Department of Behavioral Science, Penn State College of Medicine, Hershey, Pennsylvania 17033
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ABSTRACT |
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Lundy, Robert F., Jr. and Ralph Norgren. Pontine Gustatory Activity Is Altered by Electrical Stimulation in the Central Nucleus of the Amygdala. J. Neurophysiol. 85: 770-783, 2001. Visceral signals and experience modulate the responses of brain stem neurons to gustatory stimuli. Both behavioral and anatomical evidence suggests that this modulation may involve descending input from the forebrain. The present study investigates the centrifugal control of gustatory neural activity in the parabrachial nucleus (PBN). Extracellular responses were recorded from 51 single PBN neurons during application of sucrose, NaCl, NaCl mixed with amiloride, citric acid, and QHCl with or without concurrent electrical stimulation in the ipsilateral central nucleus of the amygdala (CeA). Based on the sapid stimulus that evoked the greatest discharge, 3 neurons were classified as sucrose-best, 32 as NaCl-best, and 16 as citric acid-best. In most of the neurons sampled, response rates to an effective stimulus were either inhibited or unchanged during electrical stimulation of the CeA. Stimulation in the CeA was without effect in two sucrose-best neurons, nine NaCl-best neurons, and one citric acid-best neuron. Suppression was evident in 1 sucrose-best neuron, 18 NaCl-best neurons, and 15 citric acid-best neurons. In NaCl-best neurons inhibited by CeA stimulation, the magnitude of the effect was similar for spontaneous activity and responses to the five taste stimuli. Nonetheless, the inhibitory modulation of gustatory sensitivity increased the relative effectiveness of NaCl resulting in narrower chemical selectivity. For citric acid-best neurons, the magnitude of inhibition produced by CeA activation increased with an increase in stimulus effectiveness. The responses to citric acid were inhibited significantly more than the responses to all other stimuli with the exception of NaCl mixed with amiloride. The overall effect was to change these CA-best neurons to CA/NaCl-best neurons. In a smaller subset of NaCl-best neurons (n = 5), CeA stimulation augmented the responsiveness to NaCl but was without effect on the other stimuli or on baseline activity. It appears that electrical stimulation in the CeA modulates response intensity, as well as the type of gustatory information that is transmitted in a subset of NaCl-best neurons. These findings provide an additional link between the amygdala and the PBN in the control of NaCl intake, modulating the response and the chemical selectivity of an amiloride-sensitive Na+-detecting input pathway.
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INTRODUCTION |
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Visceral signals and
experience modulate the acceptance of gustatory stimuli and,
consequently, the consumption of foodstuff. For instance, the
association of taste with gastrointestinal malaise (conditioned taste
aversion) can prevent the subsequent ingestion of the tastant
(Garcia et al. 1955; Grill 1985
;
Nachman and Ashe 1973
), while dietary sodium deficiency
can produce an avid consumption of previously avoided concentrations of
NaCl (Berridge et al. 1984
; Contreras and Hatton
1975
; Nachman 1962
; Richter
1936
). The pontine parabrachial nucleus (PBN) appears to be a
critical neural substrate for these taste-guided behaviors because
bilateral lesions of this area severely disrupt their expression
(Grigson et al. 1993
; Scalera et al.
1995
; Spector et al. 1992
). In normal animals,
both conditioned taste aversion and sodium appetite selectively alter
gustatory evoked responses in the brain stem (Chang and Scott
1984
; Nakamura and Norgren 1995
; Shimura
et al. 1997
; Tamura and Norgren 1997
;
Yasoshima et al. 1995
). One interpretation of this
modulation in response to visceral signals and experience is that
centrifugal activity normally regulates gustatory processing in the
brain stem. This is supported by the fact that chronically decerebrate
rats fail to acquire a learned taste aversion or to express a salt
appetite (Grill et al. 1986
).
The PBN distributes gustatory information to the hypothalamus,
amygdala, bed nucleus of the stria terminalis, and, via the thalamus,
to insular cortex and, in turn, receives projections from these brain
structures (Halsell 1992; Hopkins and Holstege 1978
; Norgren 1976
; Roberts 1980
;
Saper and Loewy 1980
; Veening et al.
1984
). These forebrain regions participate in autonomic and
endocrine functions, and each has been implicated in the expression of
complex taste-guided behaviors (Bermudez-Rattoni and McGaugh 1991
; Johnson et al. 1999
; Loewy and
Spyer 1990
; Roth et al. 1973
; Ruger and
Schulkin 1980
; Zardetto-Smith et al. 1994
). The
influence of descending input from these rostral structures on the
neurophysiology of brain stem gustatory neurons has received little
attention. Prior studies have shown that electrical stimulation in the
lateral hypothalamus (Bereiter et al. 1980
;
Matsuo and Kusano 1984
; Matsuo et al.
1984
; Murzi et al. 1986
) modulated
gustatory-responsive neurons in the rostral nucleus of the solitary
tract, but not in the PBN (Murzi et al. 1986
). Activity
in gustatory cortex, on the other hand, altered the ongoing spontaneous
discharge of both nucleus of the solitary tract (NST) and PBN gustatory
neurons (DiLorenzo and Monroe 1992
; Smith and Li
2000
). None of these studies tested for alterations in
gustatory evoked activity.
The choice to stimulate in the central nucleus of the amygdala (CeA)
was based on the fact that it is a major recipient of PBN gustatory
afferent axons, as well as the major source of amygdaloid efferents to
the brain stem (Halsell 1992; Norgren
1976
; Veening et al. 1984
). In addition,
neurophysiological studies examining cardiovascular and respiratory
sensitive neurons in the amygdala and PBN showed that neurons recorded
in one area were influenced by electrical stimulation in the other
(Cechetto and Calaresu 1983
; Jhamandas et al.
1996
). The effect of such forebrain stimulation on the
responses of gustatory neurons in the PBN has not been examined.
The present study characterizes the influence of the CeA on gustatory processing at the level of the PBN. We recorded responses of single PBN neurons to gustatory stimuli applied before, during, and after concurrent electrical stimulation in the CeA. Amygdala stimulation was found to modulate both ongoing spontaneous discharge and tastant-evoked responses in PBN gustatory neurons. These alterations in gustatory sensitivity mimic those produced by the induction of a sodium appetite.
A portion of this work was presented as a poster at the 2000 meeting of the Association for Chemoreception Sciences.
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METHODS |
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Subjects
Neurophysiological recordings were made in 25 male Sprague-Dawley rats weighing 360-500 g [CrL:CD (SD) BR; Charles River Breeding Laboratories]. The animals were maintained in a temperature-controlled colony room on a 12-h light/dark cycle and allowed free access to normal rat chow (Teklad 8604) and distilled water.
Amygdala stimulating electrodes
The rats were anesthetized with a 50-mg/kg injection (ip) of
pentobarbital sodium (Nembutal). Additional doses of Nembutal (0.1 ml)
were administered as necessary to continue a deep level of anesthesia.
Rectal temperature was monitored throughout a recording session and
maintained at 37 ± 0.2°C. The trachea was cannulated with
polyethylene 210 tubing, and a small suture was attached to the ventral
surface of the tongue. Animals were then secured in a stereotaxic
instrument using nonpuncture ear bars (45° taper) and a bite bar. A
midline incision was made and the skull leveled between and
.
Three 140-µm-diam stainless steel electrodes, insulated except at the
cross section of the tip, were lowered into the central nucleus of the
amygdala (CeA) using the following stereotaxic coordinates relative to
: P
1.8 mm, L 4.0 mm, and V 8.5 mm. A small hole was drilled
through the skull at the P-L coordinates, and the dura was excised. The
stimulating electrodes were lowered through the cortical tissue using a
custom-made jig that spaced the electrodes 0.5 mm apart and oriented
them in the AP plane. The electrodes were anchored with dental cement
via a stainless steel screw in the skull just posterior to the
electrodes. Electrical stimulation in the amygdala was achieved using a
Grass S 48 train generator attached to a constant current source
(PSIU6), which, in turn, was connected to a switch box that could
distribute the current to any possible pair of the 3 electrodes.
Stimulation parameters were 1-s trains of 10 pulses delivered at 0.5 Hz
for 10 s (total of 5 trains). The duration and amplitude of an
individual pulse were 0.2 ms and 0.4 mA, respectively.
Localization of PBN gustatory neurons
After drilling a small hole through the interparietal bone,
glass-insulated tungsten electrodes (resistance 2-8 M) were lowered into the PBN using a Fredrick Haer micropositioner. To avoid the transverse sinus, the electrode was oriented 20° off the vertical with the tip pointed rostrally. A drop in background activity marked
the boundary between the cerebellum and the pons, and, at that point,
any neuron encountered was tested for its sensitivity to 0.1 M NaCl
applied to the anterior two-thirds of the tongue. If a response was not
obtained with NaCl, then 0.3 M sucrose, 0.01 M citric acid, and 0.003 M
quinine hydrochloride were tested. Neural activity was amplified
(×1,000), fed through an artifact suppresser, monitored with an
oscilloscope and audiomonitor, and stored on a audio-cassette recorder
for off-line analysis. The artifact suppresser was used to adjust the
size of the electrical stimulation artifact to be easily distinguished
from the neural signal during off-line analysis. Cambridge
Electronic Design's Spike2 hardware and software were used to convert
the recorded data to digital format.
The neural signal and the artifact signal were individually classified and separated into two wavemark channels using Spike2 hardware and software. The channel with the artifact signal was used to mark the onset and offset of the electrical pulses. This procedure removes the background noise from the recording (see Fig. 5) and functions as a window discriminator in conjunction with waveform matching. The analog signal is digitized at 20,000 Hz and templates formed during an initial sampling period (60 s). Subsequently, the matching algorithm is engaged only if the digitized voltage levels reach a prespecified value. Spikes are then included in a template only if more than a user-defined percentage of the points in a spike fall within the template. The present study characterized a spike with 75 points. It then required that >60% of the points match, and that the difference in amplitude between template and spike be <20% for any potential to be counted. In two instances, Spike2 was used to separate the activity of two different action potentials recorded from the same site.
Stimulus delivery and protocols
The tongue was gently extended out from the oral cavity using the ventral tongue suture. A computer-controlled delivery system was used for taste stimulus and water presentation to the anterior tongue. For 10 s before and for longer after stimulus offset, a separate outflow nozzle delivered deionized-distilled water to the same locus on the tongue. During the rinse period, the stimulus delivery line and nozzle were flushed with water. Gustatory neurons were tested for responsiveness to 0.3 M sucrose, 0.1 M NaCl, 0.1 M NaCl mixed with 10 µM amiloride, 0.01 M citric acid (CA), and 0.003 M quinine hydrochloride (QHCl) using a water, stimulus, water procedure. Stimulus order varied from one recording session to another. Briefly, water was applied to the tongue for 10 s followed by tastant application for 10 s. The water flow that followed tastant application was for 90 s, and the total time between different stimulus applications was 2 min. The stimulation protocol was as follows: control series 1-test series-control series 2. Tastant application during a control series was without CeA stimulation. Prior to the test series, baseline activity in the absence of fluid flowing over the tongue was recorded during CeA stimulation. After another 2-3 min, the test series began and the tastants were applied during concurrent CeA stimulation. In most cases a neuron was held long enough to complete the second control series. This was done to assess the stability of the recorded neuron and to determine whether CeA stimulation produced any prolonged effects on gustatory responsiveness.
Data analysis
Corrected neural responses to a taste stimulus were calculated
by subtracting the 10-s discharge rate to each stimulus from its
preceding 10-s discharge rate to water. The 10-s response measure was
used to categorize individual neurons based on the stimulus that evoked
the greatest discharge. When a neuron was tested with both control
series 1 and 2, these response rates were averaged. During the test
series, seconds 1, 3, 5, 7, and 9 of tastant application correspond to
the time periods when pulse trains were delivered to the CeA, while
seconds 2, 4, 6, 8, and 10 correspond to the time periods between pulse
trains. Neurons in the present study were classified as inhibited or
augmented if these response measures differed from their corresponding
control rates by <25 or >25%, respectively. A difference score was
calculated by subtracting the test series response rates from the
control series response rates during seconds 1, 3, 5, 7, and 9 and 2, 4, 6, 8, and 10. The breadth of responsiveness was calculated according
to the formula H = K
pI log
pI, where K is a scaling constant (1.66 for 4 stimuli; sucrose, NaCl, CA, QHCl) and
pI is the proportion of the response
to each of the stimuli against the total response to all the stimuli
(Smith and Travers 1979
).
One- and two-way ANOVAs were performed to detect significant
differences between response rates. In some instances, post hoc contrast analyses (least significant difference) were used to determine
the source of statistically significant differences. One-sample
t-tests were used to determine whether the absolute difference scores differed from 0. A change in stimulus-evoked activity
was considered significant when it differed above or below the response
rate to water flow by 2.5 standard deviations. The results will be
shown as the mean ± SE. Data analyses were done using SPSS, and
P values <0.05 were considered significant.
Histological processing and analyses
At the end of an experiment, a small electrolytic lesion (5 µA for 30 s) was made at the site of the last recorded gustatory neuron. The rats were given a lethal dose of Nembutal (100 mg/kg ip) and perfused intracardially with 0.9% saline followed by 10% Formalin. The brain was removed, cut coronally in 50-µm sections using a freezing microtome, and stained with the cresyl Lecht violet. The lesion mark and stimulating electrode tracks were detected using a light-field microscope and the loci noted.
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RESULTS |
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Histology
Based on stereotaxic coordinates of the penetrations and the
marking lesions, gustatory neurons were isolated in the caudomedial quadrant of the PBN. Figure 1 shows a
photomicrograph of a coronal section through the PBN and line drawings
that correspond to three different levels of the PBN. The black arrow
indicates the marking lesion made following the recording of a
gustatory neuron; three electrode tracks are visible. The approximate
area in which taste-responsive neurons were isolated at various PBN
levels is illustrated by a filled oval in the line drawings
(Paxinos and Watson 1986). The present taste-responsive
zone matched closely the area reported in previous studies
(Nishijo and Norgren 1990
; Norgren and Pfaffmann 1975
; Scott and Perrotto 1980
). The
photomicrograph in Fig. 2 shows the track
of a stimulating electrode placed in the CeA. Neurophysiological data
in the present study were obtained from animals in which the
stimulating electrodes were histologically confirmed to terminate in
the CeA.
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Neuronal categorization
Fifty-one gustatory neurons were isolated in the PBN and tested
with the five taste stimuli with and without concurrent electrical stimulation in the CeA. The response profiles of these neurons are
shown in Fig. 3. The taste stimuli tested
and the activity of each neuron during the 10-s prestimulus water flow
are arranged from top to bottom and the different
neuron groups from left to right. Within each
group, neurons are arranged by their response rate to the best stimulus
in descending order. Neurons 1-3 were deemed sucrose-best,
neurons 4-35 NaCl-best, and neurons
36-51 CA-best. Although neurons 31-35 were
NaCl-best, they differed from the other NaCl-best neurons insofar as
their response to NaCl was insensitive to amiloride. They were termed
NaCl generalists (Lundy and Contreras 1999).
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The mean corrected response rates to each stimulus as a function of neuron type is shown in Fig. 4. Separate one-way ANOVA tests revealed a significant main effect for stimulus in NaCl-best (F4,135 = 42.3, P < 0.01) and CA-best neurons (F4,76 = 19.8, P < 0.01). Post hoc analyses indicated that the order of stimulus effectiveness for NaCl-best neurons was NaCl > NaCl + amiloride = CA = sucrose > QHCl. In the case of CA-best neurons, the order was CA > NaCl = NaCl + amiloride > sucrose = QHCl. Figure 4 also shows clearly that the epithelial Na+ channel antagonist amiloride was only effective in suppressing response rates to NaCl in NaCl-best neurons. In subsequent analyses, the 2 NaCl-best groups were combined because no clear differences were observed in terms of the effects of CeA stimulation on neural responsiveness.
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Amygdala stimulation
Figure 5 shows the response of a CA-best neuron to sucrose, NaCl, CA, and QHCl with and without concurrent CeA stimulation. The labels to the left of each raw record indicates the stimulus; the labels with the acronym CeA indicate stimulus applications during amygdala activation. With regard to the bottom record, the effect of CeA stimulation was assessed on the baseline activity when no fluid flowed over the tongue (Baseline/CeA). The first and third long black bar in each taste stimulus record corresponds to 10 s of water flow, and the second (middle) black bar is stimulus application. The short black bars in the records with the CeA acronym indicate the five 1-s pulse trains delivered to the CeA. The first three records show the responses to sucrose with and without concurrent CeA stimulation. To appreciate the typical signal-to-noise ratio obtained in the present study, the first two records are shown with the background noise included. Raw record 3 differs from record 2 only in that Spike2 was used to remove the background noise and extract the CeA stimulation artifacts (e.g., shorter vertical lines above pulse train marks in record 2), the spike data were unmodified. This figure shows that electrical pulses delivered to the CeA produced a significant reduction in the baseline and tastant-evoked discharge rates.
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NaCl-best neurons
Of the 32 NaCl-best neurons, electrical pulse trains delivered to
the CeA inhibited gustatory responses in 18, augmented responses in 5, and were ineffective in 9. Only the results for neurons inhibited
(amiloride sensitive and insensitive) and augmented (amiloride
sensitive) will be shown. For the neurons that were inhibited, Fig.
6A shows the responses to the
five stimuli applied before and after the applications during amygdala
stimulation. The discharge rates were nearly identical, indicating that
the recording was stable and CeA activation was without a prolonged influence on gustatory responsiveness
(F1,160 = 0.0, P = 0.99). During delivery of the pulse trains to the CeA, the response
rate to each stimulus was suppressed (Fig. 6B,
F1,176 = 10.9, P < 0.01). In contrast, the response rates to the taste stimuli between the pulse trains (seconds 2, 4, 6, 8, and 10) were comparable to the response rates during the same time periods in the control series (Fig.
6C, F1,176 = 0.2, P = 0.63). This indicates that the effect of CeA
activation rapidly reversed. The difference in response rates to the
taste stimuli and the effect of the pulse trains on baseline activity
during the same time periods are shown in Fig. 6D.
One-sample t-tests revealed that the baseline activity and
the discharge rate to each stimulus differed significantly from 0 only
during the pulse trains (P values 0.01). Although, the
degree of inhibition was comparable for spontaneous activity and
taste-evoked responses (F5,105 = 1.7, P = 0.12), CeA stimulation reduced the number of
neurons that responded significantly to sucrose from 10 to 5, to
NaCl/Amiloride from 16 to 14, to CA from 13 to 8, and to QHCl from 11 to 2.
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In another subset of NaCl-best neurons, CeA activation increased the responsiveness to NaCl. The stimulus-evoked responses in this group were stable between the two control series applications (Fig. 7A, F1,40 = 0.02, P = 0.87). Although the response rate of all five neurons to NaCl increased (range, 26-95%) during the pulse trains, the effect was not statistically significant (Fig. 7B, F1,50 = 1.1, P = 0.3). Analysis of the difference scores in Fig. 7D, however, showed that the increase in response rate to NaCl differed significantly from 0 during both concurrent CeA stimulation (t-test, P < 0.01) and between pulse trains (t-test, P = 0.01). A two-way ANOVA on the difference scores revealed a significant interaction between stimulus and measurement period (F5,60 = 6.1, P < 0.01). Post hoc analyses indicated that the increase in NaCl responsiveness between pulse trains was less than that during the pulse trains (P < 0.01). Interestingly, this amygdala-induced facilitation of NaCl-evoked responses was disrupted by amiloride and occurred in the absence of an effect on baseline activity. The number of neurons responsive to sucrose was reduced from three to two, to CA from four to two, and to QHCl from one to zero.
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CA-best neurons
With the exception of one unaffected neuron, the influence of
amygdala stimulation on CA-best neurons was always inhibitory. Before
and after CeA stimulation, the evoked responses were stable (F1,160 = 0.1, P = 0.72; Fig. 8A). Electrical
pulses delivered to the CeA dramatically suppressed the discharge rate
of PBN cells to each taste stimulus (Fig. 8B,
F1,142 = 22.3, P < 0.01). In fact, a post hoc analysis on the stimulus main effect during
CeA stimulation (F4,70 = 14.8, P < 0.01) indicated that the response rates to NaCl
and CA were no longer significantly different
(P = 0.26). During the time periods between pulse
trains, the response rates tended to recover to control levels (Fig.
8C, F1,142 = 2.7, P = 0.09). One-sample t-tests indicated that
the baseline activity and discharge rates to each tastant were
significantly different from 0 during the pulse trains (Fig.
8D, P values <0.01). The degree of
inhibition varied with stimulus effectiveness
(F5,85 = 4.6, P < 0.01); responses to CA were inhibited significantly more than the
responses to all other stimuli (P values 0.03) with the
exception of NaCl mixed with amiloride (P = 0.08).
Responses to NaCl, NaCl/Amiloride, and CA, but not to sucrose and QHCl, were inhibited to a greater degree than was the baseline activity. Between pulse trains, the inhibitory effect did not fully reverse when
NaCl or CA was the stimulus (t-test, P values
0.02). The residual inhibition of CA responses was significantly
different from sucrose, NaCl/Amiloride, QHCl, and baseline activity
(P values
0.01). As with NaCl-best neurons, the percentage
of CA-best neurons that responded to stimuli other than their best
stimulus was reduced during CeA stimulation. The number of neurons
responding to sucrose decreased from 10 to 3, to NaCl and
NaCl/Amiloride from 15 to 14, and to QHCl from 10 to 5.
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Breadth of responsiveness
The entropy measure (H value) ranges from 0 to 1. A value of
0 corresponds to a neuron that was activated by only one stimulus; a
value of 1, to a neuron activated equally by all stimuli. Amygdala stimulation reduced the entropy measure of each response category (F1,70 = 9.1, P < 0.01, Fig. 9A). Post hoc
analysis of the main effect for neuron type
(F2,70 = 10.7, P < 0.01) revealed that the H value was significantly lower for the
NaCl-best cells compared with the CA-best cells. Another way to measure
change in responsiveness in taste neurons is the percentage of overall
evoked activity elicited by each of the sapid stimuli (Fig. 9,
B-D). For the inhibited NaCl-best neurons, CeA stimulation
increased the percentage of the response produced by NaCl (60-78%)
and correspondingly decreased it for the other stimuli (Fig.
9B; sucrose, 11 to 1%; CA, 20 to 6%; QHCl, 9 to
3%).
Proportional responses did not differ significantly in the NaCl-best
neurons augmented (Fig. 9C, F1,40 = 0.0, P = 0.98), or in the CA-best neurons inhibited by CeA
stimulation (Fig. 9D, F1,96 = 0, P = 0.96).
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DISCUSSION |
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We have shown that electrical stimulation in the CeA
differentially controls gustatory neurons in the PBN. The present
findings are the first to demonstrate that a subcortical forebrain area can modulate tastant-evoked responses in brain stem gustatory neurons.
Prior studies have demonstrated that the lateral hypothalamus (LH)
(Bereiter et al. 1980; Matsuo and Kusano
1984
; Matsuo et al. 1984
; Murzi et al.
1986
) and the gustatory cortex (GC) (Smith and Li
2000
) modulate taste-responsive neurons in the nucleus of the
solitary tract (NST). Stimulation of the GC (Di Lorenzo and
Monroe 1992
), but not the LH (Murzi et al.
1986
), also has been shown to influence gustatory cells in the
PBN. Descending activity was reported to be inhibitory, excitatory, or
without effect on ongoing spontaneous discharge. Although modulation of taste-evoked responses was not assessed, these studies indicate that
the stimulation sites in the LH and the GC, which receive afferent
gustatory information, generate descending activity in the same system.
Our data are consistent with these findings and extend them by showing
how a different forebrain region modulates gustatory evoked responses
in the PBN. Similar to the LH and the GC, the CeA receives taste
information from the PBN, and the PBN, in turn, receives a reciprocal
connection from the CeA (Halsell 1992
; Norgren
1976
).
Coding
Given what is known about the central anatomy of the gustatory system, both the site and parameters of electrical stimulation were largely arbitrary. Nevertheless, we observed considerable effects, and the changes were coherent. Amygdala stimulation inhibited the spontaneous activity and the response to each taste stimulus in 18 of the 32 NaCl-best neurons and 14 of the 15 CA-best neurons. In NaCl-best neurons, the degree of inhibition on spontaneous activity and responses to the five sapid stimuli was similar, but in CA-best neurons, inhibition was greatest for the most effective stimuli (e.g., CA and NaCl). When CeA stimulation facilitated responses in NaCl-best cells (5 of 32), the effect was specific to that sapid stimulus; spontaneous activity and the responses to sucrose, CA, QHCl, and NaCl mixed with amiloride were unaffected. The remaining NaCl-best neurons (9 of 32) were not influenced by electrically stimulating the CeA. The overall effect of CeA stimulation was to sharpen the gustatory response profiles in PBN neurons.
Specifically, the breadth of responses in NaCl-best and CA-best
neurons was reduced during concurrent CeA stimulation, indicating that
these neurons increased their chemical selectivity. This shift in
chemical sensitivity was most pronounced in the NaCl-best neurons that
were inhibited by forebrain stimulation. In this subset of neurons,
sucrose, CA, and QHCl became relatively less effective during CeA
stimulation, and the percentage of the total response profile produced
by NaCl increased from 60 to 78%. At least as far as the present
NaCl-best cells are concerned, the entropy measure during CeA
stimulation (0.45 ± 0.05, mean ± SE) was very near the
value calculated for NaCl-specific PBN cells in awake behaving rats
(0.39 ± 0.05) (Nishijo and Norgren 1990). In an
anesthetized preparation, activity in the CeA modulated NaCl-best
neurons in such a way that NaCl became a more salient stimulus. In
contrast, the inhibitory effect of CeA stimulation in CA-best neurons
disrupted the differential responsiveness to CA and NaCl that is
typical of this category. The small number of sucrose-best neurons
(n = 3) precludes any conclusion concerning the
influence of CeA activation.
Possible neural connections
The differential effect of CeA stimulation on NaCl-best PBN neurons provides a hint of how forebrain activity might modulate the central gustatory system. The gustatory-evoked responses of different NaCl-best neurons isolated in the same preparation were inhibited, facilitated, or unaffected during electrical stimulation of the CeA. Modulation of spontaneous activity might underlie the inhibitory effect on gustatory responses because the degree of inhibition was similar in the presence and absence of a sapid stimulus on the tongue. In contrast, with regard to NaCl, the excitatory effect was dependent on taste-evoked activity. This differential effect of CeA stimulation on NaCl-best cells is further distinguished by the suppressive effect of amiloride on NaCl responses. The inhibited gustatory neurons included both amiloride-sensitive and amiloride-insensitive cells, while the facilitated gustatory neurons included only amiloride-sensitive cells. The excitatory effect, but not the inhibitory effect, of CeA stimulation on NaCl-evoked activity was disrupted by amiloride suppression of NaCl responses. One interpretation is that a certain amount of afferent activity might be required to engage the excitatory descending influence and that this is normally achieved only with above threshold concentrations of NaCl.
The only monosynaptic projections from the amygdala to the brain stem
originate in the CeA (Halsell 1992; Veening et
al. 1984
). Nevertheless, electrical stimulation cannot
differentiate between activation of intrinsic neurons and fibers of
passage. Using small infusions of an excitotoxin that does not activate
fibers of passage could test this possibility. If one assumes that
fibers of passage were involved, then the activation of the CeA could
have either influenced PBN neurons through direct synaptic contact or
through an indirect projection via some other forebrain region. As
already mentioned, the PBN distributes axons at least to the
hypothalamus, amygdala, and bed nucleus of the stria terminalis, and,
in turn, receives projections from each of these brain structures.
Moreover, these more rostral regions are connected with each other (see Norgren 1985
for review; Halsell 1992
;
van der Kooy et al. 1984
; Yamamoto et al.
1984
). The dissociation between these two alternative pathways
might be tested by comparing gustatory-evoked responses in the PBN
during CeA activation with and without concurrent inactivation in each
of these other rostral structures. A final possibility is that the
effects we observed in the PBN were first established in the NST and
then transmitted rostrally to the PBN. In a prior study, stimulation in
the LH modulated NST gustatory neurons, but not those in the PBN
(Murzi et al. 1986
). Any inferences drawn from these
earlier data must be tempered by the small sample of PBN neurons tested
(n = 12), and the obvious fact that centrifugal influences on brain stem gustatory neurons may differ between the LH
and the CeA. A more direct test would be to repeat the present study,
but record from gustatory neurons in the NST.
Other visceral sensory modalities also are subject to centrifugal
modulation. Many cardiovascular-sensitive cells in both the NST and PBN
respond to electrical stimulation in the hypothalamus and the amygdala
(Cechetto and Calaresu 1983; Cox et al.
1986
; Mifflin et al. 1988
; Silva-Carvalho
et al. 1995
). In the case of hypothalamic stimulation, afferent
activity evoked in the NST by inflating a balloon in the descending
aorta or the carotid sinus was either inhibited, facilitated, or
unaffected (Silva-Carvalho et al. 1995
). Interestingly,
the efficacy of afferent input was facilitated in two NST neurons
without a synaptic response (e.g., excitatory postsynaptic potential)
to the hypothalamic stimulation itself. The same types of influence,
exerted by corticofugal input, have been reported in thalamic neurons
that respond to auditory and somatosensory stimulation (Ghosh et
al. 1994
; He 1997
; Villa et al.
1991
). In the present study, electrical stimulation of the CeA
also had variable effects on somatosensory cells in the dorsal pons
(n = 15, data not shown). The spontaneous activity was
unaffected (n = 11), inhibited (n = 2),
or facilitated (n = 2).
Implications for sodium appetite
The central nucleus of the amygdala (CeA) participates in the
maintenance of body fluid balance. For instance, c-fos
expression in the central and medial divisions of the amygdala is
increased by peritoneal dialysis-induced sodium depletion
(Johnson et al. 1999). This immunocytochemical index of
neural activation indicates that cells in these two amygdala subnuclei
responded to the change in sodium balance. In addition, bi-lateral
lesions of the CeA disrupt the sodium appetite that develops following
furosemide (subcutaneous), DOCA (subcutaneous), or renin
(intracerebroventricular) injection (Galaverna et al.
1991
; Zardetto-Smith et al. 1994
). The role of
the gustatory system is to carry the neural information that signals
the presence of the Na+ ion. Specifically, the
amiloride-sensitive NaCl-best neurons are indispensable for the
discrimination between sodium and nonsodium salts (Spector et
al. 1996
) and for the expression of sodium appetite (Bernstein and Hennessy 1987
; Breslin et al.
1993
; McCutcheon 1991
).
One function of centrifugal projections to sensory nuclei might be to
extract information from the incoming barrage based on current demands.
A few electrophysiological studies have demonstrated that the induction
of a sodium appetite alters the sensitivity of the gustatory system to
sodium (Contreras and Frank 1979; Nakamura and
Norgren 1995
; Tamura and Norgren 1997
). Whether
sodium sensitivity is increased or decreased, however, seems to depend
on the method of inducing the sodium need state. For instance, dietary
sodium deprivation reduced the responsiveness to NaCl in both chorda tympani (Contreras and Frank 1979
) and NST
(Nakamura and Norgren 1995
) NaCl-best neurons. In
contrast, a sodium appetite induced with the diuretic furosemide
increased specifically the response of NaCl-best NST neurons to NaCl
(Tamura and Norgren 1997
). Our findings compliment these
prior neurophysiological studies insofar as the activation of the CeA,
a region known to be responsive to changes in sodium balance, also
alters gustatory sensory processing of sodium. Activation of the CeA
inhibited the responsiveness of some NaCl-best neurons to NaCl and
other sapid stimuli (e.g., dietary sodium deprivation), while in other
NaCl-best neurons the response to NaCl was increased selectively (e.g.,
furosemide sodium depletion).
Although the effect of this motivational state on gustatory
sensory processing in the PBN has not been examined, the anatomical connections of the CeA are well suited to modulate gustatory
information processing in the PBN based on physiological demand.
Despite a lack of knowledge about how rostral brain structures might
interpret this altered gustatory information, the present results
provide clues to a neurophysiological relationship between the CeA and the PBN in the control of NaCl intake. This centrifugal modulation might serve to increase the relative detectability and motivational properties of the Na+ ion. Indeed, electrical
stimulation in the amygdala of awake-behaving rats has been shown to
alter saline preference (Gentil et al. 1971). The
direction of the change in preference, however, seems to depend on the
particular amygdaloid subnuclei that are stimulated.
Summary
The present study demonstrated that a subcortical forebrain area modulates pontine gustatory neurons. In 38 of 51 PBN gustatory cells, descending activity generated in the CeA influenced taste-evoke responses. Specifically, taste responses were inhibited in 33 cells and facilitated in another 5 cells. This centrifugal modulation increased the chemical selectivity of pontine taste cells, particular with regard to NaCl-best cells. The NaCl message transmitted through this subset of neurons was accentuated, which tends to mimic the changes in NST taste cell responsiveness produced by a motivational state, sodium appetite. Thus the gustatory neural code is not rigid, but subject to modulation by visceral signals and experience that likely involves centrifugal input from the forebrain. In this context, we have identified one forebrain area, the central nucleus of the amygdala, that participates in endocrine functions, receives afferent gustatory input, and generates descending activity that alters gustatory sensory processing in the dorsal pons.
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ACKNOWLEDGMENTS |
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This research was supported by National Institutes of Health (NIH) Grants DC-00369, DC-00240, and MH-43787. R. Norgren was supported by NIH Senior Scientist Award MH-00653.
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FOOTNOTES |
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Address for reprint requests: R. F. Lundy Jr., Dept. of Behavioral Science, Penn State College of Medicine, 500 University Dr., Hershey, PA 17033 (E-mail: rfl6{at}psu.edu).
Received 15 July 2000; accepted in final form 30 October 2000.
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REFERENCES |
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