Download Experimentally cross-wired lingual taste nerves can restore normal

Am J Physiol Regul Integr Comp Physiol 294: R738–R747, 2008.
First published January 9, 2008; doi:10.1152/ajpregu.00668.2007.
Experimentally cross-wired lingual taste nerves can restore normal
unconditioned gaping behavior in response to quinine stimulation
Camille T. King,1 Mircea Garcea,2 Danielle S. Stolzenberg,1 and Alan C. Spector2*
1
Department of Psychology, Stetson University, DeLand, Florida; and 2Department of Psychology and Center for Smell
and Taste, University of Florida, Gainesville, Florida
Submitted 14 September 2007; accepted in final form 4 January 2008
oromotor reflex; regeneration; bitter taste; nerve injury
THE GUSTATORY SYSTEM HAS BEEN described as an ideal model to
examine the role of degenerative and regenerative processes on
sensory function due to its inherent and notable plasticity (e.g.,
25). For example, taste receptor cells normally turnover about
every 10 days (5, 11), yet somehow, in the midst of continual
synaptic rearrangement with first-order gustatory neurons, the
replaced cells seemingly sustain normal afferent messages.
Moreover, when lingual taste nerves are cut, taste buds in the
denervated oral region degenerate, either partially or completely, depending on the species, and the transected fibers
regenerate to reinnervate their native receptor field (e.g., 8, 9,
12, 13, 19, 23, 39, 55, 58, 60). Following reinnervation of the
epithelium by the nerve fibers, the taste buds return and in the
* A.C. Spector is now at the Department of Psychology; Florida State
University; Tallahassee, FL 32306-4301
Address for reprint requests and other correspondence: C. Tessitore King,
Dept. of Psychology, 421 North Woodland Blvd., Unit 8281, Stetson Univ.,
DeLand, FL 32723 USA (e-mail: [email protected]).
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rodents examined, behavioral function is restored (e.g., 3, 4, 6,
14, 27, 29, 30, 45). Such plasticity is indeed remarkable, but it
is unclear whether the recovered function is dependent upon
the tongue receptor field being reinnervated (the source of the
input) or upon the nerve and the central circuits it supplies, or
both.
Oakley (36) was the first to address this question in a
fascinating and innovative set of experiments using rats. He
transected the chorda tympani nerve (CT), which normally
innervates the anterior tongue (AT) taste buds, and formed a
cross-anastomosis with the transected glossopharyngeal nerve
(GL) such that the regenerating CT fibers reinnervated the taste
buds of the posterior tongue (PT), the normal target of the GL.
In other rats, the converse type of cross-anastomosis was
formed. Using whole-nerve electrophysiology, he showed that
it was the characteristic chemical sensitivity of the taste receptor cells of the region of the tongue reinnervated that dictated
the response properties of the regenerated nerve, not the other
way around. For example, the intact rat GL, which normally
responds well both to quinine, a bitter-tasting alkaloid, and
sodium saccharin, an artificial sweetener, and modestly to
NaCl placed on its receptor field, responded well to NaCl and
relatively poorly to quinine and saccharin when it crossreinnervated the AT taste buds. Likewise, when the CT nerve
cross-reinnervated the PT, it adopted the response profile of an
intact GL and uncharacteristically responded well to quinine
and saccharin and poorly to NaCl (36). Nejad and Beidler (34)
reached a similar conclusion when they cross-wired the CT and
the greater superficial petrosal nerve, which innervates taste
buds in the palate. In contrast, Ninomiya (35), who examined
single-fiber electrophysiological responses of cross-regenerated gustatory nerves in the mouse, found that the inhibitory
effectiveness of amiloride (which blocks epithelial sodium
channels thought to be involved with sodium taste transduction) in producing responses to NaCl was dependent on the
cross-regenerated nerve, not the receptor field it was innervating.
Of course, restoration of responsiveness in a cross-regenerated nerve to chemical stimulation of the tongue does not
guarantee that the sensory signals will be properly interpreted
by the brain and support normal taste-related behavior. Indeed,
the afferent projections of the CT and GL nerves terminate in
relatively segregated regions (but with some overlap) in the
gustatory nucleus of the solitary tract (gNST), the first central
relay in the gustatory system (22, 31, 33); and thus, in these
cross-wired animals, afferent signals may have been channeled
inappropriately into central brain circuits. In a later experiment,
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King CT, Garcea M, Stolzenberg DS, Spector AC. Experimentally cross-wired lingual taste nerves can restore normal unconditioned gaping behavior in response to quinine stimulation. Am J
Physiol Regul Integr Comp Physiol 294: R738–R747, 2008. First
published January 9, 2008; doi:10.1152/ajpregu.00668.2007.—Studies examining the effects of transection and regeneration of the
glossopharyngeal (GL) and chorda tympani (CT) nerves on various
taste-elicited behaviors in rats have demonstrated that the GL (but not
the CT) nerve is essential for the maintenance of both an unconditioned protective reflex (gaping) and the neural activity observed in
central gustatory structures in response to lingual application of a
bitter substance. An unresolved issue, however, is whether recovery
depends more on the taste nerve and the central circuits that it supplies
and/or on the tongue receptor cell field being innervated. To address
this question, we experimentally cross-wired these taste nerves,
which, remarkably, can regenerate into parts of the tongue they
normally do not innervate. We report that quinine-stimulated gaping
behavior was fully restored, and neuronal activity, as assessed by Fos
immunohistochemistry in the nucleus of the solitary tract and the
parabrachial nucleus, was partially restored only if the posterior
tongue (PT) taste receptor cell field was reinnervated; the particular
taste nerve supplying the input was inconsequential to the recovery of
function. Thus, PT taste receptor cells appear to play a privileged role
in triggering unconditioned gaping to bitter tasting stimuli, regardless
of which lingual gustatory nerve innervates them. Our findings demonstrate that even when a lingual gustatory nerve (the CT) forms
connections with taste cells in a non-native receptor field (the PT),
unconditioned taste rejection reflexes to quinine can be maintained.
These findings underscore the extraordinary ability of the gustatory
system to adapt to peripherally reorganized input for particular behaviors.
CROSS-WIRING OF GUSTATORY SYSTEM
27, 49). Transection of the GL also eliminates the characteristic
pattern of quinine-stimulated neuronal Fos expression, which
is a proxy for neural activation, in hindbrain centers involved
in gustatory processing, in particular, the medial dorsal subfield
(MD) of the gNST (24, 27, 28, 51) and the waist area of the
parabrachial nucleus (PBN; 26). Again, transection of the CT
has only marginal effects (28). Importantly, when the GL
regenerates into the PT, these functional measures return to
normal values (26, 27).
The necessity of the GL and its primary central targets, per
se, in sustaining functional rejection responses to bitter-tasting
substances is disputable, however, because taste bud cells
located in the PT prominently express receptors that bind with
bitter-tasting ligands (called T2Rs), the AT only sparsely
expresses them (1). This difference suggests that a weaker or
different “bitter” signal arises from the AT. Even so, the CT
can sufficiently mediate other quinine-stimulated taste-related
behaviors. For example, in the absence of the GL, rats are still
able to detect and discriminate quinine (46, 47); to display
quinine concentration-dependent suppression of licking in
brief-access tests (44), albeit sometimes in a slightly less
sensitive manner (32); and even to gape in response to sucrose
conditioned to be aversive (10). Collectively, these findings
have led to the suggestion that the input of the GL and its
associated central circuits play a privileged role in triggering
gapes to unconditioned taste stimuli.
Here, we rewired the adult gustatory system as Oakley (36)
did to assess for the first time whether quinine-stimulated
gaping, a reliable, stereotypical oromotor rejection response,
and the associated neuronal activation, as assessed by Fos
immunohistochemistry, in two critical brain stem regions, the
gNST and PBN, depends more on the receptor field origin of
the taste input or on the nerve that channels the signal into the
brain.
MATERIALS AND METHODS
Subjects
Forty-seven male Sprague-Dawley rats (Charles River Breeders,
Wilmington, MA) weighing ⬃400 g at the time of nerve surgery were
individually housed in polycarbonate tub cages. After surgery, they
were placed in hanging wire-mesh cages until the incision site healed.
Laboratory chow (5001; Purina Mills, Inc. St. Louis, MO) and water
were available ad libitum except where noted below. The temperature,
humidity, and light cycle (12:12-h light-dark cycle) were automatically controlled. All experimental protocols were approved by the
Institutional Animal Care and Use Committees of the University of
Florida and Stetson University.
Fig. 1. Recovery of quinine-stimulated gaping depended more on the taste
receptor cell field on the tongue than on the taste nerve transmitting the signal.
A: The mean ⫾ SE numbers of gapes (pictured in inset) elicited during the first
minute of a 30-min intraoral stimulation period with 3-mM quinine hydrochloride (0.233ml/min) on test day. One control group (SHAM-W) received
distilled water. Solid circles represent data from individual animals. Reinnervation of the posterior tongue (PT) by either its native taste nerve, the
glossopharyngeal (GL) nerve (GL3 PT), or by a non-native taste nerve, the
chorda tympani (CT) nerve (CT3 PT), maintained unconditioned gapes to
quinine. In contrast, reinnervation of the anterior tongue (AT) by either taste
nerve (GL3 AT or CT3 AT) was neither necessary nor sufficient to maintain
this gustatory reflex. B: When tested the day before quinine stimulation, none
of the groups substantially gaped to intraoral infusions of distilled water,
demonstrating that the chemical quality of the quinine and not the thermal or
mechanical properties of the fluid stimulus, was necessary to elicit gapes in the
nerve-rewired groups.
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Surgical Procedures
All surgeries were conducted in rats that were deeply anesthetized
(125 mg/kg body mass ketamine hydrochloride ⫹ 5 mg/kg body mass
xylazine im; supplemental does delivered as necessary). The rats
received subcutaneous injections of penicillin (30,000 units) and
ketorolac tromethamine (2 mg/kg body mass) for 3 days following
surgery. Wet mash (powdered chow mixed with water and supplemented with a calorically dense suspension, Nutri-Cal, Evsco Pharmaceuticals, Buena, NJ) was available ad libitum in addition to a milk
diet (1 part sweetened condensed milk in 2 parts water) for 3 days. In
some cases, the wet mash was continued until body mass displayed
signs of steady increases.
Four types of surgical manipulations of the lingual gustatory nerves
were performed bilaterally to allow for reinnervation of either the PT
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Oakley (37) did provide a crude behavioral test of taste
function in rats that had the GL routed to the AT on one side
and all of the remaining lingual gustatory nerves transected.
These animals displayed relatively normal preference-aversion
functions for sucrose, saccharin, and quinine, as assessed in
48-h two-bottle tests. The significance of such behavioral
results is undermined by the fact that the two-bottle preference
test can be influenced by nontaste factors and is not a particularly sensitive measure of gustatory competence (see Ref. 41).
In some cases, complete gustatory nerve deafferentation of the
tongue produces minor, if any, alterations in taste solution preference or aversion measured in this way (e.g., 2, 17, 40, 54).
In order for a test of the functional consequences of gustatory nerve cross-regeneration to have meaning, the taste-related behavior measured must be unequivocally disrupted by
transection of one lingual taste nerve but not the other. Moreover, the impaired behavior must recover upon normal regeneration of the transected nerve into its native receptor field.
These experimental requirements are satisfied through the
measurement of the gape (see Fig. 1A, inset), a hallmark
oromotor rejection reflex unconditionally displayed by rats and
other mammals in response to bitter solutions. Transection of
the GL severely blunts quinine-stimulated gaping in rats,
whereas transection of the CT has much less of an effect (18,
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fibers would innervate their normal targets in the AT. In these same
animals, the GL was transected in a way that prevented its regeneration (removing ⬃8 mm of the nerve), so that only the AT would be
reinnervated by its native nerve. In another control group (GL3 PT;
n ⫽ 5), the GL was simply cut and the two ends were left apposed to
promote normal reinnervation of the PT, and the CT was transected in
a way that discouraged its regeneration into the AT (removing ⬃8 mm
of the nerve).
A surgical control group included rats for which both gustatory
nerves were simply exposed. Some of these rats received quinine as
the intraoral stimulus on “test” day (SHAM-Q; n ⫽ 5) and some
received dH2O (SHAM-W; n ⫽ 6).
A final group included rats that underwent a bilateral double-nerve
transection (NoREG, n ⫽ 4), in which portions (at least 8 –10 mm) of
both gustatory nerves were removed to prevent both the regeneration
of the taste nerves and the reinnervation of their receptor fields on the
tongue.
Intraoral cannula surgeries. The intraoral cannulas through which
taste stimuli could be directly infused into the mouth (16, 28) were
bilaterally implanted in deeply anesthetized rats 2 wk before the
commencement of behavioral procedures for each rat. During this
surgery, the CT, where it passes through the middle ear, was cut in
three groups (GL3 AT, GL3 PT, and NoREG rats) to ensure that any
taste buds histologically observed 2 wk later were not due to reinnervation by the CT into the AT. The comparable surgery (cutting the
GL) in the CT3 PT, CT3 AT, or NoREG rats was not performed
because in preliminary work, there was no observable nerve tissue to
cut, and we did not want to risk losing these valuable animals. We are
confident, however, that the native GL did not regenerate into the PT
in the CT3 PT group because the pattern of neural activity observed
in the gNST did not mirror that typically found for a normally
regenerated GL nerve (see Figs. 3–5). All rats received subcutaneous
injections of penicillin (30,000 units) and ketorolac tromethamine
(2 mg/kg body mass) for 3 days following the implantation of the
intraoral cannulas, and additional injections were given as needed
throughout the recovery period. Wet mash was available ad libitum to
all rats throughout the recovery period, which lasted for 14 days. The
cannulas were cleaned daily to maintain patency and prevent
infection.
Stimulus Delivery
The behavioral procedures were based on those described previously (28). For three habituation days, the procedures emulated
exactly those described for the test day, except that all animals
received distilled water (dH2O), as the intraoral stimulus on these
habituation days. On the test day, the animal’s left cannula was
Table 1. Summary of subjects per nerve condition used for analysis
Total n Analyzed
Abbreviated in Text as
Controls
SHAM-Q
SHAM-W
Reinnervation of only the posterior tongue
GL3PT
*CT3PT
No reinnervation
NoREG
Reinnervation of only the anterior tongue
CT3AT
*GL3AT
Bilateral Surgery
NST
PBN
No nerve transection (quinine-stimulated)
No nerve transection (water-stimulated)
n ⫽5
n ⫽6
n ⫽5
n ⫽6
GL sutured to GL (⫹ CT transected)
CT sutured to GL
n ⫽5
n ⫽5
n ⫽4
n ⫽5
Both GL and CT transected
n ⫽4
n ⫽3
CT sutured to CT (⫹ GL transected)
GL sutured to CT
n ⫽4
n ⫽7
n ⫽4
n ⫽7
SHAM-Q, rats that received quinine as the intraoral stimulus; SHAM-W, control group that received distilled water; GL, glossopharyngeal nerve; PT, posterior
tongue; CT, chorda tympani nerve; NoREG, rats that underwent a bilateral double nerve transection; AT, anterior tongue; NST, nucleus of the solitary tract; PBN,
parabrachial nucleus. *Denotes cross-wired group.
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taste receptor cells or the AT taste receptor cells. In addition, one
surgery, designed to prevent the reinnervation of both taste receptor
cell fields, was performed. Two sham-surgical groups (one stimulated
with quinine, the other stimulated with water) were also included, for
a total of seven groups. All nerve surgeries were conducted 149 –231
days before behavioral testing to allow time for successful regeneration of the nerves. The sample sizes listed below under Crossanastomosis and Control nerve surgeries indicate the original number
of animals undergoing surgery in that group. Table 1 lists the total
number of animals analyzed per group and brain region. Animals were
discarded when necessary, for example, when brain sections were
damaged or when, as assessed by histological analysis of the tongue
tissue (see below), successful bilateral reinnervation of the tongue was
not observed or significant unintended regeneration occurred.
Cross-anastomosis. The cross-anastomosis procedures were modified from those described by Oakley (38) and performed bilaterally.
The CT was exposed near its junction with the lingual nerve proper by
retracting the pterygoid muscle up and laterally, and retracting the
anterior belly of the disgastric muscle in the opposite direction and
retracting the transverse manibular muscle rostrally. A small transverse cut was made in the anterior portion of the pterygoid muscle to
reveal the lingual nerve and the CT. The GL was exposed along the
external medial wall of the bulla and was followed distally for about
several millimeters after retracting the posterior belly of the digastric
muscle, the omohyoid muscle, and the sternohyoid muscle. The hyoid
was retracted rostrally. In one group (GL3 AT; n ⫽ 10), the crossanastomosis between the central portion of the GL and the peripheral
portion of the CT was achieved by first gently pulling the CT from the
bulla until it separated from the proximal end and then trimming the
distal end and carefully dissecting it from the fascia. The GL was then
exposed and stretched from the tongue and cut, and the peripheral CT
was threaded under the tendon of the digastric muscle and placed in
the field of the GL. The ends of the two nerves were then carefully
joined with 11-0 monofilament suture. In another group (CT3 PT;
n ⫽ 8), the cross-anastomosis between the central portion of the CT
and the peripheral portion of the GL was achieved by cutting the GL
distal to the tongue close to its exit from the posterior lacerated
foramen and carefully stretching the GL, under the mylohyhoid
muscle, into the area of the central stump of the CT, which was
transected close to where it joins the lingual nerve proper. The ends of
the two nerves were then sutured as described above.
Control nerve surgeries. To control for the effects of “regeneration” per se, some animals had their nerves transected in a manner that
allowed for normal reinnervation of either the AT or PT receptor
fields. In these animals, both nerves were exposed as described above,
but in one group of animals (CT3 AT; n ⫽ 6), the CT was simply cut,
and the two ends were left apposed. In this way, regenerating CT
CROSS-WIRING OF GUSTATORY SYSTEM
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attached via polyethylene tubing to a syringe on an infusion pump
(Harvard Apparatus, South Natick, MA), and the animal was placed in
a cylindrical Plexiglas chamber for a 1-h adaptation period. For the
next 30 min, 7 ml of either dH2O or 3 mM quinine-hydrochloride
were infused through the cannula at a rate of 0.233 ml/min. During the
first minute, an experimenter was present in the room to videotape the rat
for subsequent scoring of taste reactivity behaviors. Responses were
recorded using S-VHS equipment (Panasonic AW-E300 convertible
camera and SLV-R1000 video cassette recorder). At the end of the 30th
min, the infusion pump was turned off, but the animal was left in the
behavioral arena for 45 min before either being returned to its home cage
(habituation days) or anesthetized for perfusion (test day).
Brain and Tongue Histology
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Fig. 2. Cross-regeneration of the gustatory nerves differentially affected taste
bud numbers in the tongue. Values are given as means ⫾ SE. A: numbers of
taste pores in the fungiform papillae located in the AT. B: taste buds in the
circumvallate papillae located in the PT. C: taste buds in the foliate papillae
located on the lateral aspects of the tongue. Solid circles represent the data for
individual animals. Though cross-regeneration of the GL into the front of the
tongue (GL3 AT) supported a 62% return of fungiform taste bud pores, their
numbers were decreased relative to rats with normal regeneration of the CT
nerve (CT3 AT, F6,29 ⫽ 100.068, P ⬍ 0.0001; post hoc tests, P ⬍ 0.01) and
SHAM-W rats (post hoc test, P ⫽ 0.00001). The low number of fungiform
taste pores in this cross-regenerated group may relate to the low number of
fungiform papillae observed compared with SHAM-W rats (F3,18 ⫽ 11.216,
P ⬍ .0003; post hoc test, P ⫽ 0.0002), thereby reducing the number of
available targets. Cross-regeneration of the CT to the back of the tongue
(CT3 PT) supported a normal (SHAM-Q) complement of both circumvallate
(F6,29 ⫽ 69.246, P ⬍ .0001, post hoc test, P ⫽ 0.489) and foliate taste buds
(F6,29 ⫽ 99.351, P ⬍ .00001, post hoc test, P ⫽ 0.154). Moreover, this
cross-anastomosis was as successful at maintaining these taste buds as normal
regeneration of the GL (GL3 PT, post hoc tests, P ⫽ 1.00).
gNST was parceled into 6 “subfields” based on its medial-lateral and
dorsal-ventral dimensions (28, 51; see also Refs. 21, 22, 57), and the
number of Fos-labeled neurons in each subfield was tallied.
Sixteen 75-␮m sections from the caudal two-thirds of the PBN
from each subject were also used in this study. Every other of these
sections was Nissl stained, and the alternate sections were processed
for the Fos protein. The section in which Nissl-stained cell bodies
were first apparent within the central medial subdivision was identified as the most caudal PBN section for analysis in each subject. The
most rostral of these sections (the 16th section) was roughly at the
level at which the brachium abuts the mesencephalic trigeminal tract.
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Immediately after the 45-min postinfusion period on the test day,
the rats were deeply anesthetized with an overdose of pentobarbital
sodium (80 mg/kg body mass) and perfused intracardially with
chilled, heparinized 0.15 M NaCl, followed by sodium phosphatebuffered 4% paraformaldehyde. Following an overnight postfixation
at 4°C, the brains were cut in the coronal plane (75 ␮m) using a
Vibratome. Every other section was processed for the Fos protein as
follows: the sections were pretreated for 20 min with sodium borohydride (1% in potassium phosphate-buffered saline, KPBS), rinsed in
KPBS, and then incubated in rabbit polyclonal antibody [c-Fos (4):
sc-52, Santa Cruz Biotechnology, Santa Cruz, CA] at a dilution of
1:10,000 in 0.4% Triton X-100 in KPBS for 72 h at 4°C. After several
rinses in KPBS, the sections were placed in biotinylated goat antirabbit IgG (Zymed, San Francisco, CA) at a dilution of 1:600 for 4 h
at room temperature. Following several rinses in KPBS, the sections
were placed in an avidin-biotinylated peroxidase complex (ABC kit:
Vector Laboratories, Burlingame, CA) overnight at 4°C and again
rinsed in KPBS. The sections were then placed in sodium phosphate
buffer containing 0.03% diaminobenzidine, 0.008% nickel ammonium sulfate, and 0.0075% hydrogen peroxide. Finally, the reacted
sections were mounted on chrome-alum subbed slides, dehydrated,
and coverslipped. Alternate sections were mounted and stained with
0.1% thionin to aid in delineating the borders of the gNST and PBN.
The tongues were removed and postfixed in 10% buffered formalin
for several weeks. The AT of each animal was isolated, stained with
0.5% methylene blue, rinsed with distilled water to remove excess
stain, and flattened after the underlying muscle and connective tissue
were removed. The sheet of tissue was then pressed between two glass
slides for taste bud counting with light microscopy. Portions of the PT
containing the circumvallate papilla on the dorsal midline and the
foliate papillae on the lateral aspects were removed, embedded in
paraffin, sectioned at 10 ␮m, and stained with hematoxylin and eosin.
Microscopic analysis of brain and tongue tissues. The tagging of
Fos-positive cells in the NST and PBN was performed by experimenters
who were naı̈ve regarding the group to which the rats were assigned.
Brain sections were observed under ⫻4 to ⫻40 objectives with a
Zeiss (Oberkochen, Germany) Axioscope microscope equipped with a
video camera coupled to a video monitor and computer. Video images
were captured using Zeiss Image software.
Five standard sections of the NST spaced throughout its rostrocaudal extent were selected for analysis (see Ref. 28). Four of these
sections (approximately equidistant from each other) were selected
from the gNST (rostral to the area postrema). These gNST sections
were termed rostral (RgNST), intermediate rostral (IRgNST), intermediate caudal (ICgNST), and caudal (CgNST). The one section
taken at the level of the area postrema was considered nongustatory.
All Fos-positive nuclei within the borders of the NST in each of these
sections were individually identified and electronically “tagged” for
later counting. A neuron was considered Fos-positive if its nucleus
were completely stained whether the staining was dark, medium, or
light. The “tagged” sections were printed alongside their adjacent
Nissl-stained sections. Using the thionin-stained tissue as a guide, the
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For each of the eight Fos immunohistochemically stained sections, all
Fos-positive neurons within the PBN borders were electronically
tagged for later counting as described above. The Nissl-stained sections, which were printed out alongside the Fos-labeled sections, were
used to help delineate the subdivisions of the PBN.
The counting of fungiform, circumvallate, and foliate taste bud
pores was also performed by an experimenter naı̈ve to the experimental assignment of the subjects. The number of intact taste pores stained
with methylene blue on the surface of the AT relates well to the
number of morphologically intact taste buds in the fungiform papillae
(45), and so it was used as an indicator of reinnervation. In the case
of the circumvallate and foliate papillae, taste pores were also
counted. Sometimes, a taste pore was not captured by the sectioning,
but there was clear evidence of convergence of the apical membranes
of taste bud cells, and these were counted as normal.
Behavioral Analysis
One experimenter unaware of the group assignment of the subjects
viewed, in slow motion and/or frame-by-frame, the videotaped responses for the 1st min of the infusion period. On the test day, a
variety of oromotor taste reactivity behaviors, including both aversive
and ingestive domains were scored (42), but, the gape, a hallmark
oromotor rejection response, was chosen for analysis because of its
unequivocal form and its relevance to prior research. On the last water
habituation day preceding the test day, only gapes were scored. Every
occurrence of a gape was noted and summed for the 1-min epoch.
Statistical Procedures
Separate one-way ANOVAs, one for each dependent variable, were
conducted using the seven groups to assess main effects. If the
ANOVA revealed significant differences, then post hoc Bonferroni
tests were performed. The statistical rejection criterion used for
significance was P ⱕ 0.05.
RESULTS
Gaping Behavior
The results unequivocally indicated that gaping triggered by oral
stimulation with quinine was contingent upon the receptor field origin
of the taste input rather than on the nerve supplying signals to central
targets (Fig. 1; F6,29 ⫽ 26.98, P ⬍ 0.00001). After complete bilateral
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transection of the lingual gustatory nerves, reinnervation of the PT
taste buds alone, either by the native GL nerve (GL3 PT) or the
cross-regenerated CT nerve (CT3 PT), resulted in virtually as many
gapes to quinine as found in sham-operated rats (SHAM-Q; post hoc
tests, P ⫽ 1.00). On the contrary, reinnervation of the AT receptor
field alone, either by the native CT nerve (CT3 AT) or the crossregenerated GL nerve (GL3 AT) produced a striking attenuation of
gapes compared with sham-operated rats (post hoc tests, P ⬍ 0.0001).
Indeed, the quinine-stimulated gaping behavior of the GL3 AT rats
emulated the behavior elicited by water in intact rats (SHAM-W; post
hoc test, P ⫽ 1.00), confirming that the GL was not capable of
supporting normal unconditioned gaping in response to quinine stimulation when the nerve reinnervated its non-native receptor field.
Importantly, the number of gapes in response to water on the final
habituation day was near zero (Fig. 1B), and there were no differences
between the groups (F6,29 ⫽ 1.42, P ⫽ .242). Thus, the effects of the
nerve surgery conditions on gaping to quinine cannot be attributed to
the mechanical or thermal features of the stimulus. Histological
analysis of taste buds was used to confirm nerve regeneration (Fig. 2).
gNST. Figs. 3 and 4 depict the quinine-stimulated Fos response
within the MD of the gNST, a gustatory subfield that occupies the
medial dorsal 1/6 of the gNST along its rostrocaudal extent (see Fig. 5,
inset) and typically displays intense Fos expression following intraoral quinine stimulation that quantitatively and characteristically
differs from that observed following water stimulation (24, 28, 51). In
the current study, similar results were realized for both SHAM-Q rats
and rats with reinnervation of the PT by its native nerve (GL3 PT).
In these two groups, quinine-stimulated Fos expression in MD in the
RgNST (F6,29 ⫽ 31.179, P ⬍ 0.0001), IRgNST (F6,29 ⫽ 36.274, P ⬍
0.0001) and ICgNST (F6,29 ⫽ 20.894, P ⬍ 0.0001) was comparable
(post hoc tests, P ⫽ 1.00) and significantly higher than the Fos
expression in water-stimulated controls (post hoc tests, P ⬍ 0.0001).
On the other hand, in rats with cross-wired PTs (CT3 PT), the
numbers of quinine-stimulated Fos neurons were comparable to those
in SHAM-Q and GL3 PT rats but only within the rostral-most section
(RgNST) of the nucleus (Fig. 3A; post hoc tests, P ⫽ 1.00). At more
caudal levels of the nucleus, in IRgNST and ICgNST, quininestimulated Fos expression decreased in the rats with cross-wired PTs
(CT3 PT vs. SHAM-Q, P ⬍ 0.02; CT3 PT vs. GL3 PT, P ⬍
0.0004; Fig. 3, B and C), despite that all three groups gaped at roughly
equivalent rates. In fact, within ICgNST, the quinine-stimulated Fos
response was no different from the response observed following water
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Fig. 3. Cross-regeneration of the gustatory nerves
differentially affected brain stem neuronal activation
in the medial dorsal (MD) subfield of the rostral
nucleus of the solitary tract (RgNST). Photomicrographs of the left gustatory NST (gNST) show the
characteristic Fos labeling in MD (arrow) observed at
this level of the nucleus in several of the surgical
groups: A: SHAM-Q. B: SHAM-W. C: CT3 PT.
D: GL3 AT. In rats with regeneration of the CT into
the PT (CT3 PT), quinine-stimulated Fos-labeling in
MD appeared comparable to that observed in quininestimulated control rats (SHAM-Q). On the contrary,
in rats with regeneration of the GL into the AT
(GL3 AT) quinine-stimulated Fos labeling resembled
that observed in water-stimulated control rats. Scale
bar ⫽ 250 ␮m; sol, solitary tract.
CROSS-WIRING OF GUSTATORY SYSTEM
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DISCUSSION
Fig. 4. Numbers of Fos-labeled cells (means ⫾ SE) observed in MD at three
rostrocaudal levels of the gustatory NTS analyzed: A: RgNST. B: intermediate
rostral (IRgNST). C: intermediate caudal (ICgNST). Solid circles represent
data from individual animals. Reinnervation of the PT by the native GL nerve
(GL3 PT) supported the typical (SHAM-Q) number of quinine-stimulated
Fos-labeled neurons throughout the nucleus. But, reinnervation of the PT by
the cross-regenerated CT nerve (CT3 PT) only partially supported “typical”
quinine-stimulated neuronal activity. In the RgNST, the numbers of quininestimulated Fos-labeled neurons in CT3 PT rats were comparable to those
observed in both SHAM-Q rats and control-regenerated GL rats (GL3 PT),
but, at the two more caudal levels, ICgNST, and CgNST, quinine-stimulated
neural activity in this group of cross-wired rats was decreased relative to
normal intact and GL-regenerated rats. Across all levels of the gNST, reinnervation of only the AT produced few Fos-positive neurons in response to
quinine stimulation, regardless of the nerve providing the input.
stimulation (P ⫽ 0.467). Thus, reinnervation of the PT by the
non-native CT nerve only partially restored the characteristic quininestimulated Fos-pattern (Fig. 5) that is typically observed in the gNST
following intraoral application of quinine (24, 28, 51).
In groups with only reinnervation of the front of the tongue (e.g.,
GL3 AT and CT3 AT rats), quinine-stimulated neural activity in
MD was significantly lower than that observed in SHAM-Q rats
within the RgNST (post hoc tests, P ⬍ 0.00001), the IRgNST (post
hoc tests, P ⬍ 0.00001), and the ICgNST (post hoc tests, P ⱕ 0.0001).
Moreover, the Fos response in these quinine-stimulated animals was
remarkably similar to the expression pattern (Fig. 5) observed
throughout the nucleus in water-stimulated controls (SHAM-W, post
hoc tests, P ⫽1.00).
PBN. As has been reported previously (26, 50, 61, 62), many
Fos-postive neurons were observed in the taste-responsive waist
AJP-Regul Integr Comp Physiol • VOL
Cross-regeneration effects. The main purpose of this investigation was to assess whether quinine-stimulated gaping and
the associated neural activity in brain stem gustatory nuclei
depended more on the receptor field origin of the taste input or
on the nerve that channels the signal into the brain or both.
Because either taste nerve was clearly able to support normal
quinine-stimulated gaping behavior when routed into the PT
but not able to do so when routed into the AT, the specific
nerve that channels the signal appears to play a negligible role.
Instead, the data point toward the tongue receptor cell field as
the critical factor. A straightforward explanation for the more
dominant role of the PT receptor cell field in the maintenance
of the gape response lies in the more prominent expression of
T2R receptors (which bind bitter-tasting ligands) in the PT
relative to the AT (1). Presumably, upon reinnervation of the
receptor cell field, some fibers in the reinnervating nerve
(either the GL or CT) become functionally connected to these
bitter ligand-activated receptor cells, which when stimulated
by quinine, then provide sufficient neural activity to drive the
gape response. Because of the paucity of T2R receptors in the
AT, the neural signal cannot reach a critical “threshold” no
matter which nerve reinnervates that field.
This interpretation presupposes the existence of a subset of
fibers in the CT that as a rule, normally provides input, like the
GL, into the neural circuits controlling unconditioned oromotor
rejection reflexes. CT nerve fibers innervating their native
receptor cell field in the AT in conjunction with an intact
palatal taste receptor field are capable of maintaining other
types of quinine-stimulated behaviors [e.g., sensory discriminations in a normal fashion (44, 46, 47) and even gapes to
sucrose conditioned to be aversive, when GL input is removed
(10)], but unconditioned gaping to quinine is by no means
normal (18, 27, 49). Nonetheless, previous findings lend credence to the suggestion that afferent axons in the CT are
capable of mediating some aspect of quinine-stimulated gaping
behavior. For example, as previously mentioned, bilateral GL
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region of the PBN in sham-operated animals following intraoral
stimulation with quinine but not with distilled water (F6,27 ⫽ 12.150;
P ⬍ 0.0001; post hoc test, P ⫽ .0008; Figs. 6 and 7). As with MD in
the RgNST, reinnervation of the PT by either the native GL nerve
(GL3 PT) or the non-native CT nerve (CT3 PT) restored the numbers of Fos-positive quinine-stimulated neurons to normal values
(post hoc tests, P ⫽ 1.00 compared with SHAM-Q), while reinnervation of the AT by either nerve (CT3 AT or GL3 AT) did not (post
hoc tests, P ⬍ 0.006 compared with SHAM-Q). In fact, quinine
stimulation in these AT-reinnervated animals yielded numbers that
were similar to those obtained in water-stimulated controls (post hoc
tests, P ⫽ 1.00).
In both the external lateral (F6,27 ⫽ 4.892. P ⬍ 0.002) and external
medial (F6,27 ⫽ 3.391 P ⬍ 0.013) subdivisions of the PBN, areas that
have also been implicated in gustatory processing (20, 26, 50, 62),
more Fos-positive neurons were found in sham-operated rats following stimulation with quinine compared with water (post hoc tests, P ⬍
0.035; Figs. 6 and 7). The mean numbers of Fos-positive neurons in
these external subdivisions across the nerve-manipulated groups appeared to follow a similar pattern observed for the waist area (e.g.,
normal quinine-stimulated Fos response with PT reinnervation but not
with AT reinnervation), but the comparisons failed to reach statistical
significance.
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CROSS-WIRING OF GUSTATORY SYSTEM
transection (CT intact) has been shown to dramatically attenuate the number of gapes to quinine (18, 27, 49), but they were
not eliminated entirely. Moreover, bilateral CT transection (GL
intact) decreased the numbers of quinine-stimulated Fos-posi-
tive cells throughout the gNST, albeit not as unequivocally as
GL transection (28). These findings, along with the electrophysiological results of Oakley (36), suggest that when the
“quinine-responsive” CT fibers innervate the PT taste cells
Fig. 6. Cross-regeneration of the gustatory nerves
differentially affected brain stem neuronal activation
in the waist area, the classic taste region of the PBN.
Photomicrographs of the left PBN show the characteristic Fos labeling in the subnuclei that comprise the
waist area: the ventral lateral subdivision (black
arrow), the central medial subdivision (white arrow),
and the cell bridges in between in SHAM-Q
(A), SHAM-W (B), CT3 PT (C), and GL3 AT rats
(D). With regeneration of the CT nerve into the PT
(CT3 PT), the quinine-stimulated Fos-labeling in the
waist area appeared similar to that observed in
quinine-stimulated control rats (SHAM-Q). On the
contrary, in rats with regeneration of the GL nerve
into the AT (GL3 AT), quinine-stimulated Fos-labeling appeared more like that observed in water-stimulated control rats. Scale bar ⫽ 250 ␮m.
AJP-Regul Integr Comp Physiol • VOL
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Fig. 5. Cross-regeneration of the gustatory nerves differentially affected the spatial distribution of quinine-stimulated Fos-labeled neurons in the gNST. The proportion
of Fos-labeled neurons (means ⫾ SE) in each subfield for different stimulus and nerve status conditions is shown. Q-SHAM (A) and SHAM-W (B) rats expressed
distinctly different patterns of activity in the nucleus. The “typical” quinine pattern is represented by gray bars; the “typical” water pattern is represented by white bars.
In control rats, the most discriminating feature between the patterns of activity produced by stimulation with quinine vs. water is the proportion of activated neurons in
medial dorsal subfield (MD) (compare arrows). Quinine-stimulated animals lacking innervation of the PT receptor field. D: CT3 AT. F: GL3 AT, both elicit patterns
of quinine-stimulated activity that is virtually identical to that observed in water-stimulated rats. On the contrary, animals with only innervation of the PT receptor field
do show a typical quinine-stimulated pattern if the receptor field is reinnervated by the native GL nerve (GL3 PT, C). If the PT is cross-wired with the CT nerve
(CT3 PT, E), however, the pattern of neuronal activity (cross-hatched bars) falls in between those seen for the quinine- and water-stimulated sham groups. LD,
lateral-dorsal; MidD, middle-dorsal; MD, medial-dorsal; LV, lateral-ventral; MidV, middle-ventral; MV, medial-ventral (see Ref. 51).
CROSS-WIRING OF GUSTATORY SYSTEM
(with a higher density of T2R expression), they could then
become adequately stimulated by quinine and unconditioned
gapes would be effectively triggered.
That the CT and GL fibers can provide input into the same
neural circuitry is further supported by anatomical and electrophysiological data. First, although the CT and GL terminate
in somewhat segregated regions of the gNST, there is nonetheless some overlap between their terminal fields (22, 31, 33).
It is important to note that the second-order neurons themselves
do not necessarily have to be the site of the convergence;
instead, interneurons may provide communication between
these pathways. Second, some cells in the gNST (52) and in the
PBN (20) respond to stimulation of both the AT and PT fields.
And finally, in a slice preparation, Grabauskas and Bradley
(15) found cells in the gNST that could be driven by electrical
stimulation of both the VIIth nerve (of which the CT is a
branch) and GL nerve afferent fibers.
AJP-Regul Integr Comp Physiol • VOL
A caveat in our interpretation, however, stems from what is
meant by adequate stimulation. For example, what if the
cross-regenerated GL (into the AT) could somehow be adequately stimulated by the taste receptor cells located there? It
is possible, in this case, that gaping would be elicited by
quinine, and thus input from the AT receptor field would be
“sufficient” to maintain the rejection behavior. The concentration of quinine used in this experiment was quite high from a
behavioral standpoint (⬃3 orders of magnitude above the
psychophysical detection threshold), but conceivably, even
higher concentrations of quinine might have effectively generated gapes in rats that had the GL cross-wired into the AT.
Nonetheless, the current results do distinguish the PT as
playing a special role in mediating unconditioned gaping behavior. The preferential involvement of the PT in this function
likely stems from its possession of the proper receptor cells
successfully innervated by the proper type of nerve fibers, both
of which are likely critical in restoring this behavior upon
reinnervation of taste buds.
The interpretation that we have offered to explain the current
outcomes, though simple and compelling, may undervalue the
very complex and remarkable means by which functional
reconnections between nerve fibers and taste receptor cells
likely occur in the lingual epithelium. Even in normal rats, the
mechanisms that guide appropriate functional connectivity in
the tongue are unknown. It is widely assumed that because
taste receptor cells undergo constant turnover while perceptual
stability is maintained (5, 11), there must be a matching
mechanism that allows gustatory fibers to couple with their
native taste receptor cells in such a way that signals are
appropriately channeled to brain circuitry to maintain function
(35). Such a process is notable considering the fact that the
axons comprising a taste nerve represent a heterogeneous
population: some fibers respond to sweeteners, some to bitter
compounds, some to sodium or other salts, some to acids (see
Ref. 43 for a review). In short, not all axons are functionally
the same within any taste nerve or across taste nerves. The
current findings imply that a comparable and even more striking
matching mechanism operates when a regenerating taste nerve
(with its own distinct set of fibers) invades a foreign taste
receptor cell field. In this case, experimentally rewired CT
nerve fibers were able to maintain gaping behavior when they
reinnervated taste cells in a non-native receptor field in the PT.
That the gustatory system was able to mediate a normal
taste-related behavior via experimentally induced novel projections from the tongue (at least the PT) is testimony to the
plasticity inherent within the system.
Though the “signal strength” explanation for the observed
effects of cross-wiring on gaping behavior enjoys the benefit of
parsimony, we cannot completely dismiss the possibility that
our manipulations of peripheral gustatory input somehow triggered central reorganizational events (e.g., 31, 33) that played
a role in the maintenance, or lack thereof, of quinine-elicited
gaping. Reorganization of central structures as a result of
cross-regeneration is not without precedence. Sur and colleagues (48) redirected retinal projections to the auditory thalamus in neonatal ferrets, and later, these animals possessed
visually responsive cells in the auditory thalamus and cortex.
Moreover, the cross-modal projection mediated visual behavior (56). These findings led the authors to suggest that the
function of central visual structures might be specified by their
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Fig. 7. Numbers of Fos-labeled cells (means ⫾ SE) observed in three subnuclei of the PBN: the waist area (A), the external lateral subdivision (B), and the
external medial subdivision (C). Reinnervation of the PT by either its native
GL nerve (GL3 PT) or the non-native CT nerve (CT3 PT) supported
the normal (SHAM-Q) number of quinine-stimulated Fos-labeled neurons in
the waist area. Reinnervation of the AT produced few Fos-positive neurons in
response to quinine stimulation regardless of the nerve providing the input.
Though significantly more Fos-labeled neurons were found in these two
external subdivisions in sham-operated animals following intraoral stimulation
with quinine compared with water, none of the nerve manipulations appeared
to produce a significant effect on the quinine-stimulated Fos activity.
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CROSS-WIRING OF GUSTATORY SYSTEM
AJP-Regul Integr Comp Physiol • VOL
neural circuit underlying gustatory rejection behaviors; but it
remains to be seen as to what extent these relationships are
causal.
Perspectives and Significance
The importance of the current findings is highlighted by
close to 20 years of nerve transection and behavioral work
across a variety of laboratories, including ours, that suggests
that gustatory nerves in rodents are to some extent functionally
specialized. Indeed, it is accepted in the taste literature that the
GL plays a privileged role in unconditioned gaping to aversive
compounds. The present findings place limitations on this view
and demonstrate that the signals in the CT are capable of
triggering these responses when the fibers of this nerve connect
themselves to the PT taste buds.
To our knowledge, these results are the first in which the
consequences of the cross-wiring of cranial sensory nerves
have been simultaneously assessed on behavior and neural
activity in an adult mammalian model. It is remarkable that
under our cross-wired conditions normal taste-elicited rejection
reflexes were restored. There was no a priori reason to expect
a favorable behavioral outcome in any rewired state in these
adult animals; the most parsimonious prediction would have
been that none of the cross-reinnervation conditions would
support normal gaping to quinine. This, however, was not the
case. Rather, the gustatory system was able to adapt successfully
to peripherally reorganized input, at least with respect to the
specific reflex rejection behavior measured here. In the future, it
will be important to test whether other taste-related functions
disrupted by lingual gustatory nerve transection can be maintained
under conditions in which the nerves are cross-wired. The compilation of such results should help to open new and important
questions about the scope of plasticity within the gustatory system, as well its potential to recover from nerve injury.
ACKNOWLEDGMENTS
We would like to thank Angela Newth, Mandi Arnett, and Elliot James for their
technical assistance, and Dr. Michael S. King in the Department of Biology at
Stetson University for the use of his microscope imaging equipment.
GRANTS
This work was supported by a grant from the National Institute on Deafness
and Other Communication Disorders R01-DC01628.
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