1Department of Psychology and 2Neuroscience Graduate Program, University of Virginia, Charlottesville 22903; 3Department of Psychology and Neuroscience Program, Washington and Lee University, Lexington 24450; and 4Department of Physiology, Virginia Commonwealth University, Richmond, Virginia 23298
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ABSTRACT |
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Hendricks, Susan J.,
Robert E. Stewart,
Gerard L. Heck,
John A. DeSimone, and
David L. Hill.
Development of Rat Chorda Tympani Sodium Responses: Evidence for
Age-Dependent Changes in Global Amiloride-Sensitive Na+
Channel Kinetics.
J. Neurophysiol. 84: 1531-1544, 2000.
In rat,
chorda tympani nerve taste responses to Na+ salts
increase between roughly 10 and 45 days of age to reach stable, mature magnitudes. Previous evidence from in vitro preparations and from taste
nerve responses using Na+ channel blockers
suggests that the physiological basis for this developmental increase
in gustatory Na+ sensitivity is the progressive
addition of functional, Na+ transduction elements
(i.e., amiloride-sensitive Na+ channels) to the
apical membranes of fungiform papilla taste receptor cells. To avoid
potential confounding effects of pharmacological interventions and to
permit quantification of aggregate Na+ channel
behavior using a kinetic model, we obtained chorda tympani nerve
responses to NaCl and sodium gluconate (NaGlu) during receptive field
voltage clamp in rats aged from 12-14 to 60 days and older (60+ days).
Significant, age-dependent increases in chorda tympani responses to
these stimuli occurred as expected. Importantly, apical
Na+ channel density, estimated from an apical
Na+ channel kinetic model, increased
monotonically with age. The maximum rate of Na+
response increase occurred between postnatal days 12-14 and 29-31. In
addition, estimated Na+ channel affinity
increased between 12-14 and 19-23 days of age, i.e., on a time course
distinct from that of the maximum rate of Na+
response increase. Finally, estimates of the fraction of clamp voltage
dropped across taste receptor apical membranes decreased between 19-23
and 29-31 days of age for NaCl but remained stable for NaGlu. The
stimulus dependence of this change is consistent with a developmental
increase in taste bud tight junctional Cl ion
permeability that lags behind the developmental increase in apical
Na+ channel density. A significant, indirect
anion influence on apical Na+ channel properties
was present at all ages tested. This influence was
evident in the higher apparent apical Na+ channel
affinities obtained for NaCl relative to NaGlu. This stimulus-dependent
modulation of apical Na+ channel apparent
affinity relies on differences in the transepithelial potentials
between NaCl and NaGlu. These originate from differences in
paracellular anion permeability but act also on the driving force for
Na+ through apical Na+
channels. Detection of such an influence on taste depends fundamentally on the preservation of taste bud polarity and on a direct measure of
sensory function, such as the response of primary afferents.
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INTRODUCTION |
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Numerous behavioral,
neurophysiological, and electrophysiological studies indicate that the
amiloride-sensitive Na+ channel is a transduction
pathway for taste system Na+ stimuli in numerous
mammals (Brand et al. 1985; DeSimone et al. 1981
; Gilbertson et al. 1993
; Heck et al.
1984
; Hill et al. 1990
; Schiffman et al.
1983
; reviewed in Stewart et al. 1997a
)
including some primates (Hellekant et al. 1997a
,b
). In
mammals where this Na+ sensing system is
functional, passive movement of stimulus Na+ ions
across the taste cell apical membrane causes membrane depolarization and, consequently, release of neurotransmitter onto primary taste afferents (reviewed in Roper 1992
). Notably in several
species, including rat and hamster, neural responses to NaCl are not
completely suppressed by amiloride (Brand et al. 1985
;
DeSimone and Ferrell 1985
; Heck et al.
1984
; Hettinger and Frank 1990
). The
unsuppressed part of the neural response to NaCl has been termed the
amiloride-insensitive component (Formaker and Hill 1988
;
Ye et al. 1993a
).
The amiloride-insensitive component of the neural taste response to
NaCl is highly dependent on the stimulus anion (Formaker and
Hill 1988) and is thought to result from activation of
transduction sites below taste cell tight junctions (Ye et al.
1991
, 1993a
). Specifically, when Na+
salts with small, highly permeant anions, such as
Cl
, are applied to the lingual surface,
electroneutral diffusion of Na+ and the stimulus
anion through taste cell tight junctions is facilitated. In contrast,
when Na+ salts with larger, tight
junction-impermeant anions are applied to the tongue, electroneutral
diffusion of Na+ and the stimulus anion is
severely limited. Consequently, neural responses to
Na+ salts with large, organic anions, such as
acetate or gluconate, are transduced entirely by the apical pathway and
completely inhibited by amiloride (Formaker and Hill
1988
; Ye et al. 1993a
). Therefore the composite
neural response to NaCl comprises distinct apical, voltage-sensitive
and basolateral, voltage-insensitive components.
Interestingly, significant developmental alterations in gustatory
Na+ responses have been documented in several
mammalian species (Ferrell et al. 1981; Hill
1988
; Hill and Almli 1980
; Mistretta and
Bradley 1983
; Ninomiya et al. 1991
). In rats,
impressive age-dependent increases in the magnitude of whole chorda
tympani nerve responses to Na+ (and
Li+) stimuli occur (Ferrell et al.
1981
; Hill and Almli 1980
). Specifically, when
expressed relative to an NH4Cl reference
response, NaCl and LiCl response magnitudes increased progressively
between ~10 and 45 days postnatal. By ~45 days of age, NaCl and
LiCl response magnitudes reached stable, adult levels. Analyses of
single chorda tympani fiber responses during development have
established clearly that the developmental increase in rat whole chorda
tympani sensitivity to NaCl and LiCl is due specifically to increases
in single chorda tympani fiber responses to these salts (Hill et
al. 1982
). In contrast, responses to acids, ammonium chloride
and other monochloride salts are robust in early postnatal rats and
remain so during development. The fact that Na+
exhibits a unique developmental time course among taste stimuli suggests that underlying physiological changes originate at the level
of the receptor cells responsible for transduction of the Na+ taste signal. More recent work has examined
the potential mechanisms that underlie developmental changes in neural
sensitivity to Na+ stimuli.
Changes in amiloride-sensitive Na+ channel
function have been proposed to account for the developmental increase
in gustatory system Na+ sensitivity. For example,
Hill and Bour (1985) showed that the sensitivity of
whole chorda tympani NaCl responses to the suppressive effects of
amiloride increased in parallel with developmental increases in
sensitivity to NaCl and LiCl. In particular, chorda tympani responses
to NaCl and to LiCl in 12- to 13-day-old rats were unaffected by 100 µM amiloride. However, in rats aged 29-31 and 90-110 days, chorda
tympani responses were potently suppressed by amiloride. Furthermore
the degree of response inhibition caused by amiloride was proportional
to overall taste system sensitivity to these stimuli at these age
points. An implication of these results is that the apical
Na+ transduction pathway develops postnatally,
while the basolateral Na+ transduction pathway
appears to be in place and functional around the time of birth. These
findings were later replicated by Sollars and Bernstein
(1994)
. Hill and Bour (1985)
concluded that the concomitant, progressive increases in gustatory sensitivity to NaCl
(and LiCl) and to amiloride were due to an orderly increase in the
functional expression of amiloride-sensitive Na+
channels in taste cell apical membranes. With this notion in mind,
Stewart et al. (1995)
hypothesized that the
developmental increase in gustatory Na+
sensitivity was due to a progressive addition of newly synthesized, functional amiloride-sensitive Na+ channels. They
tested this hypothesis by correlating the presence of
Na+ channel-like antigen in fungiform papilla
taste buds with the developmental increase in gustatory
Na+ and amiloride sensitivity.
Surprisingly, although the rat chorda tympani does not exhibit
significant amiloride sensitivity before 7-10 days postnatal, antigenic determinants of amiloride-sensitive Na+
channel are observed in taste cells of fungiform papilla taste buds as
early as the day after birth (Stewart et al. 1995).
However, limited microscopic resolution in that study precluded
definitive localization of Na+-channel-like
immunoreactivity to the apical membrane. Likewise, Kossel and
co-workers (1997)
used whole cell recordings to identify amiloride-blockable currents in isolated, single taste cells of fungiform papilla taste buds from rats as young as 2 days postnatal. They determined that the percentage of neonatal rat taste cells that
exhibit amiloride-blockable currents is stable throughout development;
the normalized density of amiloride-sensitive channels in taste cells
is stable throughout development; and the amiloride-inhibition constant
of taste cell Na+ channels is stable throughout
development. The authors suggest that redistribution of fully
functional channels from the basolateral to apical domain and/or
maturation (i.e., progressive opening) of taste pores could account for
the developmental increase in neural Na+ and
amiloride sensitivity in rats. On the other hand, progressive pore
opening would be expected to influence the apparent development of
neural responses to all stimuli and not only those to
Na+ salts. Nonetheless these electrophysiological
and histochemical observations suggest that the
Na+ transduction apparatus is functionally mature
well before 7-10 days of age and therefore diverge considerably from
earlier neurophysiological data.
In an attempt to clarify the nature of this apparent discrepancy, we
have used in the present study in vivo lingual receptive field voltage
clamping with simultaneous whole chorda tympani recordings to resolve
age-related changes in taste cell apical Na+
channel function. This approach offers several advantages over the use
of either isolated, in vitro preparations or conventional neurophysiological recordings. First, the topological arrangement of
taste buds and, therefore segregation of apical and basolateral transduction pathways, remains intact. Second, aggregate apical membrane Na+ channel properties can be studied
quantitatively without the use of amiloride. This is especially
important because it has been suggested that restricted access of
amiloride to taste cell apical membranes caused by unopened taste pores
may contribute to the apparent lack of amiloride sensitivity in young
rats (Kossel et al. 1997). Finally, the combination of
epithelial voltage clamp with neural recording provides a measure of
how taste cells "report" exclusively taste-related membrane events.
We hypothesized that the developmental increase in gustatory
Na+ sensitivity in rats is due to the progressive
addition of functional amiloride-sensitive Na+
channels to taste cell apical membranes. The results we obtained are
consistent with such an age-related increase in the apparent number of
functional, amiloride-sensitive Na+ channels in
taste cell apical membranes. In addition, the results provide entirely
new evidence that the apparent affinity of the aggregate apical
Na+ transduction pathway also increases during
postnatal development. Finally, our results indicate that these
age-related changes in apical Na+ channel
function are temporally distinct. Therefore the onset of mature
gustatory Na+ sensing in rat appears to rely on
developmental sequelae that are more complicated than a simple,
monotonic increase in the number of functional
Na+ transduction sites. Portions of these
findings have been presented previously in preliminary form
(Hendricks et al. 1998
; Stewart et al.
1997b
).
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METHODS |
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Animals
Sprague-Dawley rats at 12-14 days of age, 19-23 days of age, 29-31 days of age, or 60 days of age and older (hereafter termed 60+ day old) were used in these studies. Rats were bred and raised in our colony with the exception of several rats in the 60+ day old group, which were obtained directly from the supplier. These adult animals, and all breeding stock, were obtained from Harlan Sprague-Dawley. Rats were provided standard laboratory chow (Purina) and water ad libitum and maintained on a 12 h:12 h light:dark photoperiod (lights on at 0700). Breeding harems (3 females and 1 male) were established, and the male remained in the cage for 14 days. After 14 days, the male was removed, and the females were segregated into individual maternity cages. Maternity cages were checked for births each day at 0800 and 1800 h. The day of birth (postnatal day 0) was recorded as the date when litters were first observed. If necessary, on the day after birth, litters were culled to 10 pups (5 female, 5 male, when possible); litters with fewer than 8 pups were not used for these studies. Generally, a single pup from each litter was sampled for each age range noted in the preceding text. However, each age range includes two data sets obtained from litter mates. Remaining offspring were weaned to the preceding diet at 21 days of age and then transferred to sex-segregated cages no later than 45 days of age.
Chorda tympani recording
Rats at the ages noted in the preceding text were deeply anesthetized by intraperitoneal injection with pentobarbital (50-65 mg/kg). Supplemental injections (20-30 mg/kg) were given as needed to maintain surgical anesthesia. Body temperature was maintained with a circulating water heating pad (K-MOD 100, Allegiance Healthcare, McGaw Park, IL). Bilateral hypoglossectomy to prevent tongue movement was performed after blunt dissection of surrounding tissue, and the trachea was cannulated to facilitate free breathing. Following placement of the animal into a nontraumatic head holder, the left or right chorda tympani nerve was exposed via an epimandibular approach. The nerve was freed of surrounding connective tissue, cut near its entrance into the tympani bulla, desheathed, and placed onto a 28-gauge platinum wire electrode. A reference electrode was placed in nearby tissue. The wound was filled with a mixture of petroleum jelly and mineral oil to prevent drying of the nerve. Neural activity was fed to a fixed gain isolation amplifier (BioAmp 100, Axon Instruments, Foster City, CA) and then to a Grass P511 preamplifier (AstroMed, West Warwick, RI). The amplified signal was monitored with an oscilloscope and audio loudspeaker. Amplified neural activity was RMS-rectified and integrated with a time constant of 1-2 s. The integrated output was recorded on one channel of a Linseis L6514 4-channel rectilinear chart recorder (Linseis, Princeton Junction, NJ).
Lingual receptive field voltage clamp
Simultaneous lingual epithelial voltage clamping and chorda
tympani neural recordings have been described in detail (Stewart et al. 1996; Ye et al. 1993a
). Briefly, a
portion of the anterior dorsal lingual epithelium was enclosed within a
vacuum-applied cast acrylic stimulation chamber. For rats aged 29-31
and 60+ days of age, the chamber enclosed a
28-mm2 patch of dorsal tongue, while the chamber
used for 12-14 and 19-23 day old rats enclosed a
12.5-mm2 patch. The outer vacuum groove in the
chamber creates a mechanically stable and electrically tight seal
between the chamber and lingual epithelium. Stimulus and rinse
solutions (see following text) were injected (4 ml; 1 ml/s) into the
chamber via tubing fitted to a dedicated port (dead space: ~0.5 ml).
The chamber was fitted with separate Ag-AgCl electrodes for measurement
of current and potential, while reference electrodes were placed
noninvasively on the ventral lingual epithelium. The current-passing
electrode in the chamber served as virtual ground, ensuring that only
current passing through the stimulated patch was collected. Command
currents and potentials were delivered by a voltage-current clamp
amplifier (VCC600; Physiologic Instruments, San Diego, CA).
Transepithelial potential and current responses were recorded on two
channels of a Linseis L6514 4-channel rectilinear chart recorder.
Stimulus solutions and stimulus application
All solutions used were made from reagent grade chemicals
(Sigma, St. Louis, MO; or Fisher, Pittsburgh, PA) dissolved in
glass-distilled H2O. The rinse solution contained
15 mM KHCO3 (pH 8.7). A
Na+-depleted Krebs-Henseleit solution (DKH) that
contained (in mM) 6 KCl, 2 CaCl2, 1.2 MgSO4, 1.3 NaH2PO4, 25 NaHCO3, and 5.6 glucose (pH 7.5) was applied
after each stimulus series to help maintain a stable transepithelial
potential. Transepithelial potential in the presence of 150 mM NaCl
rarely fluctuated more than ±5 mV during the course of an experiment.
In addition, those preparations that exhibited absolute transepithelial
potentials of greater than ±20 mV in the presence of 150 mM NaCl were
excluded from analysis. Stimuli were concentration series (50, 100, 200, and 500 mM) of NaCl and Na+ gluconate
(NaGlu). Sodium gluconate was selected as a stimulus in addition to
NaCl primarily because the lower shunt permeability of gluconate
relative to chloride significantly reduces the voltage-independent (i.e., basolateral) Na+ response component
(Ye et al. 1991, 1993a
). Therefore the chorda tympani
response to NaGlu should derive primarily from voltage-dependent, apical transduction of Na+.
Stimuli were applied under zero-current clamp (equivalent to open
circuit conditions), and steady-state potentials were recorded from the
front panel display of the voltage-current clamp amplifier. Next,
chorda tympani responses to the stimuli were obtained under voltage
clamp at +60 and 60 mV relative to the zero-current clamp potential
recorded for each stimulus. In this way, neural responses to
concentration series were obtained under three membrane potential conditions: effective open circuit, and +60 and
60 mV voltage clamp.
Each stimulus series was bracketed by the application of a 500 mM
NH4Cl reference stimulus under zero-current
clamp, and intervening data were retained for analysis only when the
magnitude of the NH4Cl reference responses before
and after the stimulus series varied by <10%. Steady-state response
magnitudes, measured as the height of each integrated chorda tympani
response 30 s after application of the stimulus, were expressed
relative to the mean NH4Cl response magnitude
bracketing a stimulus series. Ammonium chloride was selected as a
reference stimulus for several reasons. First, expression of integrated
neural data relative to NH4Cl provides a valid
and reliable measure for comparisons of multiunit taste responses among
subjects and between treatment groups (Beidler 1953
).
Importantly, because the responsiveness of the rat taste system to
NH4Cl does not change during postnatal development (Hill et al. 1982
), it also provides an
appropriate, unchanging reference response for comparisons of
Na+ responses among age groups.
The protocol for stimulation of the lingual epithelium was generally as
follows. The reference NH4Cl stimulus was applied and allowed to remain in the chamber for 40-50 s. The solution was
then rinsed from the chamber by repeated application of the KHCO3 rinse solution for 60 s. Then, the
stimuli of interest (NaCl or NaGlu) were applied as a concentration
series with each concentration allowed to remain in the chamber for
40-50 s before being rinsed from the chamber repeatedly with
KHCO3, as noted in the preceding text. About
60 s after the final stimulus in a concentration series was rinsed
from the chamber, the reference NH4Cl solution
was reapplied then rinsed. At this point, DKH was injected into the
chamber, allowed to remain on the tongue for 60 s, and then rinsed
from the chamber with repeated applications of the
KHCO3 rinse solution. Last, another reference
stimulus was applied and rinsed, and the next concentration series was begun.
Statistical analyses
Data are expressed as means ± SE unless otherwise noted. Age-dependent differences in zero-current clamp chorda tympani response magnitudes at each stimulus concentration were determined using ANOVA with Student-Newman-Keuls (SNK) post hoc comparisons where appropriate.
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RESULTS |
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Development of chorda tympani responses to NaCl
Relative to the NH4Cl reference response,
integrated chorda tympani responses to NaCl and to NaGlu under
zero-current clamp increased in a progressive manner between 12-14
days of age and adulthood. Table 1
provides summary NaCl and NaGlu response magnitude data. Because we
were primarily interested in determining whether the present results
were consistent with previously published data regarding
Na+ response development, we examined age-related
differences in response magnitude only under the zero-current clamp
condition, which is equivalent to conventional chorda tympani
recording. Significant age-related differences in NaCl response
magnitude under zero-current clamp were detected for NaCl
concentrations 50 mM (ANOVA, P < 0.05) (Table 1).
Student-Newman-Keuls posthoc comparisons indicated a significant,
progressive, age-dependent increase in mean response magnitudes to 100, 200, and 500 mM NaCl 29-31 days of age (SNK, P < 0.05)
(Table 1). At 29-31 days of age, mean chorda tympani response
magnitudes to these stimuli did not differ significantly from
60+-day-old rat response magnitudes.
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Results obtained for NaGlu were in general very similar to those
obtained for NaCl. However, significant, age-related differences in
zero-current clamp response magnitudes to NaGlu were detected only at
200 and 500 mM (ANOVA, P < 0.05). Post hoc comparisons revealed a significant age-related difference in response magnitudes to
200 mM NaGlu between 12-14 and 60+ day old rats (SNK,
P < 0.05), while significant age-related differences
in chorda tympani response magnitudes to 500 mM NaGlu were detected
among all groups, except 29- to 31- and 60+-day-old rats. The results
for zero-current clamp responses to both Na+
salts are consistent with previously published data regarding the
development of chorda tympani NaCl responses in rats (Ferrell et
al. 1981; Hill and Almli 1980
).
In conjunction with the developmental increase in
Na+ response magnitude, the sensitivity of chorda
tympani NaCl responses to applied lingual epithelial voltage
perturbations increased with age. For example, Fig.
1 depicts integrated chorda tympani responses to 100 mM NaCl under zero-current clamp and +60 and 60 mV
voltage clamp obtained from a 12- to 14- and a 29- to 31-day-old rat.
It is clear that the effect of voltage clamp on responses to this NaCl
concentration is considerably greater in the 29- to 31-day-old rat. In
general, and consistent with previously published results (Ye et
al. 1993a
,b
), chorda tympani responses to NaCl at all
concentrations were elevated under submucosal negative voltage
clamp, which increases the electrochemical driving force for
Na+ into the taste receptor cell, and suppressed
under submucosal positive voltage clamp, which decreases the driving
force for Na+ into the taste receptor cell.
However, at 250 and 500 mM NaCl, the influence of voltage clamp
appeared blunted in 29- to 31- and 60+-day-old rats (Fig.
2). Chorda tympani responses to all concentrations of NaGlu obtained under voltage clamp also exhibited marked sensitivity to applied voltage perturbations, and the
magnitude of this sensitivity appeared to increase with age. In
contrast to the NaCl case, the effect of voltage clamp on NaGlu chorda tympani response magnitudes remained robust at higher concentrations and at all ages studied (Fig. 2). The effects of lingual epithelial voltage clamp on chorda tympani response-concentration functions for
NaCl and NaGlu are shown in Figs. 3 and
4, respectively.
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Figures 3 and 4 clearly show that Na+ salt chorda
tympani responses are subject to modulation by imposed transepithelial
voltage perturbations even at the earliest age-point studied (12-14
days of age). By considering ion channel flow kinetics (Mintz et
al. 1986; Ye et al. 1993a
) and by assuming that
depolarizing Na+ ion influx into taste cells is
the rate-limiting event in the evoked neural response to
Na+, we obtain the apical channel model equation,
which is a modified form of the Beidler taste equation (Beidler
1954
)
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Figures 3 and 4 depict for each age group the best-fit parameters
obtained with the apical channel model for NaCl and NaGlu response
data, respectively, as well as plots of the theoretical functions that
follow from it. As expected for both stimuli, the channel model
equation predicted an age-related, monotonic increase in
CTmax, which is highly consistent with
the postulated developmental increase in apical
Na+ channel number. Surprisingly, predicted
values of Km decreased in an
age-related manner. This unexpected finding is also entirely consistent
with the well-reported age-related increase in rat gustatory
Na+ sensitivity. An interesting feature of these
changes is that the leftward shift in
Km along the concentration axis (i.e.,
an increase in channel affinity for Na+) occurs
between12-14 and 19-23 days of age and is superimposed on a rapid
increase in CTmax that occurs
primarily between 12-14 and 29-31 days of age. Finally, and much to
our surprise, a shift in the predicted value of occurred between
19-23 and 29-31 days of age when NaCl was the stimulus. In contrast,
when NaGlu was the stimulus, this parameter appeared to remain stable
across the developmental period studied. Using the parameters obtained in the preceding text, the chorda tympani response data shown in Figs.
3 and 4, were replotted as a function of NaCl electrochemical concentration, ce. That is, the NaCl
and NaGlu response data were replotted after transforming the
independent variables of clamp voltage and stimulus concentration into
a single variable, ce (Eq. 2).
Figures 5 and
6 depict the resulting
response-electrochemical concentration functions for NaCl and NaGlu,
respectively. It can be seen clearly that across developmental age the
three response-concentration functions (which correspond to the 3 clamp
voltage conditions) shown in each panel of Figs. 3 and 4 collapse onto
a single curve for each age group. This transformation of the data
reveals that even at the earliest age points studied the actual
stimulus intensity for Na+ is
ce, i.e., the driving force for the
ion through a passive channel. Moreover, for both NaCl and NaGlu, the
collapsed response-concentration functions for all four age groups
manifest the age-dependent increase in
CTmax and the age-dependent decrease
in Km, which is evidenced by the
increasing initial slope of the theoretical functions. Although the
channel model accounts for most of the variance in the data (minimum
R2 0.89), a notably larger
deviation is observed for the 60+-day olds.
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Such a deviation could arise from a secondary voltage-independent
transduction process and could lead to errors in estimating CTmax. In an attempt to eliminate this
as a source of error, the data were replotted as the voltage
sensitivity index (VSI, Eq. 3). Because the VSI is defined
as a difference in chorda tympani responses at two voltages,
contributions from voltage-independent processes cancel out. The
remaining, voltage-sensitive part of the chorda tympani response to
NaCl (or NaGlu) should then arise exclusively from transduction by
amiloride-sensitive Na+ channels (Ye et
al. 1993a). Consequently, the VSI can be used to provide a more
accurate estimate of the relative densities of apical
Na+ channels among experimental groups (Ye
et al. 1993b
). In the present case the VSI is
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DISCUSSION |
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In the present study, simultaneous in vivo lingual epithelial
voltage clamping and chorda tympani recording were used to examine the
increase in taste system Na+ sensitivity that
occurs during postnatal development in rat. The primary finding from
these experiments is that the age-related increase in the chorda
tympani Na+ response results from the development
of a transduction mechanism that is driven by stimulus electrochemical
concentration. This observation implicates a set of apical membrane ion
channels as the stimulus transducer and, furthermore, indicates that
the postnatal increase in chorda tympani Na+
responses is due primarily to an increase in transducer (i.e., ion
channel) relative density. These results confirm previous reports,
which, based on amiloride pharmacology, concluded that a progressive
increase in the number of functional amiloride-sensitive Na+ channels was probably the underlying
mechanism for the developmental increase in taste system
Na+ sensitivity in rat (Hill and Bour
1985; Sollars and Bernstein 1994
). Our current
results also extend those findings by revealing that in addition to the
age-dependent increase in the relative density of taste receptor cell
functional Na+ channels, the apparent
Na+ binding affinity of the amiloride-sensitive
Na+ channel (corresponding to a decrease in
Km) increases as a function of
postnatal age. Together, these two age-related changes in the intrinsic
functional properties of the Na+ sensing
apparatus contribute to the postnatal maturation of anterior tongue
taste receptor Na+ sensitivity in rat. An
especially interesting feature of these age-dependent changes in
Na+ channel properties is that they are
temporally distinct.
Results from our kinetic analyses of chorda tympani responses to NaCl and NaGlu during the postnatal period indicated differences in the timing of changes in apparent density and affinity of apical, amiloride-sensitive Na+ channels. Specifically, the age-related decrease in Km values for both Na+ salts was observed to occur between 12-14 and 19-23 days of age. Estimated Km for NaCl decreased by ~169 mM over this period and Km for NaGlu by ~120 mM. Thereafter, Km values for both salts remained stable into adulthood (Fig. 8A). In contrast, for both Na+ salts, CTmax values increased rapidly between 12-14 and 29-31 days of age, at which point the rate of growth in CTmax slowed considerably as mature, unchanging CTmax values were approached. The age-related trends in these parameters are considered in greater detail in the following sections.
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Development of chorda tympani responses to NaCl and NaGlu: parallel trends
As described in RESULTS, the quantitative trends in chorda tympani response development are in general similar for these two Na+ salts, and this similarity was fully expected at the outset of these studies. However, inspection of the data presented in RESULTS clearly indicates that there are important quantitative differences in the parameters that describe the development of the Na+ sensing apparatus with respect to these two salts. Consideration of these differences provides important insight regarding the development not only of apical, amiloride-sensitive Na+ channel function but also the development of taste bud polar epithelial topology.
By describing our data with a channel model equation, we were able to
follow, operationally, the development of the apical, voltage-dependent
Na+ sensing apparatus by obtaining changing
values in the system parameters CTmax,
Km, and . The predictive utility of
this model requires that changes in Na+ response
magnitude be the consequence of changes in apical (i.e., amiloride-sensitive) Na+ channel function. For
NaGlu, across all age points examined, it is clear that CT response
magnitude is a single-valued function of the electrochemical
Na+ concentration (e.g., Fig. 6). Moreover,
values of Rmax (the estimate of
relative channel density eliminating possible voltage-independent processes) for NaGlu predicted from consideration of the VSI revealed that the relative density of apical channels increases rapidly from
~25% of the mature density at 12-14 days of age to ~84% the mature density at 29-31 days of age. Because of the accelerating increase observed between these age points, we attempted to describe the age-dependent trend in whole system
CTmax values (predicted from Eq. 1) using a bounded-growth model
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Figure 8A also shows the theoretical bounded growth function
for the development of NaCl response
CTmax. The best fit of the model to
the data were obtained when a = 1.54, b = 7.7 days, and th = 19.0 days
(R2 = 0.98). This predicted maximum
system NaCl response is nearly identical to the value of
CTmax for 60+-day-old rats obtained from the channel model equation. Moreover, the values of b
and th are comparable to those
obtained for NaGlu, and extrapolation of
CTmax values to zero days of age,
yields a relative channel density of ~8% the adult density. This
zero-day value is of particular interest with respect to the findings
of Ye et al. (1993b), who, using the apical channel
model, determined that fungiform taste receptors of adult rats raised
under developmental dietary Na+ restriction
express a relative density of apical Na+ channels
that is
th the density in the taste receptor cells of
control animals reared on a Na+ replete diet.
However, the effect of developmental Na+
restriction appears to be unique to channel density, as no diet-related difference in apparent Km was reported
by Ye et al. (1993b)
. Those results, in conjunction with
the current findings, underscore the notion that channel density and
affinity are subject to independent regulation, and, further, raise the
intriguing possibility that developmental Na+
restriction causes truncated development of the gustatory
Na+ sensing apparatus. That is, with respect to
Na+ sensing, fungiform taste receptor cells in
developmentally Na+-restricted rats attain a
state of maturity functionally equivalent to that of a newborn. It
should be emphasized again, however, that these data relate only to the
apical Na+ sensing machinery.
With respect to the entire Na+ sensing apparatus (i.e., apical and basolateral pathways), an important distinction between the bounded growth functions for NaGlu and NaCl relates to the parallel nature of CTmax values across development. That is, system CTmax values predicted by the channel model equation and by the bounded growth model, as well as the apical Rmax values predicted by the VSI function, are larger for NaCl than for NaGlu at all age points. This reliable difference in the magnitudes of NaCl and NaGlu responses was expected and corresponds to the presence of the amiloride-insensitive component of the chorda tympani response to NaCl. It is interesting to note that despite the presence of this amiloride-insensitive response component in NaCl responses from the earliest age points studied, the fundamental hypothesis that the postnatal increase in gustatory Na+ sensitivity is due to an orderly increase in the density of functional, apical (and, therefore voltage-sensitive) Na+ channels is nonetheless validated by the apical channel model. In addition to its contribution to stimulus-dependent differences in measures of maximum response magnitude, the anion-dependent properties of the paracellular shunt in the intact taste bud contribute to differences between NaCl and NaGlu with respect to other Na+ channel kinetic parameters.
In addition to parallel, age-dependent trends in chorda tympani
CTmax for NaCl and NaGlu, parallel,
age-related changes in the apparent affinity
(Km) of Na+ for
the apical Na+ channel were seen to occur (Fig.
8B). Specifically, Km for
both salts decreased from relatively high values at 12-14 days of age to lower values at 19-23 days of age, and then remained stable across
succeeding age points. The similar trends in
Km for both salts clearly suggest that
the affinity of the apical channel for Na+ ions
increases developmentally and, thus, contributes to the overall
increase in gustatory Na+ sensitivity that is
seen during the postnatal period. We speculate that the change in
channel Km is most probably due to
developmental alteration in intrinsic channel properties. It is
possible, however, that changes in the "affinity" of processes
intermediate to Na+ binding/influx and the
generation of the neural response (e.g., taste cell neurotransmitter
release) are responsible for the apparent shift in channel affinity.
Consistent with such a view, Hill et al. (1982) observed
a developmental decrease in the latency of chemically-stimulated neural
responses in single chorda tympani fibers. On the other hand,
Hill and colleagues (1982)
found no difference in single
chorda tympani fiber response latency in rats aged 10-14 versus those
aged 24-35 days of age. In the present study, the apparent shift in
Km occurs between 12-14 and 19-23 days of age, long before Hill et al.'s latency shift, which occurred between 24-35 days of age and adulthood. Regardless of the source of
this apparent shift, the large differences in predicted
Km values observed for the two salts
raise an important question: if Km
describes the affinity of Na+ for a
Na+ specific channel, why then are the predicted
values of Km for the two salts so
divergent? That is, why, in the presence of NaCl, is the apparent
affinity of Na+ for Na+
channels so much greater than when NaGlu is the stimulus?
From the present experiments, and from previous work (Stewart et
al. 1996), it is clear that the lingual transepithelial
potential (TEP) that attends application of NaGlu is considerably more
submucosa positive compared with the potential that attends application of an equimolar concentration of NaCl. This stimulus-dependent difference in TEP results from the lower shunt permeability of the
gluconate anion relative to Cl
. Given the
polarized epithelial topology of the taste bud, it is predicted that
the lower TEP evoked by NaCl results in a greater depolarization of the
basolateral taste receptor cell membrane and in a relatively larger
hyperpolarization across the apical taste receptor cell membrane
compared with NaGlu (cf. Lindemann 1996
; Ye et
al. 1994
). Importantly, this hyperpolarization at the apical
membrane is "seen" by Na+ ions as an
increased intracellular electronegative potential and thus a relative
increase in the driving force for Na+ ions into
the taste cell across the apical amiloride-sensitive conductance. In
contrast, when gluconate is the stimulus anion, the more submucosa
positive potential creates a relative depolarization of the apical
membrane and thus a relative decrease in driving force for
Na+ ions into the taste cell. In our model
system, the net effect of these stimulus- and, hence, TEP-dependent
differences in the driving force for apical Na+
flux appears to be manifested as stimulus-dependent shifts in Km. That is, the aggregate
K values predicted by our model depend on anion- and, hence,
stimulus-related differences in TEP.
The idea that stimulus-dependent TEP can potently influence apparent
Km values for the intact system has
been demonstrated previously (Stewart et al. 1996). Such
stimulus-related modulation of the channel native Michaelis constant,
here called K, is the unique consequence of applying the
apical channel model to the intact taste system and illustrates how the
polar topology of the taste bud imposes unavoidable constraints on the
function of membrane effectors, such as ion channels. Moreover
stimulus-dependent modulation of an apparent intrinsic channel property
such as K emphasizes the importance of studying function
under conditions that maintain the essential polarity of the biological
system. Given these observations and the likelihood that
Na+ salts modulate their own apparent
Na+ channel affinity, it is perhaps most
appropriate to assign individual Na+ salt stimuli
specific K values (e.g.,
KNaCl and
KNaGlu) when such parameters are
determined in the intact system. For the present study, it is critical
to bear in mind that the consistent, stimulus-dependent difference
between KNaCl and
KNaGlu is superimposed on a large developmental change in the Na+ channel
"native" K. In addition to this clear and important
influence of the stimulus anion on apparent system
Km, the stimulus anion also
contributes to differences between NaCl and NaGlu with respect to other
system properties.
As noted in RESULTS, age-dependent changes in the fraction
of the clamp voltage, , that is sensed by Na+
ions as they traverse the apical Na+ channels
were observed. However, in contrast to parallel trends in other system
parameters, age-dependent changes in
were observed for NaCl only.
Specifically, and unexpectedly, estimated
for NaCl declined from
comparable values at 12-14 and 19-23 days of age (i.e., 0.44 and
0.45, respectively) to considerably lower values at 29-30 and 60+ days
of age (i.e., 0.25 and 0.22, respectively). By comparison, estimated
values for NaGlu were largely stable throughout the time period
studied. For all age groups,
values for NaCl are smaller than are
those for NaGlu. The consistent difference in
for the two stimuli
is a consequence of the higher shunt permeability of
Cl
relative to that of gluconate. Moreover, the
decline in
for NaCl in the two oldest age groups implies that taste
bud shunt properties change considerably as a function of postnatal
age. Specifically, the decrease in
for NaCl between 19-23 and
29-31 days of age suggests that tight junctional permeability to
mobile anions, such as Cl
, actually
increases with advancing age. That is, taste bud tight junctions do not become tighter with advancing postnatal age, they
become leakier. This decrease in tight junction resistance appears to
be unique to taste buds, as measurements of relative transepithelial
resistance and conductance did not vary as a function of postnatal age
(data not shown). The dissociation between changing
for NaCl and
stable
for NaGlu is not inconsistent and probably reflects the fact
that even a considerable increase in the shunt permeability for smaller
ions has no evident impact on permeability to large ions with low tight
junctional mobility (e.g., gluconate). This remarkable developmental
change in the topological properties of the taste bud has direct
functional correlates in the present findings.
The age-related change in for NaCl may explain in large part the
failure of the theoretical VSI function to fit accurately the data for
NaCl responses in 29- to 31- and 60+-day-old rats. While there was
excellent agreement between the observed and predicted VSIs for NaCl
for 12- to 14- and 19- to 23-day-old rats, by 29-31 days of age and
continuing into adulthood, the predicted VSI underestimated the
observed VSI at concentrations of NaCl below 200 mM and overestimated the observed VSI at the highest NaCl concentration (500 mM) (Fig. 7A). The degradation in the predictive accuracy of the model
probably derives from the loss of voltage sensitivity in chorda tympani responses to higher concentrations of NaCl in 29- to 31- and
60+-day-old rats. A striking example of this can be seen in Fig. 2. Not
surprisingly, the timing of the loss of chorda tympani response voltage
sensitivity at higher NaCl concentration coincides with the timing of
the decrement in
observed for NaCl.
The critical parameter in the VSI of the chorda tympani response to
NaCl is . That is, as
approaches unity, the observed effect of
voltage perturbations on chorda tympani responses becomes greater. In
contrast to the case of NaGlu, where
is stable and relatively
large,
appears to decline as a function of increasing NaCl
concentration, especially in the older age groups. It is possible that
in the presence of high NaCl concentrations, the paracellular diffusion
potential of the stimulus ions is increased, and, under voltage clamp
conditions, this increase in paracellular electroneutral diffusion
potential for Na+ and Cl
provides a "sink" for current injected to maintain command voltage. Essentially, the paracellular shunt becomes a lower resistance pathway
for ion movement (i.e., current) than is the apical,
amiloride-sensitive Na+ channel, and so a
relatively larger fraction of the clamp voltage is dropped across the
paracellular shunt as NaCl concentration increases. The consequences of
this age-dependent increase in the paracellular shunt permeability
(observed as a decline in
) are two. One probable consequence, which
is observed, is that dissipation of clamp voltage across the
paracellular shunt diminishes the measurable voltage sensitivity of
chorda tympani response to NaCl at high concentrations but only in
older rats that have developed the lower paracellular shunt resistance.
Another likely consequence is that as the rat matures, the relatively
increased electroneutral diffusion of Na+ and the
stimulus co-anion through the tight junctions increases the magnitude
of the voltage-independent and amiloride-insensitive response
component, particularly at high stimulus concentrations. Such a change
in shunt properties could also contribute to Na+
response development, although available evidence suggests that the
magnitude of the amiloride-insensitive component of chorda tympani NaCl
responses is stable during development in rats (Hill and Bour
1985
; Sollars and Bernstein 1994
). This
important developmental change in taste bud topological properties,
which exerts influences on several fundamental parameters that describe
system function, could only be appreciated through examination of the
intact, functioning taste system.
It is important to note that the apical channel model assumes that is a constant. However, the present data clearly indicate that the
value of
necessarily depends on the potential for paracellular electroneutral diffusion of Na+ and
Cl
, which is determined jointly by NaCl
concentration and the resistance of the shunt pathway (i.e., tight
junctions) (Ye et al. 1993a
). It should also be
emphasized here that for Na+ salt stimuli that
contain large, shunt-impermeant anions, such as gluconate, modulation
of
would not be observed. That is, modulation of
is essentially
anion dependent.
Potential mechanisms for developmental increases in apical Na+ channel number and affinity
Our current findings indicate that temporally distinct,
age-dependent changes in the density of apical, amiloride-sensitive Na+ channels and in the apparent affinity of
Na+ for those channels together provide the basis
for postnatal development of mature gustatory Na+
sensitivity of the rat chorda tympani nerve. Our results confirm previous hypotheses (e.g., Hill and Bour 1985;
Sollars and Bernstein 1994
) regarding the postnatal
increase in chorda tympani responses to Na+. The
cellular basis for the increase in the density of functional channels
remains to be elucidated, but several mechanisms could be involved. For
example, a simple increase per taste bud in the number of taste cells
that possess a fixed number of apical Na+
channels could contribute to an apparent increase in the number of
apical amiloride-sensitive Na+ channels. There
is, in fact, ample evidence that fungiform papilla taste bud size
increases postnatally (Krimm and Hill 1998
) and, furthermore, that this size increase may be due proliferation of taste
cells as opposed to hypertrophy of individual cells (Kossel et
al. 1997
). On the other hand, recent developmental studies of
taste cell proliferation revealed lower rates of cell addition to taste
buds in rats aged zero and 29-31 days compared with those aged 60+
days (Hendricks and Hill 2000). Another obvious
mechanism for an increase in the density of apical
Na+ channels would be a progressive increase in
the de novo synthesis and insertion into the apical membrane of
functional Na+ channels. However, previous
immunohistochemical results provided qualitative evidence that the
fungiform papilla taste cells of 1-day-old rats contained significant
amounts of apical Na+ channel-like immunoreactive
material (Stewart et al. 1995
). That result raises the
possibility that from early in development, rat fungiform papilla taste
receptor cells synthesize amiloride-sensitive Na+
channels, and these extant channels gradually become functional during
the postnatal period. For example, it is possible that channels are
synthesized and immediately inserted into taste cell apical membranes
but that they remain inactive. Subsequent, developmentally regulated
activation processes (e.g., covalent modification) (cf. Garty
and Palmer 1997
; Shimkets et al. 1998
) might
then act to increase the relative density of functional channels.
Alternatively, it is possible that a redistribution of
Na+ channels between basolateral and apical
membrane compartments occurs during the early postnatal period. This
potential mechanism was favored by Kossel and colleagues
(1997)
and by Stewart et al. (1995)
, who
suggested that such membrane reallocation might be promoted by
maturation of, and subsequent association of apical membrane channels
with, taste cell cytoskeletal elements. Interestingly, cytoskeletal
elements interact with and modulate the function of apical
Na+ channels (Ismailov et al.
1997
; Smith and Benos 1996
; Smith et al.
1991
). This intriguing possibility has so far evaded
elucidation in the taste system, but recent advances in the molecular
biology of apical Na+ channels in other tissues
should facilitate its evaluation (Benos et al. 1997
;
Garty and Palmer 1997
).
Strides in the understanding of the apical Na+
channel may also provide insights into the mechanism for the
considerable increase in the affinity of Na+ for
the apical channel that was observed to occur between 12-14 and 19-23
days of age. In the past several years, molecular biological techniques
have allowed the cloning of distinct genes that encode the primary
amino acid sequences for three homologous subunit proteins that in
complementation comprise an amiloride-sensitive, Na+-selective conductance with biophysical
properties similar to those of purified, native amiloride-sensitive
Na+ channels characterized from several source
tissues (Benos et al. 1997; Canessa et al.
1994
; Garty and Palmer 1997
). Specifically termed the epithelial Na+ channel, or ENaC, the
heterotrimeric channel consists of alpha, beta, and gamma subunit
proteins, all of which have been detected immunohistochemically in rat
fungiform papilla taste cells (Kretz et al. 1999
;
Lin et al. 1999
). When expressed alone, the alpha subunit comprises an amiloride-sensitive conductance, characterized by
low maximum Na+ current and low amiloride binding
affinity. However, co-expression of all three subunits results in a
highly Na+-selective, amiloride-blockable
conductance with large maximum Na+ current. It is
notable that subunit complementation patterns can significantly
influence, for example, the amiloride binding affinity and ion
selectivity of the ENaC, in addition to the maximum Na+ current that can be sustained by the channel
complex (Benos et al. 1997
). Clearly, an age-related
alteration in subunit complementation pattern could contribute to the
developmental increase in gustatory Na+
sensitivity. One striking, though fully speculative, possibility with
respect to the current results is that the age-related change in
apparent channel affinity is the result of "immature" subunit complementation patterns. However, Kossel et al. (1997)
present evidence that the inhibition constant of amiloride for blockade of whole cell inward currents in neonatal taste cells is comparable to
that obtained for adult taste cells. Given other results that suggest
alterations in subunit complementation can strongly influence ENaC
amiloride-binding affinity (Benos et al. 1997
), this
result tends to argue against an altered subunit complementation
pattern in young rat taste receptor cells. On the other hand, it is
possible that in taste receptor cells apical Na+
channel affinities for Na+ ions and for amiloride
are subject to different and distinct regulatory effectors (cf.
Garty and Palmer 1997
). Clearly, an important area for
future investigation includes resolving, in the intact, developing
taste system, whether the interaction of amiloride with the apical
Na+ channel changes significantly as a function
of postnatal age. Results from such experiments, in combination with
more molecular studies that directly assess taste receptor cell ENaC
subunit expression and complementation patterns during postnatal
development, should provide important evidence regarding the cellular
mechanisms that underlie the developmental increase in apical
Na+ channel affinity.
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APPENDIX |
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As shown in the RESULTS, the chorda tympani response
to Na+ is a saturating function of the
Na+ electrochemical concentration (cf. Eq. 1), i.e., the driving force for the flux of
Na+ through a conductive pathway. This suggests
that the form of Eq. 1 is the result of a kinetic process
involving the influx into taste receptor cells of
Na+ ions across apical membrane ion channels.
This can be demonstrated using the Na+ channel
model proposed by Mintz et al. (1986) in their
computational simulation of sodium transport across tight epithelia. It
is assumed that the apical channels have pores connecting the mucosal
solution phase with the cell interior and that each pore contains a
single Na+ ion binding site. In addition, each
Na+ ion entering the channel from either side
must overcome a potential energy barrier to reach the binding site.
While the binding site may be located at any point within the pore, we
assume here that it is located at the pore exit into the cell interior.
In this way, a Na+ ion entering the channel from
the stimulating solution will encounter the entire electrical potential
gradient across the apical membrane. The binding association reaction
between a Na+ ion originating from the mucosal
side (m) and the pore site has rate constant
fm; the similar association reaction
involving a Na+ ion originating from the cellular
side (c) of the channel has rate constant
fc. The binding dissociation
reaction releasing Na+ into the mucosal solution
has rate constant km and that
releasing Na+ into the cellular medium has rate
constant kc. Because
Na+ ions entering channels from the mucosal side
encounter the additional potential energy of the apical membrane
potential, fm and
km can be written respectively as:
fmo exp[
/2] and
kmo exp[
/2], where
is the
dimensionless apical membrane potential relative to the mucosal
solution, and fmo and
kmo are rate constants exclusive of
electrical potential effects. The values of the pairs
fmo and kmo and
fc and
kc may differ in magnitude depending
on the size of the energy barriers on each side of the pore region, but
the ratios,
kmo/fmo
and
kc/fc
are equal, defining the thermodynamic dissociation constant,
Ke, between Na+
ions and their channel binding sites.
Let N be the total number of channels per unit area of
apical membrane, po, the fraction of
pore binding sites that are unbound, and
p1, the fraction of sites with a bound
Na+ ion. Conservation of total binding sites
requires: po + p1 = 1. In the steady state, the net
Na+ flux, JNa is
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(A1) |
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(A2) |
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(A3) |
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(A4) |
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(A5) |
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ACKNOWLEDGMENTS |
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This work was supported by National Institute on Deafness and Other Communication Disorders Grants DC-00122 and DC-02422 (to J. A. DeSimone), DC-00407 and DC-02406 (to D. L. Hill), and DC-03499 (to R. E. Stewart).
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FOOTNOTES |
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Address for reprint requests: R. E. Stewart, Dept. of Psychology and Neuroscience Program, Washington and Lee University, Lexington, VA 24450 (E-mail: rstewart{at}wlu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 May 1999; accepted in final form 26 May 2000.
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REFERENCES |
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