.
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Figure 7.
Voltage-gated outward currents
(Iout) in Na/OUT cells during
postnatal development. A, Membrane currents recorded
from Na/OUT cells of the vallate papilla in mice of different ages
(PD 6, PD 11, and PD 20).
Membrane currents were elicited by a series of depolarizing pulses
between 74 mV and +86 mV, in 10 mV increments, from a holding
potential of 84 mV. Voltage-gated Na+ currents
(downward deflections in the current records) were of similar magnitude
in all three cells. On the contrary, voltage-dependent outward currents
(Iout, upward deflections in the
current records) markedly increase in their magnitude during
development. PD, Postnatal day. B,
Current-voltage relationships of Iout.
Cells were pooled into three age groups (PD 4-7,
PD 15-21, and PD > 28), and amplitude values of
Iout for each membrane potential
(Vm) were averaged within each group
(n = 44/23/44). Iout
amplitude was measured at the end of 30 msec pulses.
Iout activated at approximately 20 mV and
showed a pronounced increase during development. C,
Amplitude distribution of Iout. Amplitude
values (evaluated at +46 mV and at the end of a 30 msec voltage
pulse) are displayed in the form of a box plot (see
Materials and Methods for details) for three age groups
(PD 4-7, PD 15-21, and
PD > 28). Note that both the median
and the data range increase during development, indicating that there
is a rise in the number of Na/OUT cells possessing sizeable
Iout. n = 41 (PD
4-7), 23 (PD 15-21), 44 (PD > 28).
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Although the average peak value for
INa did not change significantly
during development, the analysis of the distribution of
INa magnitude among cells revealed a
noticeable skewness. In Figure 6B, peak
INa values are displayed in the form
of a box plot for young (PD 4-7), preweaning (PD 15-21), and adult
(PD > 28) mice. As indicated by the plot, the range of
INa magnitude was similar for all
three age groups. However, the outlined boxes show that the middle half
of the data underwent a broadening toward larger values during
developing. High INa values already in
PD 4-7 mice could be caused by variations in the onset of maturation among taste cells (Hosley and Oakley, 1987; Cooper and Oakley, 1998),
whereas low INa values in adult mice
likely reflect the appearance of new taste cells during cell turnover
(see Discussion).
We also studied Iout in Na/OUT cells
during postnatal development. Iout is
carried by K+ and
Cl and is mainly responsible for the
repolarization phase in gustatory action potentials and for
afterpotentials (Chen et al., 1996; Herness and Sun, 1999). As reported
above, the relative contribution of K+ and
Cl currents to
Iout was highly variable in taste
cells from adult mice. Because of this complexity, in a first series of
experiments we did not attempt to distinguish the two current
components, and we analyzed the outward currents as a whole. Figure
7A shows representative recordings from developing Na/OUT
cells. Although all of these cells displayed voltage-gated
Na+ currents of similar amplitude
(downward deflections in the current traces), their
Iout increased significantly with the
age of the animal. The increase of
Iout was also evident when the
averaged I-V plots were evaluated for different
age groups (Fig. 7B). At a reference potential of +46 mV,
Iout was 668 ± 113 (n = 44) for PD 4-7, whereas in adult mice (PD > 28) it was approximately three times larger (1971 ± 214;
n = 33). Interestingly,
Iout activated at approximately the
same voltage (approximately 20 mV) for all age groups tested (Fig.
7B). The increase in Iout
was not related to an increase in cell membrane surface during
development, as indicated by Cm
measurements (Fig. 5). Figure 7C shows the distribution of
Iout magnitude for young (PD 4-7),
preweaning (PD 15-21), and adult (PD > 28) mice. These plots
clearly indicate that there was an increase in the number of Na/OUT
cells possessing large Iout.
Interestingly, the lowest value of
Iout was similar for all ages. This
was likely because of the appearance of new taste cells during cell
turnover (see Discussion).
To establish whether the rise in Iout
was caused by a specific component of this current (namely, the
K+ or Cl
current), we performed a series of experiments aimed at evaluating the
effect of TEA on Iout during
development. A first comparison was made between cells in which 20 mM TEA blocked the outward currents for >80%,
that is, between cells expressing almost exclusively voltage-dependent
K+ currents
(IK). As shown by Figure
8A,
IK increased conspicuously during
development. At a reference potential of +46 mV,
IK was 742 ± 156 pA
(n = 19) for PD 4-7 mice, whereas in adult mice
(PD > 28) it was approximately four times larger (2763 ± 501 pA; n = 18). In addition to
IK, chloride currents
(ICl) also were affected by
development. As indicated by the box plot in Figure
8B, the occurrence of cells in which TEA only
partially blocked Iout was larger in
the adults than in mouse pups. The currents remaining during TEA
application were carried by chloride ions as indicated by their
sensitivity to DIDS; that is, the combination of TEA and DIDS
completely abolished the outward current (data not shown). In young
animals, the majority of taste cells (~83%; 19 of 23 cells)
possessed almost exclusively IK (PD
4-7) (Fig. 8B). On the contrary, in the adults a
large proportion of Na/OUT cells (10 of 32 cells; ~31%) had
ICl (PD > 28) (Fig.
8B) in addition to
IK. Interestingly, a few cells showed
almost exclusively ICl (~13%; 4 of
32 cells). Thus, ICl seems to be
expressed later than IK during
development. To evaluate the magnitude of chloride currents, we
measured Iout during TEA block. Figure
8C shows the I-V plots for
ICl in both young (PD 4-7) and adult
(PD > 28) mice. At a membrane voltage of +46 mV,
ICl was 396 ± 90 pA
(n = 4) in young mice, and 1274 ± 213 pA
(n = 10) in adults.
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Figure 8.
Voltage-dependent K+ and
Cl currents (IK
and ICl, respectively) in developing
Na/OUT cells of the mouse vallate papilla. A,
Current-voltage relationships for IK in
young (PD 4-7) and adult (PD > 28) mice. Current amplitude was measured at the end
of 30 msec voltage pulses. Amplitude values for each membrane potential
(Vm) were averaged within each age
group (n = 19 for PD 4-7;
n = 18 for PD > 28). Error bars represent ± SEM. Holding potential
was 84 mV. B, Box plot for the effect
of 20 mM TEA on voltage-dependent outward currents
(Iout) in Na/OUT cells from young
(PD 4-7; n = 23 cells) and adult (PD > 28;
n = 32 cells) mice. Inhibition by TEA was evaluated
on currents elicited by depolarizing the membrane to +46 mV from a
holding potential of 84 mV. In young animals, the majority of Na/OUT
cells displayed almost exclusively
IK, as indicated by the position of
the outlined box in the top of the
graph. On the contrary, in adults a large proportion of
Na/OUT cells displayed both IK and
ICl, as indicated by the broadening
of the outlined box toward the lower inhibition values.
C, Current-voltage relationships for
ICl in young (PD 4-7)
and adult (PD > 28) mice. Amplitude
values for each membrane potential
(Vm) were averaged within each age
group (n = 4 for PD 4-7;
n = 10 for PD > 28).
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IK typically exhibited a slow
inactivation during 400 msec depolarization pulses in both mouse pups
and adults (Fig. 9A), whereas
chloride currents did not inactivate (data not shown). We evaluated
inactivation properties of IK during
development by measuring the ratio between the current amplitude at 400 msec and the current peak. At +46 mV, this ratio was 0.63 ± 0.05 (n = 15) and 0.74 ± 0.03 (n = 9)
in mouse pups (PD 4-7) and adults (PD > 28), respectively. These
values were not statistically different. Figure 9B shows the
effect of holding potential (Vh) on
the inactivation of K+ currents in a
Na/OUT cell of adult mouse: currents were totally abolished by
depolarizing the cell membrane at approximately 24 mV. Similar
findings were also observed in mouse pups (data not shown).
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Figure 9.
Inactivation of voltage-dependent
K+ current (IK) in
Na/OUT cells. A, IK recorded
from cells in mice of different ages (PD 4 and PD
55). PD, Postnatal day. Currents were elicited
by a series of 400 msec depolarizing voltage pulses between 74 mV and
+86 mV, in 10 mV increments, from a holding potential of 84 mV. Note
the decrease in IK amplitude during
prolonged voltage pulses (inactivation).
INa, Voltage-gated
Na+ currents. B, Effect of holding
potential (Vh) on the inactivation of
IK in an Na/OUT cell from the vallate
papilla of a 50-d-old mouse. K+ currents (upward
deflections in the records) were elicited by a series of depolarizing
voltage pulses in 10 mV increments from different holding potentials
(Vh: 84, 64, 44, 24 mV). All records
are from the same cell. Downward deflections in the records at
Vh = 84 mV and 64 mV represent
voltage-dependent Na+ currents.
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In addition to IK and
ICl,
Ca2+-dependent
K+ currents and
Ca2+-dependent
Cl currents have been described in rat
taste cells (Chen et al., 1996; Herness and Sun, 1999). The application
of a Ca2+-free Tyrode solution (containing
3 mM Mg2+) did not
affect the outward currents in mouse Na/OUT cells (n = 4; data not shown). This result suggested that
Ca2+-dependent currents did not contribute
substantially to the outward currents. A further analysis of possible
Ca2+-dependent components of
Iout was beyond the scope of our study.
Action potentials in developing taste cells
The remarkable increase in the magnitude of repolarizing currents
(Iout), particularly
IK, was expected to have a profound effect on excitability. Because such currents tend to counteract the
depolarizing action of Na+ currents, they
modulate the shape of action potentials and the firing pattern in
excitable cells (Hille, 2001). Thus, we examined action potential
waveform in young (PD 4-7), preweaning (PD 15-21), and adult (PD > 28) mice using whole-cell current-clamp recordings. Because in mice
pups (PD 4-7) the occurrence of ICl
was very low (Fig. 8B), we studied action potential
waveforms only in Na/OUT cells possessing predominantly
IK. To correlate changes in action potential waveforms with the ionic currents underlying them, action potentials were elicited by injecting depolarizing current from a
membrane potential of approximately 80 mV, that is, close to the
holding potential from which voltage-dependent currents were produced.
Figure 10A shows
representative recordings of action potential waveforms in Na/OUT cells
from animals of different ages. These cells were electrically excitable
in young animals, but in the majority of them action potential
waveforms were distinctively different from those produced in adults.
In particular, action potential duration was typically larger in young
animals than in adults (Fig. 10A, compare PD
4 with PD 65). Although the duration of action
potential is often used to characterize its waveform (Chen et al.,
1996; Gao and Ziskind-Conhaim, 1998), we found it more convenient to
evaluate the action potential maximum rates of rise
(dV/dt+, depending on
INa) and repolarization
(dV/dt, depending on
IK) in developing taste cells. Figure
10B (left) shows the results of such an
analysis for three age groups (PD 4-7, PD 15-21, PD > 28).
dV/dt+ increased postnatally, although the differences were not statistically significant. This result was consistent with the observation that during development there was an
increase in the number of Na/OUT cells with sizeable
INa, although the magnitude range for
this current did not change (Fig. 6B). On the
contrary, dV/dt became significantly more
negative during development (Fig. 10B,
right), consistent with the large increase in
IK (Fig. 8A). Thus,
the action potential waveforms in developing Na/OUT cells (Fig.
10A) were markedly affected by the increase in
magnitude of repolarizing currents.
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Figure 10.
Action potentials recorded from Na/OUT cells of
the mouse vallate papilla. A, Action potential waveforms
in Na/OUT cells from young (PD 4), preweaning
(PD 18), and adult (PD 65) mice. Cell
membrane was held at approximately 80 mV, and two depolarizing
current pulses were injected (13 and 14 pA for PD 4; 28 and 30 pA for PD 18; 16 and 17 for PD
65). The first pulse was subthreshold and failed to elicit
action potentials. B, Action potential maximum rates of
rise (dV/dt+) and repolarization
(dV/dt) in Na/OUT cells during
development. Cells were pooled into three age groups (PD
4-7, PD 15-21, and PD > 28). dV/dt+ and
dV/dt values were averaged within each
group (n = 18/12/24). Error bars represent ± SEM. Asterisk indicates significant difference.
PD, Postnatal day. C, Firing behavior of
the Na/OUT cells shown in A. Action potential discharges
were elicited by injecting long depolarizing current pulses (1.6 and
1.8 pA for PD 4; 2.4 and 3.2 pA for PD
18; 5 and 6 pA for PD 65). The first pulse was
subthreshold, whereas the second one elicited action potentials,
although the firing pattern was different among cells. In PD 4 cells,
only one action potential could be evoked, whereas in PD 65 cells a
complex pattern of repetitive firing appeared. In PD 18 cells, an
incipient action potential was evoked after the first, full-size spike.
Vm, Membrane potential.
I, Current injected.
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We then analyzed the firing behavior of developing Na/OUT cells. In
young animals (PD 4-7), only 4 of 18 cells (~22% of tested cells)
were capable of repetitive firing (at least two action potentials per
discharge) after stimulation with a long depolarizing pulse. On the
contrary, in adults, a complex pattern of repetitive firing could be
evoked in 12 of 23 cells (~52%) by long-lasting depolarizations.
Figure 10C shows the firing pattern in the same Na/OUT cells
of Figure 10A. These findings were also consistent with the significant increase in the magnitude of
IK during development. In conclusion,
the fourfold postnatal increase of IK
density resulted in a higher speed of repolarization of action
potential and in an increased firing capability by Na/OUT cells.
-Gustducin immunoreactivity during postnatal development
To establish whether electrophysiological changes in mouse taste
buds occurred concomitantly with variations in other properties of
taste cells, such as the expression of specific proteins involved in
the early events of sensory transduction, we used immunofluorescence to
quantify the distribution of gustducin immunoreactivity (IR). Gustducin
is the -subunit of a G-protein considered to be a potent marker of
chemosensitive cells (Boughter et al., 1997; Sbarbati et al.,
1999).
We first evaluated the number of taste buds containing
gustducin-positive cells during postnatal development. As shown in Figure 11A,
gustducin-IR was distributed throughout the cytoplasm of taste cells
(Boughter et al., 1997; Cho et al., 1998; Sbarbati et al., 1999; Yang
et al., 2000). Quantitative analysis of gustducin expression at
different postnatal ages revealed a significant increase in the number
of immunolabeled taste buds from PD 4 to PD 14. By PD 14, the number of
gustducin-positive buds reached the value found in adults (~120)
(Fig. 11B). In addition, we found that the number of
gustducin-positive cells in single taste buds (Fig.
12A) increased
markedly during the first 2 weeks after birth and reached the value
found in the adult (~10 cells per bud) by PD 14 (Fig.
12B). Thus, our findings indicated that the
chemotransduction system involving -gustducin developed rapidly
during the first 2 postnatal weeks (Figs. 11, 12). On the contrary,
membrane excitability matured more slowly in Na/OUT cells (Fig. 7). In
addition, the abrupt rise in the number of leaky cells among patched
cells occurred after the gustducin expression had reached its maximum
(compare Fig. 4 with Figs. 11 and 12).
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Figure 11.
Gustducin immunoreactivity in developing vallate
papillae of the mouse. A, Confocal images (1 and 3 µm
thick at PD 4-8 and PD 14-adult, respectively) through whole-mount
preparations of the vallate papilla from animals at different ages
showing gustducin immunoreactivity visualized with FITC-conjugated
secondary antibodies. Gustducin-positive taste buds are clearly
distinguishable at all ages. B, Quantitative evaluation
of the number of taste buds containing gustducin-positive cells during
postnatal development. Mean number (±SEM) of gustducin-immunoreactive
taste buds from five animals for each age. This number significantly
increased from PD 4 to PD 14 (p < 0.001),
with no further changes until adulthood. Asterisks
indicate significant differences. PD, Postnatal
day.
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Figure 12.
Gustducin-immunoreactive taste cells in
developing taste buds of the mouse vallate papillae. A,
Confocal images (0.5 and 1 µm thick at PD 4-8 and PD 14-adult,
respectively) through taste buds in whole-mount preparations from
animals of different ages showing gustducin immunoreactivity visualized
with FITC-conjugated secondary antibodies. Gustducin immunoreactivity
appears to be distributed throughout the cytoplasm of labeled cells.
B, Quantitative evaluation of the number of
gustducin-positive cells per each taste bud during postnatal
development. Mean number (±SEM) of gustducin-immunoreactive cells from
30-40 measurements for each age. This number significantly increased
from PD 4 to PD 14 (p < 0.001), with no
further changes until adulthood. Asterisks indicate
significant differences. PD, Postnatal day.
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DISCUSSION |
Development of membrane excitability in putative taste
sensory cells
In this study, we used patch-clamp recordings to examine the
membrane properties of mouse vallate taste cells during postnatal development. The main finding is that a specific functional subset of
taste cells (the Na/OUT cells, thought to be sensory) undergoes an
electrophysiological differentiation after birth. Specifically, we
found that the magnitude of voltage-dependent outward currents (Iout, repolarizing currents composed
of IK and
ICl) increased approximately three
times in 5 weeks. This increase was not dependent on the cell surface
area. In other words, it was an actual increase in current density. The
pharmacological analysis allowed us to establish that the rise of
Iout was caused by both components of
this current, namely, IK and
ICl (Fig.
13). Interestingly, the development of
IK seemed to precede the appearance of
ICl. Consistent with the significant
postnatal increase in IK, we found
that Na/OUT cells became more excitable, as indicated by the
characteristics of their action potentials and firing capability.
Indeed, voltage-dependent potassium channels permit excitation because
they do not interfere with the rise to the threshold for action
potential firing, and they actively promote recovery and rapid
refiring. The "resting" potential of Na/OUT cells, estimated as
V0, becomes more negative during
development, and this is also consistent with the increased excitability of Na/OUT during development. The postnatal increase of
IK and
ICl might reflect an increase in the
number of K+ and
Cl channels, assuming that
single-channel conductances and their open probability remain
unchanged. Indeed, inactivation properties of
IK did not change significantly during
development.
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Figure 13.
Schematic overview of the development of
voltage-dependent K+ and Cl
currents (IK and
ICl, respectively) in Na/OUT cells of
the mouse vallate papilla. Line thickness is proportional to the
average amplitude of ion currents in taste cells for each age group.
Percent values refer to the relative occurrence among patched cells.
PD, Postnatal day.
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Unlike our findings, Kossel et al. (1997) established that
voltage-gated outward currents undergo little if any changes during postnatal development in taste cells from rat fungiform papillae. Thus,
the maturation processes affecting the functional properties of taste
cells during postnatal development can differ considerably, even in
related mammalian species such as rat and mouse. These findings add
further complexity to the physiology of the peripheral taste system and
could be related to the variety of transduction mechanisms adopted by
different vertebrate species (Lindemann, 1996).
Several questions about membrane excitability in developing mouse taste
cells remain to be answered. Among other things, the development of
ICl and its impact on membrane firing
capabilities represents one of the future directions of our research.
Indeed, the occurrence of some taste cells possessing almost
exclusively ICl in the adults suggests
an electrophysiological diversification among Na/OUT cells during
development (Fig. 13). It is interesting to note that in rat vallate
taste cells chloride currents represent only a small portion of the
outward current (Herness and Sun, 1999), whereas in mouse vallate taste
buds some cells exhibit large ICl. It
is likely that these differences may have a profound effect on the
transduction capabilities of taste cells in mouse as compared with rat.
Our data indicate that variations in membrane excitability occurred
during several days after birth (at least 4 weeks), whereas gustducin-IR matured quite rapidly in mouse taste buds (~2 weeks). Thus, the expression of specific components of transduction mechanisms, such as voltage-dependent ion channels and G-proteins, takes place on
different time scales during postnatal development in the mouse.
Membrane excitability and taste transduction
Changes in the membrane excitability of taste cells may have a
profound impact on the functioning of taste buds during development. Several lines of evidence suggest that taste buds are complex chemosensory structures. For example, not all chemosensitive cells are
innervated (Ohtubo et al., 2001), and lateral interactions mediated by
gap junctions (electrical synapses) have been documented between taste
cells (Bigiani and Roper, 1993, 1995). Accordingly, the sensory output
to the nerve terminals most likely represents the integrated activity
of a population of cells rather than the activity of single, separate
cells (Roper, 1989, 1993). It is then tempting to speculate that the
increase in the number of spiking cells among Na/OUT cells could imply
variation in the sensitivity of the taste transduction system during
development, because firing might provide a significant boost in
signaling inside taste buds. In addition, it is well established that
certain transduction mechanisms involve the modulation of
voltage-dependent K+ currents (Cummings et
al., 1996; Gilbertson et al., 1997). Finally, Cl currents seem to play a role in
sensory adaptation and in efferent control by nerve terminals (Taylor
and Roper, 1994; Herness and Sun, 1999). In conclusion, the increased
magnitude of repolarizing currents (IK
and ICl) during development is likely
to affect markedly the transduction properties of mouse taste cells.
Functional populations of taste cells during
postnatal development
In addition to membrane excitability changes in putative sensory
cells, we found a functional rearrangement of taste cell subsets in the
vallate papilla during postnatal development. In particular, the
frequency of the so-called leaky cells among the patched cells
increases noticeably in the beginning of the third postnatal
week. This change may actually reflect the presence of a larger number
of leaky cells in taste buds. It has been suggested recently that leaky
cells represent glia-like elements (Bigiani, 2001). Thus, the
appearance of these cells during postnatal development could be related
to the increased excitability of putative sensory cells (the Na/OUT
cells). Ionic fluxes associated with action potential firing are
expected to increase, and this may require a more efficient
"glia-like system" inside taste buds to control ion concentrations,
most notably K+ concentration, in the
extracellular space.
In addition to putative sensory cells (Na/OUT cells) and glia-like
cells (leaky cells), we identified a third subset of taste cells in the
vallate papilla of adult mouse. These cells, called OUT cells,
possessed only voltage-gated outward currents carried by
K+ and were not electrically excitable
(data not shown). OUT cells occurred at all ages. Their specific role
in taste bud physiology is unknown. According to work on
Necturus taste cells (Bigiani and Roper, 1993; Delay et al.,
1994; Mackay-Sim et al., 1996), unexcitable cells endowed with
potassium current might represent putative stem basal cells or a
transitional state during the continuous turnover of taste sensory
cells (see below).
Postnatal development and cell turnover
Adult taste buds consist of a renewing population of
neuroepithelial cells (Roper, 1989). The life span of taste cells is ~10 d in fungiform and vallate papillae of the adult rat (Beidler and
Smallman, 1965; Farbman, 1980). Many mature taste cells are electrically excitable and are thought to develop from unexcitable, undifferentiated cells within the taste buds (Delay et al., 1986, 1994). As a consequence, variations in the membrane properties of taste
cells in adult animals are expected to occur. Strong support for this
hypothesis has been provided by findings on the electrophysiological
properties of Necturus taste cells (Mackay-Sim et al.,
1996). Our results on the taste cells of the adult mouse are consistent
with cell turnover. For example, the amplitude of
INa and
Iout is quite variable in "adult"
taste cells. Moreover, as indicated by the error bars in Figure
10B, variability in the action potential waveform do
occur in the taste cells of adult mice. This is consistent also with
previous studies reporting different types of action potentials (long
and short) in rat taste cells (Béhé et al., 1990; Chen et
al., 1996).
An obvious issue is whether changes in electrophysiological properties
of mouse taste cells during postnatal development are caused by the
normal cell turnover or by other developmental processes switched on
before adulthood. Recent studies on taste cell proliferation have
revealed lower rates of cell turnover in newborn and juvenile (PD
0-21) rats compared with adults (Hendricks and Hill, 1999). This
finding suggests that taste cells might have a longer life span in
juveniles than in adults. It is then tempting to speculate that the
maturation of IK and
ICl that occurs over several weeks during postnatal development could reflect a slow cell turnover. Consistent with this is the observation that the lowest value of
Iout distribution does not change
during postnatal development. However, this interpretation cannot
explain adequately the abrupt rise in the occurrence of leaky cells
during the third postnatal week. Leaky cells could result from a
conversion of the membrane properties of Na/OUT cells (or OUT cells).
Recently it has been shown that protein kinase A (PKA) mediates the
reversible conversion of potassium leak into a voltage-dependent
channel in hippocampal neurons (Bockenhauer et al., 2001). PKA is
expressed in taste cells (Avenet et al., 1988). Thus, PKA regulation in
developing Na/OUT cells (or OUT cells) could induce changes in their
membrane properties, leading to the appearance of leaky cells. Also,
the rapid development of gustducin-IR during the first 2 postnatal weeks is not explained satisfactorily by a slow cell turnover. Therefore, it is likely that in addition to cell turnover, other age-related processes take place during postnatal development of mouse
taste cells.
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FOOTNOTES |
Received July 26, 2001; revised Sept. 27, 2001; accepted Oct. 19, 2001.
This study was supported by Ministero dell'Università e della
Ricerca Scientifica e Tecnologica (MURST, Cofin 1998 and 2000 to
A.B. and P.B.) and by Banca Popolare dell'Emilia Romagna (scholarship to F.F.).We thank Fausto Vaccari and Giuseppe Nespoli (Università di Modena e Reggio Emilia) for their excellent technical assistance.
Correspondence should be addressed to Dr. Albertino Bigiani,
Dipartimento di Scienze Biomediche, Sezione di Fisiologia,
Università di Modena e Reggio Emilia, via Campi 287, 41100 Modena, Italy. E-mail: bigiani{at}unimo.it.
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TOP
ABSTRACT
INTRODUCTION
