Citation
Rats Relearn a Sodium Chloride vs. Potassium Chloride Discrimination after Chorda Tympani Transection Using Remaining Gustatory Input

Material Information

Title:
Rats Relearn a Sodium Chloride vs. Potassium Chloride Discrimination after Chorda Tympani Transection Using Remaining Gustatory Input
Copyright Date:
2008

Subjects

Subjects / Keywords:
Chorda tympani nerve ( jstor )
Gustatory discrimination ( jstor )
Gustatory perception ( jstor )
Learning ( jstor )
Nerves ( jstor )
Rats ( jstor )
Sensory discrimination ( jstor )
Signals ( jstor )
Sodium ( jstor )
Taste buds ( jstor )

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Embargo Date:
7/12/2007

Downloads

This item has the following downloads:


Full Text





RATS RELEARN A SODIUM CHLORIDE VS. POTASSIUM CHLORIDE
DISCRIMINATION AFTER CHORDA TYMPANI TRANSACTION
USING REMAINING GUSTATORY INPUT


















By

GINGER BLONDE


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

































2007 Ginger Blonde









TABLE OF CONTENTS

Page

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

CHAPTER

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









TABLE
Table page


2-1 Training and Testing Param eters ............................................... ............................ 17









LIST OF FIGURES

Figure page

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

By

Ginger Blonde

May, 2007

Chair: Alan Spector
Major: Psychology

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

new discrimination.









CHAPTER 1
INTRODUCTION

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.









CHAPTER 2
METHODS

Subjects

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.

Apparatus

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.

Trial Structure

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.

Training

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.

Presurgical Testing

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.









Postsurgical Testing

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.

Surgery

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.

Histology

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

surgical condition.

Data Analysis

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


Phase
Training
Spout
Side
Alternation
Discrimination I
Discrimination II
Discrimination III


LH
Sessions (s)


N/A
180
15
10
10
5


Stimuli


N/A H20
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


Stimulus
Presentation

Constant
Constant
Criteriona
Semi-randomb
Semi-random
Semi-random


Testing
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.









CHAPTER 3
RESULTS

Histology

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;

SHAM-INT, n=7.

Behavioral Testing

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.










PRESURGICAL
CONTROL SESSIONS


I-
w
2 90
O
0
0 80

70
6 70
Z
O 60
50
50


PRESURGICAL
AMILORIDE SESSIONS


GROUP GROUP
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^













POSTSURGICAL
FIRST FOUR WEEKS


-- SHAM
-- SHAM-INT
-- CTx
-- CTx + GLx
-A- 7x


1 2 3 4

POSTSURGICAL
TESTING WEEK

Figure 3-2. Overall performance during initial postsurgical testing.
Mean performance (+ SE) by group on control sessions during postsurgical testing weeks 1-4.










POSTSURGICAL
CONTROL SESSIONS


1J-
(~


-;k G\0


POSTSURGICAL
AMILORIDE SESSIONS


GROUP GROUP
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
100
I-

80
^ 80
0
0 60

40
z
o 20
0


RAT
Figure 3-4. Water control test. Overall performance for individual rats during the water control
test. *: performance was significantly greater than 50% overall.









CHAPTER 4
DISCUSSION

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

others.

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.









LIST OF REFERENCES


1. Geran LC, Guagliardo NA, Spector AC (1999) Chorda tympani nerve transaction, but not
amiloride, increases the KC1 taste detection threshold in rats. Behav Neurosci 113(1):
185-95.

2. Kopka SL, Spector AC (2001) Functional recovery of taste sensitivity to sodium chloride
depends on regeneration of the chorda tympani nerve after transaction in the rat. Behav
Neurosci 115(5):1073-85.

3. Slotnick BM, Sheelar S, Rentmeister-Bryant H (1991) Transection of the chorda tympani
and insertion of earpins for stereotaxic surgery: equivalent effects on taste sensitivity.
Physiol & Behav 50:1123-27.

4. Spector AC, Schwartz G, and Grill H. J. (1988). Chemospecific deficits in taste
detection following bilateral chorda tympani section in rats. Chem Senses 13: 738.

5. Spector AC, Schwartz GJ, Grill HJ (1990) Chemospecific deficits in taste detection after
selective gustatory deafferentation in rats. Amer J Physiol 258(3 Pt 2):R820-6.

6. Kopka SL, Geran LC, Spector AC (2000) Functional status of the regenerated chorda
tympani nerve as assessed in a salt taste discrimination task. Amer J Physiol: Regul,
Integ and Comp Physiol 278:R720-731.

7. Spector AC, Schwartz GJ, Grill HJ (1990) Chemospecific deficits in taste detection after
selective gustatory deafferentation in rats. Amer J Physiol 258(3 Pt 2): R820-6.

8. St. John SJ, Markison S, Spector AC (1995) Salt discriminability is related to the number
of regenerated taste buds after chorda tympani nerve transaction in rats. Amer J Physiol:
Regul, Integ, and Comp Physiol 269:R141-153.

9. St. John SJ, Markison S, Spector AC (1997) Chorda tympani nerve transaction disrupts
taste aversion learning to potassium chloride, but not sodium chloride. Behav Neurosci
111(1):188-194.

10. Yasumatsu K, Katsukawa H, Sasamoto K, Ninomiya Y (2003) Recovery of amiloride-
sensitive neural coding during regeneration of the gustatory nerve: behavior-neural
correlation of salt taste discrimination. J Neurosci 23(10):4362-8.

11. Breslin P.A., Spector A.C., Grill H.J. (1993). Chorda tympani section decreases the
cation specificity of depletion-induced sodium appetite in rats. Amer J Physiol, 264(2 Pt
2): R319-23.

12. Breslin PA, Spector AC, Grill HJ (1995) Sodium specificity of salt appetite in Fischer-
344 and Wistar rats is impaired by chorda tympani nerve transaction. Amer J Physiol
269(2Pt2):R350-6.











13. Roitman MF, Bernstein IL (1999) Amiloride-sensitive sodium signals and salt appetite:
multiple gustatory pathways. Amer J Physiol: Regul, Integ, Comp Physiol 276:R1732-
R1738.

14. Grill HJ, Schwartz GJ, & Travers JB (1992) The contribution of gustatory nerve input to
oral motor behavior and intake-based preference. I. Effects of chorda tympani or
glossopharyngeal nerve section in the rat. Brain Res 573:95-104.

15. Chappell JP, St John SJ, Spector AC. (1998) Amiloride does not alter NaCl avoidance in
Fischer-344 rats. Chem Senses 23(2):151-7.

16. Sollars SI, Bernstein IL (1994) Gustatory deafferentation and desalivation: effects on
NaCl preference of Fischer 344 rats. Amer J Physiol 266(2Pt2):R510-7.

17. Pfaffmann C (1952) Taste preference and aversion following lingual denervation. J Comp
Physiol Psychol 45:393-400.

18. Beidler, LM (1953) Properties of chemoreceptors of tongue of rat. J Neurophys 16: 595-
607.

19. Boudreau JC, Hoang NK, Oravec J, & Do LT (1983) Rat neurophysiological taste
responses to salt solutions. Chem Senses i:131-150.

20. Contreras RJ, Frank M (1979) Sodium deprivation alters neuronal responses to gustatory
stimuli. J Gen Phys 73:569-594.

21. Frank ME, Contreras RJ, Hettinger TP (1983) Nerve fibers sensitive to ionic taste stimuli
in chorda tympani of the rat. J Gen Phys 50(4):941-60.

22. Nejad MS (1986) The neural activities of the greater superficial petrosal nerve of the rat
in response to chemical stimulation of the palate. Chem Senses 11: 283-93.

23. Ninomiya Y, Tonosaki K, Funakoshi M (1982) Gustatory neural response in the mouse.
Brain Res 244(2):370-3.

24. Ogawa H, Sato M, Yamashita S (1968) Multiple sensitivity of chorda tympani fibers of
the rat and hamster to gustatory and thermal stimuli. J Physiol 199:223-240.

25. Pfaffman C (1955) Gustatory nerve impulses in rat, cat and rabbit. J Neurophys
18(5):429-40.

26. Frank ME, Bieber SL, Smith DV (1988) The organization of taste sensibilities in hamster
chorda tympani fibers. J Gen Phys 91(6):861-96.









27. Hettinger TP, Frank ME (1990) Specificity of amiloride inhibition of hamster taste
responses. Brain Res 513(1):24-34.

28. Ninomiya Y, Funakoshi M (1988) Amiloride inhibition of responses of rat single chorda
tympani fibers to chemical and electrical tongue stimulations. Brain Res 451(1-2):319-25.

29. Sollars SI, Hill DL (2005) In vivo neurophysiological recordings from geniculate ganglia:
taste response properties of individual greater superficial petrosal and chorda tympani
neurons. J Physiol (Lond) 877-893.

30. Brand JG, Teeter JH, Silver WL (1985) Inhibition by amiloride of chorda tympani
responses evoked by monovalent salts. Brain Res 334(2):207-14.

31. DeSimone JA, Ferrell F (1985) Analysis of amiloride inhibition of chorda tympani taste
response of rat to NaC1. Amer J Physiol 249(1 Pt 2):R52-61.

32. Heck GL, Mierson S, DeSimone JA (1984) Salt taste transduction occurs through an
amiloride-sensitive sodium transport pathway. Science 223(4634):403-5.

33. Ye Q, Heck GL, DeSimone JA (1994) Effects of voltage perturbation of the lingual
receptive field on chorda tympani responses to Na+ and K+ salts in the rat: implications
for gustatory transduction. J Gen Phys 104(5):855-907.

34. Blonde GD, Jiang E, Garcea M, Spector AC (2006) Salt discrimination in rats with cross-
regenerated lingual gustatory nerves. Chem Senses 31(5): A123.

35. Harada S, Takashi Y, Keiko Y, and Yasuo K (1997) Different Characteristics of
Gustatory Responses Between the Greater Superfical Petrosal and Chorda Tympani
Nerves in the Rat. Chem Senses 22:133-14.

36. Sollars SI, Hill DL (1998). Taste responses in the greater superficial petrosal nerve:
substantial sodium salt and amiloride sensitivities demonstrated in two rat strains. Behav
Neurosci, 112(4):991-1000.

37. Doolin RE, Gilbertson TA (1996) Distribution and characterization of functional
amiloride-sensitive sodium channels in the rat. J Gen Phys 107(4):545-54.

38. Blonde GD, Garcea M, Spector AC (2006) The Relative Effects of Transection of the
Gustatory Branches of the Seventh and Ninth Cranial Nerves on NaCl Taste Detection in
Rats. Behav Neurosci 120(3):580-9.

39. Geran LC, Garcea M, Spector AC (2002) Transecting the gustatory branches of the facial
nerve impairs NH(4)C1 vs. KC1 discrimination in rats. Amer J Physiol: Regul, Integ,
Comp Physiol 283(3):R739-47.









40. Markison S, St John SJ, Spector AC (1995) Glossopharyngeal nerve transaction does not
compromise the specificity of taste-guided sodium appetite in rats. Amer J Physiol 269(1
Pt 2):R215-21.

41. Geran LC (2003) The psychophysics of salt taste transduction pathways (Doctoral
dissertation, University of Florida, 2003). Retrieved October 1, 2005, from the
University of Florida's Health Science Center Library catalog.

42. Colbert CL, Garcea M Spector AC (2004) Effects of selective lingual deafferentation on
suprathreshold taste intensity discrimination of NaCl in rats. Behav Neurosci
118(6):1409-17.

43. Frank ME (1991) Taste-responsive neurons of the glossopharyngeal nerve of the rat. J
Neurophys 65(6): 1452-63.

44. Ogawa H (1972) Taste response characteristics in the glossopharyngeal nerve of the rat.
Kumamoto Med J 25(4): 137-147.

45. Yamada K (1966) Gustatory and thermal responses in the glossopharyngeal nerve of the
rat. Jap J Physiol 16(6):599-611.

46. Formaker BK, Hill DL (1991) Lack of amiloride sensitivity in SHR and WKY
glossopharyngeal taste responses to NaC1. Physiol & Behav 50(4):765-9.

47. Kitada Y, Mitoh Y, Hill DL (1998) Salt taste responses of the Ixth nerve in Sprague-
Dawley rats: lack of sensitivity to amiloride. Physiol & Behav 63(5):945-9.

48. Lin W, Finger TE, Rossier BC, Kinnamon SC (1999) Epithelial Na+ channel subunits in
rat taste cells: localization and regulation by aldosterone. J Comp Neuro 405(3):406-20.

49. Miller IJ Smith D (1984) Quantitative taste bud distribution in the hamster. Physiol &
Behav 32:275-285.

50. Travers, SP, Nicklas K (1990) Tast bud distribution in the rat pharynx and larynx. The
Anat Rec, 227:373-379.

51. Bradley RM (2000) Sensory receptors of the larynx. Amer J Med 108:47-50.

52. St. John SJ, Spector AC (1998) Behavioral discrimination between quinine and KC1 is
dependent on input from the seventh cranial nerve: implications for the functional roles
of the gustatory nerves in rats. J Neurosci 18(11):4353-4362.

53. Lundy RF, Jr., Contreras RJ (1994) Neural responses of thermal-sensitive lingual fibers
to brief menthol stimulation. Brain Res 641:208-216.









54. Spector AC, Kopka SL (2002) Rats fail to discriminate quinine from denatonium:
implications for the neural coding of bitter-tasting compounds. J Neurosci 22(5):1937-41.

55. Tapper DN, Halpem BP (1968) Taste stimuli: a behavioral categorization. Science
161:708-710.

56. Nowlis GH (1974) Conditioned stimulus intensity and acquired alimentary aversions in
the rat. J Comp Physiol Psychol 86:1173-1184.

57. Sollars SI, Tracy CJ, Bernstein IL (1996) Retention of conditioned taste aversion to NaCl
after chorda tympani transaction in Fischer 344 and Wistar rats. Physiol Behav 60(1):65-
69.

58. Cooper SJ (2005) Donald O. Hebb's synapse and learning rule: a history and commentary
Neurosci Behav Rev 28:851-874.

59. May OL, Hill DL (2006) Gustatory terminal field organization and developmental
plasticity in the nucleus of the solitary tract revealed through triple-fluorescence labeling.
J Comp Neurol 497(4):658-69.









BIOGRAPHICAL SKETCH

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

Honor Society.

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.




Full Text

PAGE 1

1 RATS RELEARN A SODIUM CHLORI DE VS. POTASSIUM CHLORIDE DISCRIMINATION AFTER CHORDA TYMPANI TRANSECTION USING REMAINING GUSTATORY INPUT By GINGER BLONDE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

PAGE 2

2 © 2007 Ginger Blonde

PAGE 3

3 TABLE OF CONTENTS Page LIST OF TABLES ...........................................................................................................................4 LIST OF FIGURES .........................................................................................................................5 ABSTRACT ............................................................................................................................... ......6 CHAPTER 1 INTRODUCTION ....................................................................................................................8 2 METHODS .............................................................................................................................11 Subjects ............................................................................................................................... ....11 Apparatus ............................................................................................................................... .11 Trial Structure .........................................................................................................................12 Training ............................................................................................................................... ....12 Presurgical Testing .................................................................................................................13 Postsurgical Testing ................................................................................................................14 Surgery ............................................................................................................................... .....14 Histology ............................................................................................................................... ..15 Data Analysis ..........................................................................................................................16 3 RESULTS ............................................................................................................................... 18 Histology ............................................................................................................................... ..18 Behavioral Testing ..................................................................................................................18 Water Control Test .................................................................................................................19 4 DISCUSSION .........................................................................................................................25 LIST OF REFERENCES ...............................................................................................................31 BIOGRAPHICAL SKETCH .........................................................................................................36

PAGE 4

4 TABLE Table page 2-1 Training and Testing Parameters .......................................................................................17

PAGE 5

5 LIST OF FIGURES Figure page 3-1 Overall performance during presurgical testing ................................................................21 3-2 Overall performance during initial postsurgical testing .....................................................22 3-3 Overall performance during postsur gical control and amiloride sessions .........................23 3-4 Water control test ...............................................................................................................24

PAGE 6

6 Abstract of Thesis Presen ted 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 CHLORI DE VS. POTASSIUM CHLORIDE DISCRIMINATION AFTER CHORDA TYMPANI TRANSECTION USING REMAINING GUSTATORY INPUT By Ginger Blonde May, 2007 Chair: Alan Spector Major: Psychology When the chorda tympani nerve (CT), which i nnervates 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, ra ts lacking input from the CT were initially impaired but able to significan tly improve in an NaCl vs. KC l 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-re sponse operant task to discriminate NaCl and KCl on the basis of taste. They were then split into five groups: CT transection, (CTx, n=7); CT and glossopharyngeal nerve (GL) tran section, (CTx+GLx, n=4); CT and greater superficial petrosal nerve (GSP) transection, (CTx + GSPx, n=8), or sham surgery (SHAM, n=7; and SHAM-INT, n=7). SHAM-INT ra ts were tested postsurgically with lower stimulus concentrations to determine whether th e decrease in performan ce was due to a reduction in stimulus intensity after surgery. In the firs t week of postsurgical te sting, performance by the rats with nerve transections was impaired. Howe ver, 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

PAGE 7

7 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 transections on performance can be explained by lower stimulus intensities af ter surgery. It instead seems that the taste quality of one or both of the salts changes after CT tran section. However, the signal arising from the GSP, but not the GL, is suffici ently different between the two salts to allow the rats to learn a new discrimination.

PAGE 8

8 CHAPTER 1 INTRODUCTION The chorda tympani nerve (CT), a gustatory branch of the sevent h cranial nerve that innervates taste buds in the an terior tongue, has been shown to be important for normal tasteguided behavior related to salts. When the CT is transected, detection of salts, particularly NaCl, 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 transection 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 s how that in rodents the CT is highly responsive to salts, especially sodium salts (e.g., 18-25). A subpopulati on of nerve fibers in the rodent CT, called Nfibers, responds robustly and specifi cally to sodium salts relative to other tastants. In contrast, a separate subpopulation called H-fibers is more electrolyte-general, resp onding to both sodium and nonsodium salts as well as acids and quini ne (26-30). Additionally, taste buds in the fungiform papillae contain epitheli al 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 ge neral, and N-fibers in particular, to sodium salts. There seems to be little effect on the CT response to KCl, or responsiveness of H-fibers, with the addition of amiloride. The distinct N-fiber transmission pathway specifically representing sodium in the periphery, as comp ared to the more general H-fiber pathway for electrolytes, seems to be the basis for NaCl vs . KCl discriminability, and the CT incorporates both pathways (e.g., 28-29, 31-33). The results of behavioral and electrophys iological studies all suggest that the CT is important for sa lt detection and discrimination in rats.

PAGE 9

9 Most of the behavioral studies described a bove tested animals over a short period of time postsurgically. In one previous study in our la boratory, rats with a bilaterally transected CT performed poorly postsurgically in a NaCl versus KCl disc rimination, as seen in other experiments. However, with longer testing, so me rats were able to improve their overall performance in the task, approach ing 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 transection, some degree of sodium detectability remains. It has been suggested that the greater s uperficial petrosal nerv e (GSP), the branch of the seventh cranial nerve that innervates ta ste buds in the nasoincisor ducts, geshm acksstreifen, and soft palate in the rat, is responsible for the remain ing ability of rats to perform th ese tasks after CT transection. Electrophysiological recording experiments have s hown that the GSP also responds well to salts, although it responds best to sugars (22, 35-36). A dditionally, the taste bud fields innervated by the GSP seem to contain ENaCs; lingual appl ication of amiloride decreases overall GSP responsiveness to sodium salts (36-37). The results of behavioral studi es also suggest that the GSP has a role in taste-guided behaviors related to sodium. Af ter combined CT and GSP transection, eliminating all gustatory input from the seventh cranial ne rve, the detection threshold fo r 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 gloss opharyngeal 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 detect ion or recognition of sodium by the rat. After the GL is transected, there is no effect on the specificity or vigor of a depletion-i nduced sodium appetite,

PAGE 10

10 salt discrimination performance, or intensity difference thresholds for NaCl (13, 40-42). Electrophysiological findings demonstrate that th e GL responds best to bitter-tasting stimuli such as quinine hydrochloride. It has a weaker res ponse to sodium salts (25, 43-45), and it has been suggested that the circumvallate taste buds of the rat have no f unctional 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 transection in our previous study to improve performance in a NaCl versus KCl discriminatio n postsurgically. We also tested whether the GL, which was also left intact in those rats with CT transection, was providing sufficient information to support recovery of performance over the postsur gical testing period. A control group was included in the design that allowed us to assess whethe r the decrease in discriminability is related to a decrease in intens ity of the stimuli that would follow after some of nerve transection surgeries. We hypothesized that given the elec trophysiological response properties of the GSP along with the fact that combined CT and GSP transection often has greater effect on performance than transection of the CT alone, it is likely that the GSP, and not the GL, is responsible for the level of performance seen after CT transection.

PAGE 11

11 CHAPTER 2 METHODS Subjects 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. Th e 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 traini ng, they were given oil mash (5 parts powdered rodent chow to 2 parts vegetable oil) in prepar ation for the presentation of this palatable and lubricated diet during postsurg ical 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 testi ng, the rats obtained purified water as part of their 40-minute testing sessions. Throughout te sting, no rat ever dropped belo w 85% of its free-feeding body weight. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Florida. Apparatus Training and testing took place in a gustome ter described in Blonde et al, 2006 (38). Briefly, rats were placed in a sound attenuati on chamber fitted with a central sample spout positioned behind an access slot and two reinforcemen t spouts, one to either side of the sample spout. A cue light was placed above each reinfo rcement spout. Stimuli were contained in pressurized reservoirs connected to solenoid valves calibrated to deliver ~5 µL of fluid, and were

PAGE 12

12 delivered through the sample spout af ter it was initially fille d at the start of a trial. After each trial, the sample spout was ro tated over a funnel, rinsed with purified water and blown dry. Trial Structure Each trial began when the rat licked the samp le 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 recei ved up to 10 licks of purified water in 8-s from the reinforcement spout. During amiloride sessions, 100 µM amiloride replaced water in the reinforcem ent spouts. If the rat responded incorrectly or failed to respond during the limited hold, it received a 30-s time-out, when all light s were extinguished and it had no access to water. The reinforcement or timeout was followed by a 6-s intertrial interval, during which the sample spout was rinsed and drie d. 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 tr ials as possible during each 40-min session. Training The rats were trained to associate one rein forcement spout with 0.2 M NaCl and the other with 0.2 M KCl (both reagent grad e chemicals; Fisher Scientific , Orlando, FL). The assignment was counterbalanced, so that for ha lf of the rats the left spout re presented NaCl, and for the other half it represented KCl. The training and testing schedule is depicted in Table 2-1.

PAGE 13

13 Spout training. The rats were trained to lick each spout. One 40-min session was devoted to each spout (sample, left reinforcem ent, right reinforcemen t), and rats received purified water ad libitum . Side training. The rats were trained to associate one reinforcement spout with NaCl and the other with KCl. 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 avai lable was alternated each day. Alternation. For this phase, both 0.2 M NaCl and 0.2M KCl were presented in each session. The rat was required to make a predet ermined number of correct responses to each stimulus before the other stimulus was pr esented. This criteri on number was reduced systematically over three sessions, from 6 responses to 2. Discrimination Training I. In this phase, 0.2 M NaCl a nd 0.2 M KCl were presented randomly. The probability of receiving either st imulus was 0.5. The rat was required to perform > 80% overall on trials with a response to m ove 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 ti me-out was increased. As in Discrimination Training I, the rat had to perform > 80% overall to move on to the next phase. Presurgical Testing During presurgical testing, th e rats were tested on Monday, Wednesday, and Friday with 0.1 M, 0.2 M, and 0.4 M NaCl and KCl in control sessions. For the Tuesday and Thursday sessions, the salt stimuli were dissolved in 100µM amiloride. Testing was conducted for two weeks, for a total of six control sessions and four amiloride sessions.

PAGE 14

14 Postsurgical Testing Testing resumed 28-30 days after surgery. For four weeks, animals were tested Monday through Friday in the same manner as during pres urgical control sessions. During weeks 5 and 6 of postsurgical testing, the rats we re tested identically to presurgi cal testing. Three days after the last day of testing, a water control test was cond ucted in which all reserv oirs were filled with water. This test helped to determine whether th e performance of the rats relied on chemical cues of the stimuli as opposed to other extraneous cues. Surgery Surgical groups were balanced by overall performance, performance to each salt, body weight, and gustometer. All rats were anesthetiz ed with an intramuscular injection of ketamine hydrochloride (125 mg/kg body weight) mixed with xylazine hydrochloride (5 mg/kg body weight). For rats receiving bila teral CT transection (CTx), th e external auditory canal was retracted and the tympanic membrane was punc tured. The chorda tympani was exposed and transected where it disappears behind the ma lleus. 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 transection (CTx + GLx), the above procedure was performed to transect the CT. Then, to transect the GL, we retracted the sublingual and submaxillary salivary glands a nd the sternohyoid, omohyoi d, and posterior belly of the digastic muscles. The fascia underl ying 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.

PAGE 15

15 For rats receiving bilateral combined CT a nd GSP transection (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 dril l. The tympanic membrane, ossicles, and CT were removed. The tensor tympani muscle a nd a small piece of temporal bone were then removed with microforceps, exposing the GSP. It was transected, and the e nds 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 tr ansected. Additionally, an incision dorsal to the pinna was made, the external ear was retract ed, 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 r eceived Penicillin G Procaine suspension (30,000 units sc) and ketorolac tromethamine (2 mg/kg body mass sc) fo r 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 sweetene d 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, de monstrated by a stable body weight of at least 90% of an animalÂ’s presurgical weight, which took a minimum of 5 and a maximum of 25 days. Histology 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%

PAGE 16

16 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, a nd 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 µm sect ions. These sections were mounted on slides, stained with hematoxylin and eosin, and taste buds were counted under the microscope. All tissue sections were code d so that the counters were uninformed of the surgical condition. Data Analysis The overall percentage correct for trials with a response was cal culated. Individual performance was calculated for control and amilo ride sessions (separatel y for both presurgical and postsurgical testing). Perf ormance during the first four w eeks of postsurgical testing was compiled by week (4-5 sessions pe r week) for each animal. Thes e data were compared by group using matched t -tests and analyses of variance (ANO VAs) 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 percen tage 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 posit ive differences from chance with the one-tailed normal approximation of the binomial distribution.

PAGE 17

17 Table 2-1. Training an d testing parameters Phase Sessions LH (s) TO (s) Stimuli Stimulus Presentation Training Spout 3 N/A N/A H2O Constant Side 6 180 0 0.2M NaCl or KCl Constant Alternation 3 15 10 0.2M NaCl and KCl Criteriona Discrimination I 4-5 10 10 0.2M NaCl and KCl Semi-randomb Discrimination II 3-4 10 20 0.1, 0.2, and 0.4 M NaCl and KCl Semi-random Discrimination III 3-4 5 30 0.1, 0.2, and 0.4 M NaCl and KCl Semi-random Testing Presurgical 10 5 30 0.1, 0.2, and 0.4 M NaCl and KCl [with and without 100µM amiloride] Semi-random Postsurgical 29-31 5 30 0.1, 0.2, and 0.4 M NaCl and KCl [with and without 100µM 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 s econds) for incorrect responses. a: the alternation criteria used: 6, 4, 2. b: Stimuli were presented in randomized blocks of 6 without replacement.

PAGE 18

18 CHAPTER 3 RESULTS Histology Stained tissues were semi-quan titatively analyzed to determine the success of surgery. If it appeared that the taste bud fi eld was not intact (i.e., the nerve innervating it had been transected), the number of tast e 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 regene ration in those animals. Their data were not included in the analyses. One rat in the CTx + GSPx group had 20 taste buds in the naso incisor 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; CT x + GLx, n=4; CTx + GSPx, n=8; SHAM, n=7; SHAM-INT, n=7. Behavioral Testing During presurgical control sessions, all gr oups were performing at high levels overall (left panel, Figure 1). Even af ter the removal of rats due to nerve regeneration, there was no difference between groups during presurgical contro l sessions, with no main effect of group in a two-way ANOVA (F(4,28)=1.87, p=0.144). As expecte d, the addition of amiloride significantly decreased performance presurgically (main effect of AMILORIDE: F(1,28)=1927.509, p<0.001; no effect of the inte raction GROUP x AMILORIDE: F(4,28) =0.332, p=0.854; right panel, Figure 1), although performance for the SHAM and CTx gr oups were significantly higher than chance (p>0.05 for both).

PAGE 19

19 In the first week of postsurgi cal testing (i.e., the first 5 sess ions), all grou ps with nerve transections were significantly impaired comp ared to their presurgical performance (all pvalues<0.05; Figure 2). Additionally, rats with CTx and CTx + GLx surgeries were performing at similar levels to each other (p>0.05). The pe rformance of rats with CTx + GSPx surgeries was at chance (p = 0.781) and signi ficantly lower than that for CTx and CTx + GLx rats (p<0.021), suggesting that the GSP was contributing to perf ormance in this discrimination. Rats in the SHAM-INT group, however, were performing similarl y to SHAM animals, despite being tested with concentrations that were 1.0 log10 unit lower (p>0.05). Their performance suggests that the impairment seen in rats with nerve transections 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 w eek (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 through out postsurgical testing (p>0.05 for Postsurgical Weeks 1-4; p = 0.02 duri ng Postsurgical Control sessions; see Figures 2 & 3). As with presurgical testi ng, all groups performed at or near chance levels when tested with amiloride (Figure 3). Statis tically speaking, the SHAM gr oup 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 comp ared to control sessions (p<0.05 for both).

PAGE 20

20 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 ch ance levels. Also, cons idering 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.

PAGE 21

21 PRESURGICAL CONTROL SESSIONS GROUP S H A M S H A M I N T C T x C T x + G L x 7 x PERCENTAGE CORRECT 50 60 70 80 90 100 PRESURGICAL AMILORIDE SESSIONS GROUP S H A M S H A M I N T C T x C T x + G L x 7 x * * Figure 3-1. Performance dur ing presurgical testing. Overall percentage correct (+ SE) by group for presurgical co ntrol sessions (left panel) and amiloride sessions (right panel). *: group pe rformance was significantly higher than chance (50% overall) during amiloride sessions.

PAGE 22

22 POSTSURGICAL FIRST FOUR WEEKS POSTSURGICAL TESTING WEEK 1234 SHAM SHAM-INT CTx CTx + GLx 7x Figure 3-2 . Overall performance during initial postsurg ical testing. Mean performance (+ SE) by group on control sessions duri ng postsurgical testing weeks 1-4.

PAGE 23

23 POSTSURGICAL CONTROL SESSIONS GROUP S H A M S H A M I N T C T x C T x + G L x 7 x OVERALL PERCENTAGE CORRECT 50 60 70 80 90 100 POSTSURGICAL AMILORIDE SESSIONS GROUP S H A M S H A M I N T C T x C T x + G L x 7 x * Figure 3-3 . Overall performance during postsurgi cal control and amiloride sessions. Mean overall performance (+ SE) by group during postsurgical c ontrol sessions (left panel) and amiloride sessions (right panel). *: group perf ormance was significantly above 50% overall during amiloride sessions.

PAGE 24

24 WATER CONTROL TEST RAT PERCENTAGE CORRECT 0 20 40 60 80 100 ** Figure 3-4. Water control test. Overall performance for individual rats during the water control test. *: performance was signifi cantly greater th an 50% overall.

PAGE 25

25 CHAPTER 4 DISCUSSION The recovery from the impaired performance in salt discrimination, caused by CT transection, that occurs with pr olonged postsurgical testing clear ly depends on the input of the GSP, not the GL. Not only were CTx + GLx ra ts able to significantly improve over the six weeks of postsurgical testi ng, their overall performance level increa sed at the same rate as rats in the CTx group, with an intact GL. This indicates that the GSP is supplyi ng the signal that rats use to discriminate NaCl from KCl after input from the CT is lost. Additionally, when rats lacked input from both the CT and the GSP, th ey performed more poorly than the CTx and CTx + GLx groups, and their performance remained virt ually at chance levels during the six weeks of testing. Thus, the GSP is both necessary and suffi cient for the rats to improve in this salt discrimination task when the CT has been dama ged. 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 thes e rats. Because the CTx + GSPx group performed at or near 50% throughout postsurgical testi ng, 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 gr oups. Based on the position of the taste buds it innervates and its general respons e properties, it has been suggested that the primary role of the SLN is the protection of the airway (see 51). Alt hough 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 discrimina tion corresponds with other studies done by our laborator y. Rats with this combined CT and GSP transection also performed at chance levels when tested with a NH4Cl vs. KCl discrimination (39). Also, while

PAGE 26

26 still able to detect very high c oncentrations of NaCl, rats lack ing 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 discri mination of gustatory stimuli (see 52). Additionally, the overall improve ment shown by CTx and CTx + GLx rats in this study seems to require the amiloride-sensitive transduc tion pathway. With the addition of amiloride, performance by the CTx and CTx + GLx groups signi ficantly decreased. Mo reover, it decreased to the level seen during presurgica l amiloride sessions. This corre sponds with the ot her 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 sa me 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 leve ls throughout postsurgical testing, indicating that the GL is al so not sufficient. It is possibl e 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 th e discrimination. Also, despite the fact that transection of the GL has never been shown to affect performan ce in salt-based gustatory tasks (13, 40-42), the possibility that the GL could sup port improvement after CT transection if other stimuli were used cannot be dismissed. As an example, transection of the CT has also been shown to diminish performance by rats in a KCl vs. quinine discrimination (52). Because the GL does not seem to have functional ENaCs in the rat, it is unlikely to support discriminations involving sodium salts, but it does respond well to bitter stimuli such as quin ine hydrochloride.

PAGE 27

27 It is possible that, given the el ectrophysiological response properti es of the GL, it would be able to provide a discriminable signal to allo w overall improvement in that task. In contrast to groups with nerve trans ections, the SHAM-INT group performed similarly to SHAM animals throughout postsur gical testing, despite receivi ng considerably lower stimulus concentrations. Because the SHAM-INT rats were relatively unaffected by decreasing stimulus concentrations it suggests that the diminished performance by the ra ts with nerve transections is not simply due to a decline in stimulus intens ity after gustatory denerv ation. Rather, it seems that the rats are effectively learning a new discrimination, possi bly because the rats do not perceive the stimuli the same after the loss of inpu t from the CT. For instance, transection of the CT impairs the specific increase in intake of sodi um by sodium-deplete rats , indicating that rats do not recognize sodium as such (7) – suggesting, pe rhaps, that sodium is not perceived the same by rats after the CT is transected. The same coul d be true for KCl, but would be more difficult to determine because there is no comparable deplet ion-induced specific appetite for potassium. It should be noted that while there wa s 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 mi ght be true that intensity is ha ving some effect on the impairment in performance seen in the groups with nerve tran sections. However, it w ould 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 KCl for the SHAM-INT rats are already below the detection thre shold for intact rats seen previously in the literature (1). These rats might actually be using a difference in in tensity to discriminate the two stimuli, since the NaCl concentrations used here are all above the detect ion threshold of intact rats (e.g., 2, 38). Also, since detect ion threshold is technically a st atistical term th at denotes the

PAGE 28

28 concentration at the midpoint of a psychophys ical function, it is also possible that discriminability continues below that point. The 1 log10 unit decrease in stim ulus concentrations for the SHAM-INT rats was designed to emulate the lowered detection thresholds seen after nerve transection. As with the SHAM-INT rats, at least the lowest concentr ation of KCl is below the reported detection threshol d for CTx rats (1). The concentrations of NaCl used are technically above, but approachi ng threshold for those rats, as well (e.g., 2). Only 0.4 M NaCl was above the reported detection threshold for CT x + GSPx rats (38), although KCl detection has not been explicitly tested in these rats. As such, all of the groups were tested postsurgically with some subthreshold concentrations. One might e xpect, then, that the ne rve-transected groups could show a similar level of performance to the SHAM-INT rats, if the only effect of nerve transections were to decrease the stimulus intens ity, although it might still not be possible for the CTx + GSPx group. Similarly, it would be difficult to increase c oncentrations postsurgically for the groups with nerve transections, since th ere 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 performan ce immediately after surgery, followed by a rapid improvement in both the CTx and CTx + GLx gr oups 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 KCl, and then replaced quinine with NaCl. At first, the rats performed poorly to the new NaCl vs. KCl

PAGE 29

29 discrimination, but quickly improved to near the leve l of their initial testi ng with quinine (54). While those rats did not receive nerve transecti on surgery, the pattern of performance is similar to that seen in this study. Given that sodium is an essential nutrit ional element, it is perhaps not surprising that even after the loss of input n ecessary to perform this discrimination normally, remaining gustatory input would continue to a llow the discrimination of sodium salts from others. In the future, use of a generalization paradigm , such as presenting test stimuli in a licking or intake test after presurgically conditioning a ta ste aversion to either KCl or NaCl, would help determine whether the qualitative percepts of th ese stimuli change after nerve transection. 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 conditi oned stimulus, but will also decrease intake of other stimuli; this generali zation is thought to be an index of perceptual similarity between the test st imulus and the conditioned stimulus (e.g., 55-56). It might be possible to condition an aversion to either NaCl or KCl, 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 su rgery. While this has been do ne for NaCl in F344 and Wistar rats, which suggested that there is no qualitative difference in th e perception of NaCl after CTx (57), it has never been tested Spra gue-Dawley rats, or for KCl. It should also be noted that the experiment did not test a wide range of stimu li, which might explain why an effect of CT transection was not shown.

PAGE 30

30 These results show that not only is the se venth cranial nerve crucial for normal salt discrimination, the GSP itself is capable of suppor ting the relearning of th is discrimination after the CT is damaged. It is unknown whether the GSP is inherently capab le 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 learni ng (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 possibl e that in this area of overlap the strong signal from the CT corre lates with a potentially weaker signal from the GSP during presurgical exposure to the stimuli, st rengthening the signal from the GSP. Then, once the input from the CT is lost, the GSP provide s sufficient information for the rats to relearn the discrimination. One way to test this woul d be to perform surger y before training, thus avoiding the presurgical experien ce. However, given the poor performance by the rats with CT transections 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 discriminati on of taste stimuli, and further indicate that signals from remaining gustatory nerves can suppor t relearning of a salt discrimination even after the loss of necessary gusta tory 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 discriminations after nerve damage.

PAGE 31

31 LIST OF REFERENCES 1. Geran LC, Guagliardo NA, Spector AC (1999) Chorda tympani nerve transection, but not amiloride, increases the KCl ta ste detection threshold in rats. Behav Neurosci 113(1): 185-95. 2. Kopka SL, Spector AC (2001) Functional recovery of taste sensitivity to sodium chloride depends on regeneration of the chorda tympani nerve after transection in the rat. Behav Neurosci 115(5):1073-85. 3. Slotnick BM, Sheelar S, Rentmeister-Bryant H (1991) Transection of the chorda tympani and insertion of earpins for st ereotaxic surgery: equivalent effects on taste sensitivity. Physiol & Behav 50:1123-27. 4. Spector AC, Schwartz G, and Grill H. J. (1988). Chemospecifi c deficits in taste detection following bilatera l chorda tympani section in rats. Chem Senses 13: 738. 5. Spector AC, Schwartz GJ, Grill HJ (1990) Chemos pecific deficits in taste detection after selective gustatory deaffere ntation in rats. Amer J P hysiol 258(3 Pt 2):R820-6. 6. Kopka SL, Geran LC, Spector AC (2000) Func tional status of the regenerated chorda tympani nerve as assessed in a salt taste di scrimination task. Amer J Physiol: Regul, Integ and Comp Physiol 278:R720-731. 7. Spector AC, Schwartz GJ, Grill HJ (1990) Chemos pecific deficits in taste detection after selective gustatory deaffere ntation in rats. Amer J P hysiol 258(3 Pt 2): R820-6. 8. St. John SJ, Markison S, Spector AC (1995) Salt discriminability is related to the number of regenerated taste buds after chorda tympani nerve transection in rats. Amer J Physiol: Regul, Integ, and Comp Physiol 269:R141-153. 9. St. John SJ, Markison S, Spector AC (1997) C horda tympani nerve transection disrupts taste aversion learning to potassium chloride , but not sodium chlo ride. Behav Neurosci 111(1):188-194. 10. Yasumatsu K, Katsukawa H, Sasamoto K, Ninomiya Y (2003) Recovery of amiloridesensitive neural coding duri ng regeneration of the gustato ry nerve: behavior-neural correlation of salt taste discrimi nation. J Neurosci 23(10):4362-8. 11. Breslin P.A., Spector A.C., Grill H.J. (1993) . Chorda tympani section decreases the cation specificity of depletion-induced sodium appetite in rats. Amer J Physiol, 264(2 Pt 2): R319-23. 12. Breslin PA, Spector AC, Grill HJ (1995) Sodi um specificity of salt appetite in Fischer344 and Wistar rats is impaired by chorda tympani nerve transection. Amer J Physiol 269(2Pt2):R350-6.

PAGE 32

32 13. Roitman MF, Bernstein IL (1999) Amiloride-sensitive sodium signals and salt appetite: multiple gustatory pathways. Amer J Physiol: Regul, Integ, Comp Physiol 276:R1732R1738. 14. Grill HJ, Schwartz GJ, & Travers JB (1992) Th e contribution of gustatory nerve input to oral motor behavior and intake-based pref erence. I. Effects of chorda tympani or glossopharyngeal nerve section in the rat. Brain Res 573:95-104. 15. Chappell JP, St John SJ, Spector AC. (1998) Am iloride does not alter NaCl avoidance in Fischer-344 rats. Chem Senses 23(2):151-7. 16. Sollars SI, Bernstein IL (1994) Gustatory d eafferentation and desa livation: effects on NaCl preference of Fischer 344 rats . Amer J Physiol 266(2Pt2):R510-7. 17. Pfaffmann C (1952) Taste preference and aver sion following lingual denervation. J Comp Physiol Psychol 45:393-400. 18. Beidler, LM (1953) Properties of chemorecep tors of tongue of rat. J Neurophys 16: 595607. 19. Boudreau JC, Hoang NK, Oravec J, & Do LT (1983) Rat neurophysiological taste responses to salt solutions. Chem Senses i:131-150. 20. Contreras RJ, Frank M (1979) Sodium deprivat ion alters neuronal re sponses to gustatory stimuli. J Gen Phys 73:569-594. 21. Frank ME, Contreras RJ, Hettinger TP (1983) Ne rve fibers sensitive to ionic taste stimuli in chorda tympani of the rat. J Gen Phys 50(4):941-60. 22. Nejad MS (1986) The neural activities of the gr eater superficial petros al nerve of the rat in response to chemical stimulation of the palate. Chem Senses 11: 283-93. 23. Ninomiya Y, Tonosaki K, Funakoshi M (1982) Gustatory neural response in the mouse. Brain Res 244(2):370-3. 24. Ogawa H, Sato M, Yamashita S (1968) Multiple sensitivity of chorda tympani fibers of the rat and hamster to gustatory and thermal stimuli. J Physiol 199:223-240. 25. Pfaffman C (1955) Gustatory nerve impulse s in rat, cat and rabbit. J Neurophys 18(5):429-40. 26. Frank ME, Bieber SL, Smith DV (1988) The orga nization of taste sensibilities in hamster chorda tympani fibers. J Gen Phys 91(6):861-96.

PAGE 33

33 27. Hettinger TP, Frank ME (1990) Specificity of amiloride inhibition of hamster taste responses. Brain Res 513(1):24-34. 28. Ninomiya Y, Funakoshi M (1988) Amiloride in hibition of responses of rat single chorda tympani fibers to chemical and electrical tongue stimulations. Br ain Res 451(1-2):319-25. 29. Sollars SI, Hill DL (2005) In vivo neurophysiol ogical recordings from geniculate ganglia: taste response properties of indiviual greater superficial petrosal and chorda tympani neurons. J Physiol (Lond) 877-893. 30. Brand JG, Teeter JH, Silver WL (1985) I nhibition by amiloride of chorda tympani responses evoked by monovalent salts. Brain Res 334(2):207-14. 31. DeSimone JA, Ferrell F (1985) Analysis of am iloride inhibition of chorda tympani taste response of rat to NaCl. Amer J Physiol 249(1 Pt 2):R52-61. 32. Heck GL, Mierson S, DeSimone JA (1984) Salt taste transduction occurs through an amiloride-sensitive sodium transport pathway. Science 223(4634):403-5. 33. Ye Q, Heck GL, DeSimone JA (1994) Eff ects of voltage perturbation of the lingual receptive field on chorda tympani responses to Na+ and K+ salts in the rat: implications for gustatory transduction. J Gen Phys 104(5):855-907. 34. Blonde GD, Jiang E, Garcea M, Spector AC ( 2006) Salt discrimination in rats with crossregenerated lingual gustatory nerv es. Chem Senses 31(5): A123. 35. Harada S, Takashi Y, Keiko Y, and Yas uo K (1997) Different Characteristics of Gustatory Responses Between the Greater Superfical Petrosal and Chorda Tympani Nerves in the Rat. Chem Senses 22:133-14. 36. Sollars SI, Hill DL (1998). Taste responses in the greater superfic ial petrosal nerve: substantial sodium salt and amilo ride sensitivities demonstrated in two rat strains. Behav Neurosci, 112(4):991-1000. 37. Doolin RE, Gilbertson TA (1996) Distribu tion and characteriza tion of functional amiloride-sensitive sodium channels in the rat. J Gen Phys 107(4):545-54. 38. Blonde GD, Garcea M, Spector AC (2006) The Relative Effects of Transection of the Gustatory Branches of the Seventh and Ninth Cranial Nerves on NaCl Taste Detection in Rats. Behav Neurosci 120(3):580-9. 39. Geran LC, Garcea M, Spector AC (2002) Transec ting the gustatory branches of the facial nerve impairs NH(4)Cl vs. KCl discrimination in rats. Amer J Physiol: Regul, Integ, Comp Physiol 283(3):R739-47.

PAGE 34

34 40. Markison S, St John SJ, Spector AC (1995) Gl ossopharyngeal nerve tr ansection does not compromise the specificity of taste-guided s odium appetite in rats. Amer J Physiol 269(1 Pt 2):R215-21. 41. Geran LC (2003) The psychophysics of salt taste transduction pathways (Doctoral dissertation, University of Florida, 20 03). Retrieved October 1, 2005, from the University of FloridaÂ’s Health Science Center Library catalog. 42. Colbert CL, Garcea M Spector AC (2004) Eff ects of selective lingual deafferentation on suprathreshold taste intensity discrimina tion of NaCl in rats. Behav Neurosci 118(6):1409-17. 43. Frank ME (1991) Taste-responsive neurons of the glossopharyngeal ne rve of the rat. J Neurophys 65(6): 1452-63. 44. Ogawa H (1972) Taste response characteristics in the glossopharyngeal nerve of the rat. Kumamoto Med J 25(4):137-147. 45. Yamada K (1966) Gustatory and thermal respons es in the glossophar yngeal nerve of the rat. Jap J Physiol 16(6):599-611. 46. Formaker BK, Hill DL (1991) Lack of amiloride sensitivity in SHR and WKY glossopharyngeal taste responses to NaCl. Physiol & Behav 50(4):765-9. 47. Kitada Y, Mitoh Y, Hill DL (1998) Salt tast e responses of the Ixth nerve in SpragueDawley rats: lack of sensitivity to amiloride. Physiol & Behav 63(5):945-9. 48. Lin W, Finger TE, Rossier BC, Kinnamon SC (1999) Epithelial Na+ channel subunits in rat taste cells: localization and regulation by aldosterone. J Comp Neuro 405(3):406-20. 49. Miller IJ Smith D (1984) Qu antitative taste bud distribution in the hamster. Physiol & Behav 32:275-285. 50. Travers, SP, Nicklas K (1990) Tast bud di stribution in the rat pharynx and larynx. The Anat Rec, 227:373-379. 51. Bradley RM (2000) Sensory receptors of the larynx. Amer J Med 108:47-50. 52. St. John SJ, Spector AC (1998) Behavioral discrimination between quinine and KCl is dependent on input from the se venth cranial nerve: implicat ions for the f unctional roles of the gustatory nerves in ra ts. J Neurosci 18(11):4353-4362. 53. Lundy RF, Jr., Contreras RJ (1994) Neural re sponses of thermal-sensitive lingual fibers to brief menthol stimulation. Brain Res 641:208-216.

PAGE 35

35 54. Spector AC, Kopka SL (2002) Rats fail to discriminate quinine from denatonium: implications for the neural coding of bitt er-tasting compounds. J Neurosci 22(5):1937-41. 55. Tapper DN, Halpern BP (1968) Taste stimu li: a behavioral categorization. Science 161:708-710. 56. Nowlis GH (1974) Conditioned stimulus intens ity and acquired alimentary aversions in the rat. J Comp Physiol Psychol 86:1173-1184. 57. Sollars SI, Tracy CJ, Bernstein IL (1996) Rete ntion of conditioned taste aversion to NaCl after chorda tympani transection in Fische r 344 and Wistar rats. Physiol Behav 60(1):6569. 58. Cooper SJ (2005) Donald O. HebbÂ’s synapse and learning rule: a hi story and commentary Neurosci Behav Rev 28:851-874. 59. May OL, Hill DL (2006) Gustatory terminal field organization and developmental plasticity in the nucleus of the solitary tr act revealed through triple-fluorescence labeling. J Comp Neurol 497(4):658-69.

PAGE 36

36 BIOGRAPHICAL SKETCH Ginger Blonde was born on July 28, 1982 in Car bondale, 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 Honor Society. While still an undergraduate, she began working in a Behavioral Neuroscience laboratory, studying the peripheral gu statory system as it related to taste-guided behavior. She joined the Psychology DepartmentÂ’s graduate program in August, 2004.