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RATS RELEARN A SODIUM CHLORIDE VS. POTASSIUM CHLORIDE
DISCRIMINATION AFTER CHORDA TYMPANI TRANSACTION
USING REMAINING GUSTATORY INPUT
A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
2007 Ginger Blonde
TABLE OF CONTENTS
L IS T O F T A B L E S ........................................................................... 4
LIST OF FIGURES .................................. .. ..... ..... ................. .5
A B S T R A C T ......... ....................... .................. .......................... ................ .. 6
1 INTRODUCTION ................................................... ..............................8
2 M E T H O D S ............................................................. ...... .......................................... 1 1
S u b je cts ...................................................................................................... . ...... 1 1
A p p a ra tu s ................................................................ ................................................1 1
T ria l S tru ctu re ................................................................................................................... 12
T raining ....... ............................................................... 12
P re su rg ic al T e stin g ........................................................................................................... 13
Postsurgical Testing................................................. 14
S u rg e ry ................... ...................1...................4..........
H isto lo g y ................... ...................1...................5..........
D ata A analysis ................................................... 16
3 R E S U L T S ..............................................................................................18
H isto lo g y ................... ...................1...................8..........
B behavioral Testing ................................................. 18
W after C o n tro l T e st ........................................................................................................... 19
4 D IS C U S S IO N ........................................................................................................2 5
L IST O F R E F E R E N C E S ..................................................................................................... 3 1
B IO G R A PH IC A L SK E T C H ................................................................................................... 36
2-1 Training and Testing Param eters ............................................... ............................ 17
LIST OF FIGURES
3-1 Overall performance during presurgical testing ..................................... .................21
3-2 Overall performance during initial postsurgical testing..................................................22
3-3 Overall performance during postsurgical control and amiloride sessions.........................23
3-4 W after control test .................. .................. ................. ............ .. ............. 24
Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science
RATS RELEARN A SODIUM CHLORIDE VS. POTASSIUM CHLORIDE
DISCRIMINATION AFTER CHORDA TYMPANI TRANSACTION
USING REMAINING GUSTATORY INPUT
Chair: Alan Spector
When the chorda tympani nerve (CT), which innervates taste buds in the anterior tongue,
is transected, the ability of rats to discriminate sodium from nonsodium salts is severely affected
but remains above chance. In a recent study, rats lacking input from the CT were initially
impaired but able to significantly improve in an NaCl vs. KC1 discrimination with continued
testing. This study was designed to determine what remaining gustatory input supported the
improvement. Rats were presurgically trained and tested in a two-response operant task to
discriminate NaCl and KC1 on the basis of taste. They were then split into five groups: CT
transaction, (CTx, n=7); CT and glossopharyngeal nerve (GL) transaction, (CTx+GLx, n=4); CT
and greater superficial petrosal nerve (GSP) transaction, (CTx + GSPx, n=8), or sham surgery
(SHAM, n=7; and SHAM-INT, n=7). SHAM-INT rats were tested postsurgically with lower
stimulus concentrations to determine whether the decrease in performance was due to a reduction
in stimulus intensity after surgery. In the first week of postsurgical testing, performance by the
rats with nerve transactions was impaired. However, over the course of six weeks, the CTx and
CTx+GLx groups were able to increase their overall percentage correct to near-presurgical levels
and were affected by amiloride similarly to SHAM rats. The CTx + GSPx group never
improved. Also, the SHAM-INT group performed similarly to the SHAM group throughout
postsurgical testing. Thus, it does not appear that the effect of nerve transactions on performance
can be explained by lower stimulus intensities after surgery. It instead seems that the taste
quality of one or both of the salts changes after CT transaction. However, the signal arising from
the GSP, but not the GL, is sufficiently different between the two salts to allow the rats to learn a
The chorda tympani nerve (CT), a gustatory branch of the seventh cranial nerve that
innervates taste buds in the anterior tongue, has been shown to be important for normal taste-
guided behavior related to salts. When the CT is transected, detection of salts, particularly NaC1,
discrimination of sodium versus nonsodium salts, expression of sodium appetite, and ingestive
oromotor responses are impaired (e.g., 1-14). In some cases, NaCl preference is also impaired
after CT transaction in the rat (e.g., 15-16), but it has generally been unaffected by the
denervation when measured by intake tests (e.g., 14, 17).
Electrophysiological findings show that in rodents the CT is highly responsive to salts,
especially sodium salts (e.g., 18-25). A subpopulation of nerve fibers in the rodent CT, called N-
fibers, responds robustly and specifically to sodium salts relative to other tastants. In contrast, a
separate subpopulation called H-fibers is more electrolyte-general, responding to both sodium
and nonsodium salts as well as acids and quinine (26-30). Additionally, taste buds in the
fungiform papillae contain epithelial sodium channels (ENaCs) that are thought to be the primary
transduction mechanism for sodium detection. Lingual application of amiloride, which blocks
ENaCs, diminishes responsiveness of the CT in general, and N-fibers in particular, to sodium
salts. There seems to be little effect on the CT response to KC1, or responsiveness of H-fibers,
with the addition of amiloride. The distinct N-fiber transmission pathway specifically
representing sodium in the periphery, as compared to the more general H-fiber pathway for
electrolytes, seems to be the basis for NaCl vs. KC1 discriminability, and the CT incorporates
both pathways (e.g., 28-29, 31-33). The results of behavioral and electrophysiological studies all
suggest that the CT is important for salt detection and discrimination in rats.
Most of the behavioral studies described above tested animals over a short period of time
postsurgically. In one previous study in our laboratory, rats with a bilaterally transected CT
performed poorly postsurgically in a NaCl versus KC1 discrimination, as seen in other
experiments. However, with longer testing, some rats were able to improve their overall
performance in the task, approaching presurgical levels (34). This suggests that the remaining
gustatory input is capable of providing sufficient information to the rat, allowing it to accurately
perform the discrimination when provided adequate postsurgical testing to relearn the task.
Indeed, even after CT transaction, some degree of sodium detectability remains. It has
been suggested that the greater superficial petrosal nerve (GSP), the branch of the seventh cranial
nerve that innervates taste buds in the nasoincisor ducts, geshmacksstreifen, and soft palate in the
rat, is responsible for the remaining ability of rats to perform these tasks after CT transaction.
Electrophysiological recording experiments have shown that the GSP also responds well to salts,
although it responds best to sugars (22, 35-36). Additionally, the taste bud fields innervated by
the GSP seem to contain ENaCs; lingual application of amiloride decreases overall GSP
responsiveness to sodium salts (36-37).
The results of behavioral studies also suggest that the GSP has a role in taste-guided
behaviors related to sodium. After combined CT and GSP transaction, eliminating all gustatory
input from the seventh cranial nerve, the detection threshold for NaCl and performance on salt
discrimination tasks are more affected than when the CT is transected alone (38-39).
In contrast to the CT and GSP, the glossopharyngeal nerve (GL), a branch of the ninth
cranial nerve which innervates the taste buds of the posterior tongue, has been shown to be
unnecessary in behavioral detection or recognition of sodium by the rat. After the GL is
transected, there is no effect on the specificity or vigor of a depletion-induced sodium appetite,
salt discrimination performance, or intensity difference thresholds for NaCl (13, 40-42).
Electrophysiological findings demonstrate that the GL responds best to bitter-tasting stimuli such
as quinine hydrochloride. It has a weaker response to sodium salts (25, 43-45), and it has been
suggested that the circumvallate taste buds of the rat have no functional ENaCs on the basis that
application of amiloride has no effect on GL responsiveness (46-47; but see 48).
This study was designed to explicitly test whether the GSP, considering its proposed
contribution to salt taste, allowed the rats with CT transaction in our previous study to improve
performance in a NaCl versus KC1 discrimination postsurgically. We also tested whether the
GL, which was also left intact in those rats with CT transaction, was providing sufficient
information to support recovery of performance over the postsurgical testing period. A control
group was included in the design that allowed us to assess whether the decrease in
discriminability is related to a decrease in intensity of the stimuli that would follow after some of
nerve transaction surgeries. We hypothesized that given the electrophysiological response
properties of the GSP along with the fact that combined CT and GSP transaction often has
greater effect on performance than transaction of the CT alone, it is likely that the GSP, and not
the GL, is responsible for the level of performance seen after CT transaction.
Thirty-six Sprague-Dawley rats served as subjects. The rats were housed individually in
polycarbonate cages during training and testing. While recovering from surgery, rats were
housed in stainless-steel hanging wire cages. The room was maintained with a 12-hr light-dark
cycle with temperature automatically controlled; lights came on at 6:30 a.m. Rats had ad-lib
access to laboratory rodent chow (Rodent Diet 5001; PMI, Brentwood, Missouri) in their home
cages. For one week prior to the start of training, they were given oil mash (5 parts powdered
rodent chow to 2 parts vegetable oil) in preparation for the presentation of this palatable and
lubricated diet during postsurgical recovery. During training and testing, water bottles with
purified water (Millipore Elix 10; Bellerica, Massachusetts) were available Friday afternoon after
the last testing session, until Sunday afternoon when they were removed. Monday through
Friday during training and testing, the rats obtained purified water as part of their 40-minute
testing sessions. Throughout testing, no rat ever dropped below 85% of its free-feeding body
weight. All procedures were approved by the Institutional Animal Care and Use Committee at
the University of Florida.
Training and testing took place in a gustometer described in Blonde et al, 2006 (38).
Briefly, rats were placed in a sound attenuation chamber fitted with a central sample spout
positioned behind an access slot and two reinforcement spouts, one to either side of the sample
spout. A cue light was placed above each reinforcement spout. Stimuli were contained in
pressurized reservoirs connected to solenoid valves calibrated to deliver -5 tL of fluid, and were
delivered through the sample spout after it was initially filled at the start of a trial. After each
trial, the sample spout was rotated over a funnel, rinsed with purified water and blown dry.
Each trial began when the rat licked the sample spout twice in 250-ms, to ensure that the
rat was engaged in spout licking when the stimulus was delivered. During the sample phase, the
rat was allowed 5 licks of the stimulus or 3-s, whichever came first. The decision phase began
immediately: the sample spout rotated away from the access slot, the house lights were
extinguished and the cue lights illuminated. The rat had 5-s (limited hold) to lick from one of the
two reinforcement spouts. If the rat responded correctly, it received up to 10 licks of purified
water in 8-s from the reinforcement spout. During amiloride sessions, 100 iM amiloride
replaced water in the reinforcement spouts. If the rat responded incorrectly or failed to respond
during the limited hold, it received a 30-s time-out, when all lights were extinguished and it had
no access to water. The reinforcement or time-out was followed by a 6-s intertrial interval,
during which the sample spout was rinsed and dried. The sample spout was then rotated back to
the access slot, the house lights were turned on, and the animal could initiate another trial. The
rats were allowed to initiate as many trials as possible during each 40-min session.
The rats were trained to associate one reinforcement spout with 0.2 M NaCl and the other
with 0.2 M KC1 (both reagent grade chemicals; Fisher Scientific, Orlando, FL). The assignment
was counterbalanced, so that for half of the rats the left spout represented NaC1, and for the other
half it represented KC1. The training and testing schedule is depicted in Table 2-1.
Spout training. The rats were trained to lick each spout. One 40-min session was
devoted to each spout (sample, left reinforcement, right reinforcement), and rats received
purified water ad libitum.
Side training. The rats were trained to associate one reinforcement spout with NaCl and
the other with KC1. In each session, only one stimulus was presented via the sample spout, and
only the reinforcement spout associated with that stimulus was available. The stimulus presented
and the reinforcement spout available was alternated each day.
Alternation. For this phase, both 0.2 M NaCl and 0.2M KC1 were presented in each
session. The rat was required to make a predetermined number of correct responses to each
stimulus before the other stimulus was presented. This criterion number was reduced
systematically over three sessions, from 6 responses to 2.
Discrimination Training I. In this phase, 0.2 M NaCl and 0.2 M KC1 were presented
randomly. The probability of receiving either stimulus was 0.5. The rat was required to perform
>80% overall on trials with a response to move on to the next phase of training.
Discrimination Training II-III. The number of stimuli was increased to six. The
limited hold was systematically reduced, and the time-out was increased. As in Discrimination
Training I, the rat had to perform >80% overall to move on to the next phase.
During presurgical testing, the rats were tested on Monday, Wednesday, and Friday with
0.1 M, 0.2 M, and 0.4 M NaCl and KC1 in control sessions. For the Tuesday and Thursday
sessions, the salt stimuli were dissolved in 100lM amiloride. Testing was conducted for two
weeks, for a total of six control sessions and four amiloride sessions.
Testing resumed 28-30 days after surgery. For four weeks, animals were tested Monday
through Friday in the same manner as during presurgical control sessions. During weeks 5 and 6
of postsurgical testing, the rats were tested identically to presurgical testing. Three days after the
last day of testing, a water control test was conducted in which all reservoirs were filled with
water. This test helped to determine whether the performance of the rats relied on chemical cues
of the stimuli as opposed to other extraneous cues.
Surgical groups were balanced by overall performance, performance to each salt, body
weight, and gustometer. All rats were anesthetized with an intramuscular injection of ketamine
hydrochloride (125 mg/kg body weight) mixed with xylazine hydrochloride (5 mg/kg body
weight). For rats receiving bilateral CT transaction (CTx), the external auditory canal was
retracted and the tympanic membrane was punctured. The chorda tympani was exposed and
transected where it disappears behind the malleus. The ossicles were removed, and the
remaining tympanic membrane as well as the surrounding rim of the ear canal was cauterized,
which generates the production of cerumin that fills the middle ear to reduce the likelihood of
nerve regeneration. For rats receiving bilateral CT and GL transaction (CTx + GLx), the above
procedure was performed to transect the CT. Then, to transect the GL, we retracted the
sublingual and submaxillary salivary glands and the sternohyoid, omohyoid, and posterior belly
of the digastic muscles. The fascia underlying the hypoglossal nerve was dissected near the
external medial wall of the bulla to expose the GL. It was cut with microscissors and
approximately 10 mm of the nerve was removed. The incision was closed with nylon sutures.
For rats receiving bilateral combined CT and GSP transaction (CTx + GSPx), an incision
was made dorsal to the pinna and the external ear was retracted, exposing the ear canal. A small
slit was made in the canal. The fascia was dissected to expose the auditory meatus, and the
surrounding musculature was blunt dissected and retracted. The opening of the bony meatus was
enlarged with a high-speed pneumatic dental drill. The tympanic membrane, ossicles, and CT
were removed. The tensor tympani muscle and a small piece of temporal bone were then
removed with microforceps, exposing the GSP. It was transected, and the ends were cauterized.
The incision was closed with wound clips.
The remaining rats received sham surgery (SHAM, and SHAM-INT). For these animals,
the GL was exposed as described above but not transected. Additionally, an incision dorsal to
the pinna was made, the external ear was retracted, a slit was made in the ear canal, and the
tympanic membrane was punctured.
During recovery, rats were individually housed in hanging wire cages until their incisions
had healed. Each animal received Penicillin G Procaine suspension (30,000 units sc) and
ketorolac tromethamine (2 mg/kg body mass sc) for 3 days following surgery. All rats also
received a supplemental diet of oil mash (5 parts powdered 5001 chow to 2 parts oil) and diluted
sweetened condensed milk (1:1 Carnation sweetened condensed milk to water with 1 mL
Polyvisol multivitamin supplemental drops, Enfamil, Eversville, IN). This diet of oil mash and
milk was provided until the rats had recovered, demonstrated by a stable body weight of at least
90% of an animal's presurgical weight, which took a minimum of 5 and a maximum of 25 days.
Two to three days after the water control test, the rats were deeply anesthetized with
sodium pentobarbital (60 mg/kg, ip) and transcardially perfused with saline, followed by 10%
buffered formalin. The tongue, soft palate, and nasoincisor ducts of each rat were removed and
stored in 10% buffered formalin. The anterior portion of the tongue from the intermolar
eminence to the tip was placed in purified water for 20 min, dipped in 0.5% methylene blue until
saturated (-30 sec), then rinsed with purified water. The epithelium was removed from the
underlying tissue, pressed between two slides, and the total number of fungiform papillae and
taste pores were counted under a light microscope. The foliate and circumvallate papillae and
the nasoincisor ducts were embedded in paraffin and cut into 10 .im sections. These sections
were mounted on slides, stained with hematoxylin and eosin, and taste buds were counted under
the microscope. All tissue sections were coded so that the counters were uninformed of the
The overall percentage correct for trials with a response was calculated. Individual
performance was calculated for control and amiloride sessions (separately for both presurgical
and postsurgical testing). Performance during the first four weeks of postsurgical testing was
compiled by week (4-5 sessions per week) for each animal. These data were compared by group
using matched t-tests and analyses of variance (ANOVAs) with Tukey's honestly significant
difference procedure test. Since the probability of the presentation of either salt was 0.5, chance
performance is considered an overall percentage correct of 50%. Group performance was
compared to chance levels using one-sample t-tests. The performance of each rat on the water
control test was statistically tested for positive differences from chance with the one-tailed
normal approximation of the binomial distribution.
Table 2-1. Training and testing parameters
0 0.2M NaCl or KCl
10 0.2M NaC1 and KCl
10 0.2M NaC1 and KC1
20 0.1, 0.2, and 0.4 M NaCl and KCl
30 0.1, 0.2, and 0.4 M NaCl and KCl
0.1, 0.2, and 0.4 M NaCl and KCl
Presurgical 10 5 30 [with and without 100[tM amiloride] Semi-random
0.1, 0.2, and 0.4 M NaCl and KCl
Postsurgical 29-31 5 30 [with and without 100lM amiloride] Semi-random
Note: LH = limited hold, the amount of time (in seconds) the rat has to respond after sampling
the stimulus. TO = time out (in seconds) for incorrect responses. a: the alternation criteria used:
6, 4, 2. b: Stimuli were presented in randomized blocks of 6 without replacement.
Stained tissues were semi-quantitatively analyzed to determine the success of surgery. If
it appeared that the taste bud field was not intact (i.e., the nerve innervating it had been
transected), the number of taste buds were counted. Three rats in the CTx + GLx group were
removed because the anterior tongue had more than 30% of its fungiform papillae intact,
indicating some level of CT regeneration in those animals. Their data were not included in the
analyses. One rat in the CTx + GSPx group had 20 taste buds in the nasoincisor ducts; however,
based on its performance it does not appear that the taste buds were sufficient to support
discrimination. The statistical outcomes of the analyses were unaffected by whether this rat was
included or not; therefore, that rat was not discarded. After discarding rats on histological
grounds the final group sizes were CTx, n=7; CTx + GLx, n=4; CTx + GSPx, n=8; SHAM, n=7;
During presurgical control sessions, all groups were performing at high levels overall
(left panel, Figure 1). Even after the removal of rats due to nerve regeneration, there was no
difference between groups during presurgical control sessions, with no main effect of group in a
two-way ANOVA (F(4,28)=1.87, p=0.144). As expected, the addition of amiloride significantly
decreased performance presurgically (main effect of AMILORIDE: F(1,28)=1927.509, p<0.001;
no effect of the interaction GROUP x AMILORIDE: F(4,28)=0.332, p=0.854; right panel, Figure
1), although performance for the SHAM and CTx groups were significantly higher than chance
(p>0.05 for both).
In the first week of postsurgical testing (i.e., the first 5 sessions), all groups with nerve
transactions were significantly impaired compared to their presurgical performance (all p-
values<0.05; Figure 2). Additionally, rats with CTx and CTx + GLx surgeries were performing
at similar levels to each other (p>0.05). The performance of rats with CTx + GSPx surgeries was
at chance (p = 0.781) and significantly lower than that for CTx and CTx + GLx rats (p<0.021),
suggesting that the GSP was contributing to performance in this discrimination. Rats in the
SHAM-INT group, however, were performing similarly to SHAM animals, despite being tested
with concentrations that were 1.0 logo unit lower (p>0.05). Their performance suggests that the
impairment seen in rats with nerve transactions is not simply due to a reduction in stimulus
intensity after surgery.
By the second week of testing, rats in the CTx and CTx + GLx groups had significantly
improved compared to their performance in the previous week (p<0.035 and p<0.004,
respectively), though those groups still performed below presurgical levels (p<0.05 for both).
They continued to improve slightly throughout postsurgical testing. These two groups were
never significantly different from each other. The CTx + GLx group actually reached
presurgical levels by the start of amiloride testing in Weeks 5 & 6 (p>0.2). Rats in the CTx +
GSPx group, on the other hand, remained at or near chance levels throughout postsurgical testing
(p>0.05 for Postsurgical Weeks 1-4; p = 0.02 during Postsurgical Control sessions; see Figures 2
& 3). As with presurgical testing, all groups performed at or near chance levels when tested with
amiloride (Figure 3). Statistically speaking, the SHAM group was significantly above 50%
overall during amiloride sessions (p<0.001 and p<0.03, respectively), but these groups were
clearly impaired when performance was compared to control sessions (p<0.05 for both).
Water Control Test
Two rats performed significantly better than chance on the water control test (Figure 4).
Performance for those rats, however, was significantly lower than their performance when tested
with chemical stimuli and was very close to chance levels. Also, considering that the CTx +
GSPx rats were all performing at chance levels, it is unlikely that the rats were using a consistent
extraneous cue to guide performance.
Figure 3-1. Performance during presurgical testing.
Overall percentage correct (+ SE) by group for presurgical control sessions (left panel) and
amiloride sessions (right panel). *: group performance was significantly higher than chance
(50% overall) during amiloride sessions.
I II A
-A^' o~o c cA^ o^< ^
st^Ds\ 0^ ^' 0^
FIRST FOUR WEEKS
-- CTx + GLx
1 2 3 4
Figure 3-2. Overall performance during initial postsurgical testing.
Mean performance (+ SE) by group on control sessions during postsurgical testing weeks 1-4.
Figure 3-3. Overall performance during postsurgical control and amiloride sessions.
Mean overall performance (+ SE) by group during postsurgical control sessions (left panel) and
amiloride sessions (right panel). *: group performance was significantly above 50% overall
during amiloride sessions.
WATER CONTROL TEST
Figure 3-4. Water control test. Overall performance for individual rats during the water control
test. *: performance was significantly greater than 50% overall.
The recovery from the impaired performance in salt discrimination, caused by CT
transaction, that occurs with prolonged postsurgical testing clearly depends on the input of the
GSP, not the GL. Not only were CTx + GLx rats able to significantly improve over the six
weeks of postsurgical testing, their overall performance level increased at the same rate as rats in
the CTx group, with an intact GL. This indicates that the GSP is supplying the signal that rats
use to discriminate NaCl from KC1 after input from the CT is lost. Additionally, when rats
lacked input from both the CT and the GSP, they performed more poorly than the CTx and CTx
+ GLx groups, and their performance remained virtually at chance levels during the six weeks of
testing. Thus, the GSP is both necessary and sufficient for the rats to improve in this salt
discrimination task when the CT has been damaged. One caveat, however, is that the superior
laryngeal nerve (SLN, a branch of CN X that in rodents innervates taste buds in the laryngeal
epithelium; see 49-50) remained intact in these rats. Because the CTx + GSPx group performed
at or near 50% throughout postsurgical testing, the SLN is clearly not sufficient for this
discrimination. However, it might play a role, along with the GSP, to support the recovery of
performance shown by the CTx and CTx + GLx groups. Based on the position of the taste buds
it innervates and its general response properties, it has been suggested that the primary role of the
SLN is the protection of the airway (see 51). Although for the reasons stated it is unlikely that
the SLN is contributing to the improvement seen in this study, its contribution cannot be entirely
ruled out at this time.
The chance performance by CTx + GSPx rats in this discrimination corresponds with
other studies done by our laboratory. Rats with this combined CT and GSP transaction also
performed at chance levels when tested with a NH4C1 vs. KC1 discrimination (39). Also, while
still able to detect very high concentrations of NaC1, rats lacking input from both the CT and the
GSP have a higher NaCl detection threshold than rats missing only the CT (38). The results of
this study therefore add to the suggestion in the literature that the seventh cranial nerve is
essential for normal detection and discrimination of gustatory stimuli (see 52).
Additionally, the overall improvement shown by CTx and CTx + GLx rats in this study
seems to require the amiloride-sensitive transduction pathway. With the addition of amiloride,
performance by the CTx and CTx + GLx groups significantly decreased. Moreover, it decreased
to the level seen during presurgical amiloride sessions. This corresponds with the other literature
which suggests that amiloride-sensitive ENaCs are crucial for normal sodium discrimination.
Unlike the GSP, however, the GL seems to be neither necessary nor sufficient. Rats in
the CTx and CTx + GLx groups improved at the same rate, never differing significantly from
each other, suggesting that the GL is not providing a necessary signal. Rats with an intact GL
but without an intact CT and GSP performed at or near chance levels throughout postsurgical
testing, indicating that the GL is also not sufficient. It is possible that the GL would be able to
support performance if tested for a longer period of time; although, given that rats in the CTx +
GSPx group were performing at or near chance for 6 weeks of testing, it is more likely that they
would lose stimulus control before relearning the discrimination. Also, despite the fact that
transaction of the GL has never been shown to affect performance in salt-based gustatory tasks
(13, 40-42), the possibility that the GL could support improvement after CT transaction if other
stimuli were used cannot be dismissed. As an example, transaction of the CT has also been
shown to diminish performance by rats in a KC1 vs. quinine discrimination (52). Because the GL
does not seem to have functional ENaCs in the rat, it is unlikely to support discrimination
involving sodium salts, but it does respond well to bitter stimuli such as quinine hydrochloride.
It is possible that, given the electrophysiological response properties of the GL, it would be able
to provide a discriminable signal to allow overall improvement in that task.
In contrast to groups with nerve transactions, the SHAM-INT group performed similarly
to SHAM animals throughout postsurgical testing, despite receiving considerably lower stimulus
concentrations. Because the SHAM-INT rats were relatively unaffected by decreasing stimulus
concentrations it suggests that the diminished performance by the rats with nerve transactions is
not simply due to a decline in stimulus intensity after gustatory denervation. Rather, it seems
that the rats are effectively learning a new discrimination, possibly because the rats do not
perceive the stimuli the same after the loss of input from the CT. For instance, transaction of the
CT impairs the specific increase in intake of sodium by sodium-deplete rats, indicating that rats
do not recognize sodium as such (7) suggesting, perhaps, that sodium is not perceived the same
by rats after the CT is transected. The same could be true for KC1, but would be more difficult to
determine because there is no comparable depletion-induced specific appetite for potassium.
It should be noted that while there was not a significant effect of decreasing
concentrations on performance in the SHAM-INT group, they were slightly lower than SHAM
rats (Figure 2). It therefore might be true that intensity is having some effect on the impairment
in performance seen in the groups with nerve transactions. However, it would be difficult to test
using lower concentrations for the SHAM-INT rats because there is a risk of dropping further
below the normal threshold for these stimuli. The two lowest concentrations of KC1 for the
SHAM-INT rats are already below the detection threshold for intact rats seen previously in the
literature (1). These rats might actually be using a difference in intensity to discriminate the two
stimuli, since the NaCl concentrations used here are all above the detection threshold of intact
rats (e.g., 2, 38). Also, since detection threshold is technically a statistical term that denotes the
concentration at the midpoint of a psychophysical function, it is also possible that
discriminability continues below that point. The 1 logo unit decrease in stimulus concentrations
for the SHAM-INT rats was designed to emulate the lowered detection thresholds seen after
nerve transaction. As with the SHAM-INT rats, at least the lowest concentration of KCl is below
the reported detection threshold for CTx rats (1). The concentrations of NaCl used are
technically above, but approaching threshold for those rats, as well (e.g., 2). Only 0.4 M NaCl
was above the reported detection threshold for CTx + GSPx rats (38), although KC1 detection has
not been explicitly tested in these rats. As such, all of the groups were tested postsurgically with
some subthreshold concentrations. One might expect, then, that the nerve-transected groups
could show a similar level of performance to the SHAM-INT rats, if the only effect of nerve
transactions were to decrease the stimulus intensity, although it might still not be possible for the
CTx + GSPx group.
Similarly, it would be difficult to increase concentrations postsurgically for the groups
with nerve transactions, since there is a risk of NaCl stimulating the trigeminal system with
higher concentrations (e.g., 53). Thus, intensity cannot be ruled out completely. However, it
remains true that the GSP, and not the GL, is supporting the improvement in performance seen in
CTx rats, and that a decrease in stimulus intensity cannot fully explain the change in
performance postsurgically for rats lacking certain gustatory input.
Indeed, the marked decrease in performance immediately after surgery, followed by a
rapid improvement in both the CTx and CTx + GLx groups is similar to that seen previously in
our laboratory when animals learn a particular discrimination and then are given new stimuli.
Spector and Kopka (2002) initially trained rats to discriminate quinine from KC1, and then
replaced quinine with NaC1. At first, the rats performed poorly to the new NaCl vs. KCl
discrimination, but quickly improved to near the level of their initial testing with quinine (54).
While those rats did not receive nerve transaction surgery, the pattern of performance is similar
to that seen in this study. Given that sodium is an essential nutritional element, it is perhaps not
surprising that even after the loss of input necessary to perform this discrimination normally,
remaining gustatory input would continue to allow the discrimination of sodium salts from
In the future, use of a generalization paradigm, such as presenting test stimuli in a licking
or intake test after presurgically conditioning a taste aversion to either KC1 or NaC1, would help
determine whether the qualitative percepts of these stimuli change after nerve transaction. In
conditioned taste aversions studies, exposure to a novel stimulus is paired with, usually, an
intraperitoneal injection of lithium chloride, which induces visceral malaise. An animal then
associates that stimulus, sometimes after only one pairing, with malaise, and avoids it on
subsequent presentations. Animals will not only ingest less of the conditioned stimulus, but will
also decrease intake of other stimuli; this generalization is thought to be an index of perceptual
similarity between the test stimulus and the conditioned stimulus (e.g., 55-56). It might be
possible to condition an aversion to either NaCl or KC1, and then transect the CT One could
then compare the degree of avoidance in CTx rats to that of SHAM rats, to determine whether
there is some difference after surgery. While this has been done for NaCl in F344 and Wistar
rats, which suggested that there is no qualitative difference in the perception of NaCl after CTx
(57), it has never been tested Sprague-Dawley rats, or for KC1. It should also be noted that the
experiment did not test a wide range of stimuli, which might explain why an effect of CT
transaction was not shown.
These results show that not only is the seventh cranial nerve crucial for normal salt
discrimination, the GSP itself is capable of supporting the relearning of this discrimination after
the CT is damaged. It is unknown whether the GSP is inherently capable of supporting this
discrimination, or if the signal from the GSP is strengthened because it is correlated with the
signal from the CT during presurgical testing, in a form of Hebbian learning (for recent review,
see 58). There is overlap in the terminal fields of the CT and GSP in the nucleus of the solitary
tract, which is the first site of synapse for the gustatory system (e.g., 59). It is possible that in this
area of overlap the strong signal from the CT correlates with a potentially weaker signal from the
GSP during presurgical exposure to the stimuli, strengthening the signal from the GSP. Then,
once the input from the CT is lost, the GSP provides sufficient information for the rats to relearn
the discrimination. One way to test this would be to perform surgery before training, thus
avoiding the presurgical experience. However, given the poor performance by the rats with CT
transactions immediately after surgery, it would probably be difficult to effectively train the
animals at that point.
These results contribute to the body of literature that suggests that the seventh cranial
nerve is necessary for detection and discrimination of taste stimuli, and further indicate that
signals from remaining gustatory nerves can support relearning of a salt discrimination even after
the loss of necessary gustatory input. Further studies should be done to determine whether this
phenomenon extends to other stimuli and nerves, as well as to define the underlying neural basis
for relearning discrimination after nerve damage.
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Ginger Blonde was born on July 28, 1982 in Carbondale, Illinois. She grew up in Tampa,
Florida, graduating from C. Leon King High School in 2000. She earned her B.S. from the
University of Florida in Gainesville, Florida, in 2004. She is a member of the Golden Key
While still an undergraduate, she began working in a Behavioral Neuroscience
laboratory, studying the peripheral gustatory system as it related to taste-guided behavior. She
joined the Psychology Department's graduate program in August, 2004.