Group Title: Molecular Pain 2008, 4:43
Title: Characterization of mouse orofacial pain and the effects of lesioning TRPV1-expressing neurons on operant behavior
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Title: Characterization of mouse orofacial pain and the effects of lesioning TRPV1-expressing neurons on operant behavior
Series Title: Molecular Pain 2008, 4:43
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Creator: Neubert JK
King C
Malphurs W
Wong F
Weaver JP
Jenkins AC
Rossi HL
Caudle RM
Publication Date: 39722
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Research

Characterization of mouse orofacial pain and the effects of lesioning
TRPV I-expressing neurons on operant behavior
John K Neubert* 1,4,5, Christopher King', Wendi Malphurs1, Fong Wong3,
James P Weaver', Alan C Jenkins', Heather L Rossil,4 and
Robert M Caudle2,4,5


Address: 'Department of Orthodontics, College of Dentistry, University of Florida, Gainesville, FL, USA, 2Department of Oral Surgery, College of
Dentistry, University of Florida, Gainesville, FL, USA, 3Department of Prosthodontics, College of Dentistry, University of Florida, Gainesville, FL,
USA, 4Department of Neuroscience, College of Medicine, University of Florida, Gainesville, FL, USA and 5Evelyn F. and William L. McKnight Brain
Institute, University of Florida, Gainesville, FL, USA
Email: John K Neubert* jneubert@dental.ufl.edu; Christopher King cking@dental.ufl.edu; Wendi Malphurs pontomaniac@yahoo.com;
Fong Wong fwong@dental.ufl.edu; James P Weaver jpweaver@ufl.edu; Alan C Jenkins ajenkins@dental.ufl.edu;
Heather L Rossi hrossi@dental.ufl.edu; Robert M Caudle caudle@ufl.edu
* Corresponding author



Published: I October 2008 Received: 20 August 2008
Molecular Pain 2008, 4:43 doi:10.1 186/1744-8069-4-43 Accepted: I October 2008
This article is available from: http://www.molecularpain.com/content/4/1/43
2008 Neubert et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Rodent models of orofacial pain typically use methods adapted from manipulations to hind paw; however,
limitations of these models include animal restraint and subjective assessments of behavior by the experimenter. In
contrast to these methods, assessment of operant responses to painful stimuli has been shown to overcome these
limitations and expand the breadth of interpretation of the behavioral responses. In the current study, we used an
operant model based on a reward-conflict paradigm to assess nociceptive responses in three strains of mice (SKH I -Hrhr,
C57BU6J, TRPV I knockout). We previously validated this operant model in rats and hypothesized in this study that wild-
type mice would demonstrate a similar thermal stimulus-dependent response and similar operant pain behaviors.
Additionally, we evaluated the effects on operant behaviors of mice manipulated genetically (e.g., TRPVI k.o.) or
pharmacologically with resiniferatoxin (RTX), a lesioning agent for TRPVI-expressing neurons. During the reward-
conflict task, mice accessed a sweetened milk reward solution by voluntarily position their face against a neutral or heated
thermode (37-55oC).
Results: As the temperature of the thermal stimulus became noxiously hot, reward licking events in SKH I-Hrhr and
C57BU6J mice declined while licking events in TRPVI k.o. mice were insensitive to noxious heat within the activation
range of TRPVI (37-52oC). All three strains displayed nocifensive behaviors at 55oC, as indicated by a significant decrease
in reward licking events. Induction of neurogenic inflammation by topical application of capsaicin reduced licking events
in SKH I -Hrhr mice, and morphine rescued this response. Again, these results parallel what we previously documented
using rats in this operant system. Following intracisternal treatment with RTX, C57BU6J mice demonstrated a block of
noxious heat at both 48 and 55oC. RTX-treated TRPVI k.o. mice and all vehicle-treated mice displayed similar reward
licking events as compared to the pre-treatment baseline levels. Both TRPVI k.o. and RTX-treated C57BU6J had
complete abolishment of eye-wipe responses following corneal application of capsaicin.
Conclusion: Taken together, these results indicate the benefits of using the operant test system to investigate pain
sensitivity in mice. This ability provides an essential step in the development of new treatments for patients suffering from
orofacial pain disorders.


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Background
Orofacial pain encompasses a multitude of disorders,
including temporomandibular disorders (TMDs), trigem-
inal neuralgia, headaches, and myofascial pain, and
affects an estimated 20% of the U.S. population [1]. Some
distinct orofacial pain disorders, such as trigeminal neu-
ralgia and migraine headache have no analogous counter-
parts in other parts of the body (e.g., below the neck).
While systems underlying the orofacial pain pathway
appear to be similar to those elsewhere in the body, using
the same receptors, neurocircuitry (e.g., c-fibers, A6 fib-
ers), and neurotransmitters (e.g., substance P, glutamate),
it is unclear, however, whether trigeminal pain processing
is mediated differently. Therefore, investigating mecha-
nisms affecting the trigeminal system may provide unique
treatment options for those suffering from orofacial pain.

To this end, most pain models in rodents, which have typ-
ically targeted the hindpaw, have been adapted to the oro-
facial region. These models involve: the induction of
inflammation [2-4]; production nerve injuries [5-7]; neu-
rogenic inflammatory agents (e.g., capsaicin) [8,9]; for-
malin [10,11]; Complete Freund's adjuvant (CFA) [12].
One major limitation in these studies, though, involves
the assessment and interpretation of the behavioral
responses. Testing in the facial region provides a unique
set of hurdles, as many of the behavioral assays require
restraining the animals and are often associated with a
great degree of time and training on the part of the exper-
imenter. For example, assessment of mechanical sensitiv-
ity typically involves holding an animal while applying a
von Frey filament to the surface of the skin, but there are
visual anticipatory cues to contend with if the animal's
eyes are unshielded. Additionally, what experimenter
defines as a "painful response" can be as varied as a head
withdrawal to freezing, whereby an animal becomes com-
pletely unresponsive, which is indistinguishable from a
fear response. The investigator can overcome some of
these limitations by using a "hands-free" operant design
to assess facial pain behaviors.

Operant systems utilizes a reward-conflict paradigm,
whereby an animal can decide between receiving a reward
or escaping an aversive stimulus [13,14]. In this case, the
animal controls the amount of nociceptive stimulation
and can modify its behavior based on cerebral processing
[15,16]. We recently made great strides in bridging the gap
in orofacial thermal testing with the development of a
novel operant orofacial testing paradigm. We validated
and characterized an operant thermal assessment para-
digm in rats with both hot [17] and more recently cold
[18] stimuli, and evaluated this system using a number of
pain models, including capsaicin-induced neurogenic
inflammation [19], carrageenan-induced inflammation
[17], and menthol-induced sensitization [18]. However,


thus far our assessment of thermal processing has been
limited to rats.

The use of mice to evaluate nociception has gained
momentum, as most pain induction methods and behav-
ioral assays traditionally applied to rats can be easily
scaled for application in mice. The advent of transgenic
technology in the mouse has allowed an unprecedented
look into basic mechanisms relating to pain modulation.
There are many major classes of pain related molecules
that have been targeted using transgenic manipulations,
including neurotrophins, peripheral factors, opioids,
intracellular signal transducers, and non-opioid systems
[20]. However, behavioral assessments of pain in mice
still primarily use reflex-based methods. Operant tests for
mice such as the conditioned place preference task (CPP)
have been used extensively in field of addiction research
[21] and, to some degree, in the field of pain research [22].
Recent work in transient receptor potential channel mel-
astatin 8 (TRPM8) knockout mice has also used prefer-
ence tasks to evaluate the cold processing in the hindpaw
[23,24]. However, these assessments focus on pain induc-
tion in the hindpaw. Therefore, we sought to adapt our
operant orofacial assay, previously used with rats, for use
with mice. Although the nociceptive pathways are gener-
ally similar in rats and mice, some behaviors seen in rats
are not reliably noted in mice, such as the flinching behav-
ior following injection of formalin [25 Other differences
may exist between the two species that could affect per-
formance on our behavioral assay, such as level of atten-
tiveness, tendency to engage in exploratory behavior, and
the rewarding potential of the milk solution.

The current study had several goals. The first goal of this
study was to characterize the operant behavior of mice
exhibited in the presence of a range of neutral to hot stim-
uli and evaluate if their behavior is strongly influence by
the nociceptive potential of the stimulus, as is true of rats
[17]. Additionally, we wanted to evaluate the responses of
wild-type (w.t.) mice following induction of pain with
and without analgesic intervention. Once we established
this behavioral profile in genetically w.t. mice, our second
goal was to characterize operant behavior of TRPV1 k.o.
mice to thermal stimuli. Original behavioral characteriza-
tion of this strain has focused on withdrawal latency and
innate nocifensive behaviors, but no studies have assessed
the behavioral phenotype of this knock out in an operant
context. In addition, it is becoming apparent that the
encoding of thermal sensations relies on co-expression
and interaction among the thermal TRP channels, as well
as others. Combining genetic manipulations with phar-
macological manipulations can provide insights to the
how changes in these channels influence behavior
towards thermal stimuli. Thus, our final goal was to com-
pare the behavior exhibited by TRPV1 k.o. mice to w.t.


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C57BL/6J counterparts treated with resiniferatoxin (RTX),
a potent TRPV1 agonist used for lesioning agent TRPV1-
expressing neurons. This work will serve to enhance the
understanding of how nociceptive processes in the trigem-
inal system are integrated and will be critical for the
advancement of the field of orofacial pain research for the
transition into clinical treatments.

Materials and methods
General
Hairless SKH1-Hrhr, TRPV1 k.o., and age-matched wild-
type controls (C57BL/6J) mice were obtained from Jack-
son Laboratories (Bar Harbor, ME). TRPV1 k.o. and
C57BL/6J mice were lightly anesthetized with isoflurane
(1-2.5%, inhalation) and their fur was bilaterally
removed from the orofacial region using depilatory cream
at least 1 day prior to behavioral testing. All mice were
food fasted for 15 hrs prior to each facial testing session
and provided with standard food chow at the completion
of testing. Mice were tested at the same time of the day and
weighed weekly to monitor health. A recovery day was
included between the testing sessions to minimize nutri-
tional deficits due to fasting. Water was also made availa-
ble ad libitum before and after testing sessions. The mice
were placed into the behavioral procedure room 30 min
prior to testing and allowed to acclimate to the tempera-
ture and ambient noise of the room. Animal testing pro-
cedures and general handling complied with the ethical
guidelines and standards established by the Institutional
Animal Care & Use Committee at the University of Florida
and all procedures complied with the Guide for Care and
Use of Laboratory Animals [26].

General activity assessment rearing behavior
A potential confounding factor for completing an operant
behavioral test (see below) includes strain differences in
general activity or due to a specific treatment. For exam-
ple, if a drug produces significant sedation, one would
expect an animal to perform poorly on the operant task,
which could provide a false negative result in terms of
analgesic-potency for that drug. While no analgesic drug
were evaluated in the current study in terms of altered
rearing activity, we modified our rat rearing chamber
design [27] to accommodate the testing of the mice to
measure rearing activity as an assessment of general
behavior. An acrylic cylinder (11 cm diameter x 17.6 cm
height) was constructed with aluminum sheets placed
both on the floor and on 6.3-cm above the floor. The
metal siding was connected to a DC power supply and, in
series, to a multi-channel data acquisition module
(DATAQ Instruments, Inc) and the floor served as the
ground for the circuit. Unrestrained mice were placed sep-
arately into each cylinder and the data acquisition system
was activated. During a rearing event, the animal would
place its front paw on the metal side of the cylinder and


this would complete an electrical circuit that registered in
the computer. Each session lasted 10 min and the number
of contacts, duration of contacts, and the duration of each
contact was determined. Animals were tested 7 sessions at
the same time of the day (afternoon) over a period of two
and a half weeks.

Thermal testing
To change the effective size of the test box, we modified
our existing rat operant test chamber, which consisted of
a 20.3 cm W x 20.3 cm D x 16.2 cm H acrylic box, by plac-
ing an acrylic insert (7 cm W x 7 cm D x 8 cm H) onto an
elevated platform (6 cm off the floor) to effectively reduce
the size of the internal dimensions to better accommodate
mice. The existing opening (4 x 6 cm) was which was
lined with grounded metal (aluminum) tubing was also
reduced to a 1" x 1" opening using an acrylic face plate.
This again served as a stimulus thermode when connected to
a heated circulating water pump (Model RTE-7 D+,
Thermo Electron) [17]. The stimulus temperature was
adjusted from neutral to very hot (37 to 550C) and the
stimulus thermode temperature was verified for each
experiment using a contact thermometer (Fluke, Model
5411).

Unrestrained animals were placed separately in each test-
ing cage insert and the reward bottle containing diluted
(1:2 with water) sweetened condensed milk solution
(Nestle, room temperature) was positioned in proximity
to the cage such that the animal will be allowed access to
the reward bottle when simultaneously contacting the
thermode with its face (Figure 1; Additional file 1). The
metal spout on the watering bottle was connected to a DC
power supply and, in series, to a multi-channel data
acquisition module (WinDaq Data Acq DI-710-UH,
DATAQ Instruments, Inc). When the mouse completed
the task and drank from the bottle, the animal's tongue
contacted the metal spout on the water bottle, completing
an electrical circuit (Figure 1A, upper trace). The closed
circuit was registered in the computer and each spout con-
tact was recorded as a "licking" event. The threshold for
detection was set to eliminate noise and minimize arti-
facts. A separate circuit was established from the alumi-
num thermode to the animal by grounding the floor with
an aluminum sheet for recording of "facial contact" events
(Figure 1A, lower trace). The latter circuit is necessary to
determine if the animal is discouraged by the thermal
stimulus. The investigator monitored online data acquisi-
tion to ensure that each recorded licking event from the
first circuit corresponded to a recorded facial contact on
the tubing (the second circuit) to ensure that the animal
did not access the reward while avoiding the thermode,
and it minimized false-positive recordings of licks. A com-
plete session lasted 20 min. During offline data analysis,
the threshold for detection of the licking contacts was set


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lI oil, 11


1E IT' E LIC MG



11IRM )D STI JS

600
Time ( c)


I NTACI




120TA
1200


Reward bottle


Figure I
Mouse facial thermal operant assay. (A) Reward licking contacts (upper trace) and facial thermode stimulus contacts (lower
trace) acquired in a typical 20 min test session at 37oC. (B) Example of a hairless SKHI-Hrhr mouse completing the task by con-
tacting the thermode (left) while simultaneously licking from the reward bottle (right).


above background noise, to minimize false positive event
registration and events typically registered as > 5.0 V. An
event was registered when the signal went above thresh-
old and ended when the signal dropped below threshold.
Data analyses were completed using custom-written rou-
tines (generously provided by Dr. Charles Widmer, Uni-
versity of Florida) in LabView Express (National
Instruments Corporation) and Excel (Microsoft). The
total number of reward licking events was determined for


each mouse and compared across experimental condi-
tions. Room temperature was maintained at 22 + 1 C for
all behavioral tests.
Animals were first trained to drink milk while contacting
metal tubing in a training box that was of similar dimen-
sions to the thermal testing box. The training boxes do not
have connections to the water bath and hence the metal
contact was equal to the room temperature. This lead-in


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0


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"-


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cc


ill


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0


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Thermode


Molecular Pain 2008, 4:43







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training period was necessary to acquaint the animals
with the task of locating the reward bottle. Animals were
considered trained when they achieved a total of 2 1000
licks in the training box. To evaluate the effects of food
fasting, a subset of animals were tested first without fast-
ing for three training sessions and then with overnight
fasting for three sessions over a period of 2 weeks.

Induction of neurogenic inflammation in the face
We induced pain and nociceptive sensitization in the oro-
facial region using the neurogenic inflammatory agent,
capsaicin as described previously by our group [19].
Briefly, capsaicin cream (0.035%, Chattem, INC, Chat-
tanooga, TN) was liberally applied to facial region of anes-
thetized (isoflurane, inhalation, 2%) SKH1-Hrhr mice (N
= 8) and left on for 5 min. The capsaicin was then
removed using a moist paper towel, followed by a 70%
EtOH wipe and animals were tested 30 min post-capsaicin
application at 47 C. One set of animals (N = 5) was
treated in the same manner with capsaicin, but also was
administered morphine (s.c., 0.5 mg/kg, 100 il) between
the scapulae immediately post-capsaicin application (30
min prior to testing). Note that the 0.5 mg/kg dose was
chosen to minimize side effects such as sedation, as this
may significantly impact completion of the operant task
[27].

To evaluate the effects of morphine on feeding and reward
behavior at a non-noxious stimulus temperature, we
tested a separate group of SKH1-Hrhr mice (N = 10) at
37C. Five animals received morphine (s.c., 0.5 mg/kg,
100 il) and five received vehicle (dH20, s.c., 100 il) and
then were evaluated 30' post-injection using the operant
system. The treatments were crossed-over for a second ses-
sion 1-week later and the treatment groups combined for
final analysis.

Resiniferatoxin (RTX) administration
Resiniferatoxin (RTX) is an ultrapotent TRPV1-specific
agonist that we have used as a molecular neurosurgical
agent to selectively lesion TRPV1-positive afferent neu-
rons [28,29]. Central administration of RTX or vehicle
(0.25% Tween 80 in phosphate buffered saline (PBS),
0.05% ascorbic acid) was achieved by injection into the
cisterna magna in TRPV1 k.o. (N = 5/treatment) and their
wild-type CB57BL/6 control mice (N = 5/treatment). The
skin overlying the occipital and cervical regions of the
head and neck was disinfected with Betadine. A 30-gauge
needle tip connected to Hamilton syringe via PE-10 tub-
ing was carefully directed to touch the mid- to lower por-
tion of the occiput. Note that contacting the bony surface
provided distinct tactile feedback as the tip was sequen-
tially moved caudally until the needle punctured the
atlanto-occipital membrane overlying the cisterna magna.
RTX (100 rig, 1 il) or vehicle (1 il) was injected slowly


over 10 seconds to allow the solutions to mix with the cer-
ebrospinal fluid (CSF). Animals recovered for 1-week
prior to behavioral testing.

A capsaicin eye-wipe test was completed [30] prior to and
following the injections to assess TRPV1 functionality.
Briefly, a 0.1% capsaicin solution (20 il) was placed onto
the cornea of the eye of gently restrained animals and the
number of eye wipes was counted for 1 min. A negative
response (no wipes) verified RTX-lesioning of TRPV1
afferent neurons and only animals that were negative were
used in the final operant comparisons. Capsaicin eye-
wipe was also completed on TRPV1 k.o. mice as a negative
control for comparison.

Statistical analysis
Statistical analyses were completed (SPSS Statistical soft-
ware, SPSS Inc) including Student's T-tests and ANOVAs
to evaluate the effects of temperature or treatment on the
reward licking outcome measure, and a general linear
model for multivariate analysis was used to assess the
effects of time and strain on rearing behavior. When sig-
nificant differences were found, post-hoc comparisons
were made using the Tukey HSD. *P < 0.05 was consid-
ered significant in all instances.

Results
General activity and rearing behavior
We used a rearing chamber with automated data acquisi-
tion to assess the overall activity of three strains of mice.
The number of rearing events significantly decreased for
SKH1-Hrhr and CB57B1/6J strains over the two-week test
period, but not TRPV1 k.o. mice (Table 1A). The duration
of rearing events was not significantly different for any of
the strains (Table 1B). When comparing the number of
rearing events between the strains over the 7 sessions, we
found that there was a significant time effect (F,31 = 5.65,
P < 0.001), but not a significant time*strain interaction
(F12,64 = 1.59, P = 0.116). This was also true of rearing
duration: time (F6,23= 3.99, P= 0.007); time*strain (F12,48
= 0.89, P = 0.568). Tests of within-subjects effects demon-
strated a significant time effect (F6,216 = 5.82, P < 0.001),
but not a significant time*strain interaction (F12,26 =
1.74, P = 0.059). This was again similar for the duration
of rearing: time (F6,168 = 3.18, P = 0.006); time*strain
(F12,168 = 0.79, P = 0.655). Tests of between-subjects
effects on number of events demonstrated a significant
difference between the strains (F2,36 = 3.92, P = 0.029),
with post-hoc tests revealing that the hairless SKH1-Hrhr
mice had significantly greater rearing activity as compared
to the C57BL/6J strain. The duration of rearing followed
in a similar fashion (F2,28 = 4.18, P = 0.026). For both
measures, the TRPV1 had an intermediate level of activity,
but were not significantly different from either of the
other two strains.


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Table I: Rearing behavior.


http://www.molecularpain.com/content/4/1/43


STRAIN (Mean SEM)


A. EVENTS
Session

I
2
3
4
5
6
7
Significance level

B. DURATION
Session

I
2
3
4
5
6
7
Significance level


Hairless


103.3 9.6
77.8 12.8
*58.7 6.4
70.1 8.0
72.5 10.5
*58.0 8.2
*51.0 5.8
(F,176 = 3.5, P = 0.003)


Hairless


57.4 6.1
59.5 8.5
45.5 7.0
52.1 7.3
52.1 7.3
46.9 7.5
51.2 7.5
(F6,168= 0.46, P = 0.837)


C5 7BL/6J


50.6 5.3
50.6 5.5
34.1 5.1
44.6 7.8
3 1.8 3.2
33.0 4.7
28.9 5.2
(F,69 = 2.9, P = 0.014)


C5 7BL/6J


38.6 5.0
38.5 3.1
3 1.3 4.2
41.6 5.3
34.6 3.2
30.2 2.8
26.0 4.0
(F,,69 = 1.9, P= 0.093)


TRPVI k.o.


53.1 3.8
71.6 10.4
54.8 14.1
48.3 6.8
40.1 9.7
52.4 8.3
37.5 6.3
(F6,60= 1.5, P = 0.201)


TRPVI k.o.


48.5 5.7
56.0 7.2
41.7 7.1
54.6 9.1
36.3 8.3
39.4 7.1
37.2 7.9
(F,60 = 1.2, P= 0.324)


General activity of the three strains of mice (C57BL/6J, TRPVI k.o., hairless SKH I-HRHR) was assessed by determining the number of rearing
events and duration of the rearing events over the 7-sessions. There was a significant decrease in rearing events for the hairless and C57BL/6J
strains (ANOVA, *P < 0.05).


When we evaluated the average rearing (e.g., duration/
event ratios) across seven testing sessions, there was a sig-
nificant effect of time on this outcome (F6,23 = 4.06, P =
0.006), but not a significant time*strain effect (F12,48 =
1.40, P = 0.200). The hairless (F6,16 = 3.13, P = 0.006) and
CB57B1/6J (F6,69 = 3.8, P = 0.003) mice had significantly
higher ratios over time, but each strain had values that
were not significantly different from each other by the sev-
enth session (Figure 2). The within-subject effects of time
were significant (F6,16 = 5.41, P < 0.001), but time*strain
effects were not significant (F12,168 = 1.36, P = 0.191).
There was no significant between-subjects effects for strain
(F2,28 = 1.94, P = 0.162). Overall, the decrease in the rear-
ing events and duration across testing sessions indicate
that all three strains habituate to the testing environment.
The rearing duration/event ratio indicates that while there
may initially be differences in activity level between
strains, these differences are no longer significant with
repeated testing.

2. Effects of thermal stimuli on operant behavior (Figure 3)
The mice completed the reward-conflict task in a fashion
similar to rats [17], whereby they would contact a ther-
mode with their face to access a reward bottle, generating
an electrical signal that was acquired for analysis (see Fig-
ure 1A, Babove). There was a significant increase in reward
licking events when animals were fasted overnight (Figure
3A). During the unfasted sessions, there was a significant


effect of testing day (F2,72 = 16.95, P < 0.001). The first ses-
sion had significantly lower licks than the second and
third sessions. This initial increase may be due to animals
learning the task; however, the outcomes during the fasted
sessions were relatively stable, as there was not a signifi-
cant effect due to test session on licks for the fasted ses-
sions, indicating that the behavior was relatively stable
with fasting and not indicative of further learning-related
increases.

There was a significant effect of temperature on licking
behavior for all three strains: SKH1-Hrhr (F4,49 = 23.61, P <
0.0001), C57BL/6J (F3,9 = 40.26, P < 0.0001), TRPV1 k.o.
(F3,19 = 5.89, P = 0.007). Post-hoc analyses revealed that
the SKH1-Hrhr and C57BL/6J mice displayed the normal
operant pain behavior of significantly decreasing reward-
licking performance as the temperature increases into the
noxious hot range (Figure 3B, C). The TRPV1 k.o. mice
(Figure 3D); however, exhibited a rather flat response
with stimulus temperatures of 37-52 C, with no signifi-
cant decrease in reward licking across this range. There
was a significant decrease in reward licking only at the
highest temperature tested, 55 C.

We used the SKH1-Hrhr strain extensively in this and other
studies; however, we realize that this is not the typical
strain used to study pain, unlike the C57BL/6J or 129
strains. Therefore as part of the thermal-stimuli compari-



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1.4-


1.2-


1.0-



C,

0.6-
0E

-- 0.4-


0.2-


0.0-


1 2 3 4 5 6 7
Test Session
Figure 2
Rearing behavior. General activity of the three strains of mice (C57BL/6J, TRPVI k.o., hairless SKHI-Hrh') was assessed by
monitoring rearing activity. The amount of time (sec) per rearing event was calculated and plotted over several test sessions,
completed over the course of 2.5 weeks. There was a significant increase in the time/event ratio for the hairless and C57BL/6J
strains over time; however, there were no between strain differences at the later time points, indicating a similar level of
accommodation for the strains. *denotes values significantly higher than the first session (P < 0.05).


son, we evaluated the response of the SKH1-Hrhr strain as
compared to the C57BL/6J strain, relative to their respec-
tive baseline 37 C results (Figure 4). We found that there
are raw value differences when testing different strains at
these stimulus temperatures (Figure 4A); however, the
direction and magnitude of the response to nociceptive
stimuli was the same between the strains, as indicated
when licking events are normalized to 370C (Figure 4B).
The hairless strains tend to consume more at each temper-
ature, but the relative change in performance with a pain-
ful stimulus is similar for both strains of mice.

3. Effects of neurogenic inflammation on operant behavior
We previously tested the pain sensitivity of rats following
capsaicin-induced neurogenic inflammation and evalu-
ated the effects of a low-dose of morphine on this model
of thermal hyperalgesia [19]. Here we evaluated the effects
of capsaicin and capsaicin/morphine on operant licking
reward behavior in the SKH1-Hrhr strain, tested at 47C
(Figure 5). We found that there was a significant treatment
effect on reward licking when animals were treated with


capsaicin and capsaicin/morphine (F2,22 = 7.70, P =
0.003). Post-hoc analyses reveal that the capsaicin-treated
animals had significantly lower licking events as com-
pared the baseline session (no treatment) and this hyper-
algesic response was reversed with treatment of morphine.
The capsaicin/morphine-treated animals had licking
events similar to baseline levels, but significantly higher
than the capsaicin-treated animals.

4. Effects of intracisternal RTX on w.t. mice and TRPVI-
k.o. mice
We wanted to compare the behavioral effects of pharma-
cological removal of TRPV1 using RTX with the genetic
removal of TRPV1. Not surprising, TRPV1 k.o. mice were
completely insensitive to corneal application of capsaicin,
as they had a significantly lower eye-wipe response as coz-
mpared to untreated w.t. C57BL/6J mice (Figure 6A). In
fact, these animals were completely unresponsive, with
the exception of an animal or two removing the liquid
with a few wipes. The w.t. C57BL/6J mice treated with
i.c.m. RTX also had a significantly lower response com-


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* C57BL/6J

* TRPV1 knockout
* Hairless SKH1-


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A. Hairless SKH1- training EUnfasted Fa B. Hairless SKH1-
E Unfasted BFasted

3000 3000 *
2500h 2500
4 2000 4 2000
a 1500 ** 1500
W 1000 1000
A' 100
500 + *. 500
0 0


1 2 3 4 5
Training Session
C. C57BL/6J


3000
2500
2000
1500
1000
500
0


I I


*- I


m m


N.T.


37 46 49 52 55
Temp (C)


37 46


i 4 i


T 4


4 4.


F---
--

r I r-^


49 52 55


Temp (C)
D. TRPV1 knock-out
3000
2500
w 2000
a 1500
W 1000
500
0
37 46 49 52 55
Temp (0C)


Figure 3
Operant reward licking events for three different strains of mice. Hairless mice significantly increased their reward
licking events (A) when fasted overnight (N = 13-27/session). Unfasted animals had significantly lower (+P < 0.05) licking
events in session I as compared to session 2, but there was no difference between session 2 and 3, and overall the fasted ses-
sions produced significantly higher performance as compared to the unfasted sessions. Hairless (B) and C57/BL6 (C) mice dem-
onstrated a stimulus response such that increasing temperatures significantly reduced the number of reward licking events,
indicating aversion to the more noxious stimuli. The TRPVI knock-out mice (D) were relatively insensitive to temperatures <
52oC, as their responses in the noxious heat range of 46-52oC produced responses similar to baseline 37oC testing conditions.
These knock-out mice only demonstrated a significant decrease in reward licking events when they were tested at the highest
stimulus temperature of 55oC. A total of N = 10 hairless, N = 5 CB57BL6, and N = 5 TRPV I knock-out mice were used to
generate these data. N.T. = not tested. *Significantly higher (P < 0.05). Note that there were no significant differences between
male and female mice when comparing the number of licking events during the unfasted and fasted sessions, therefore their
data was pooled.


pared to naive and vehicle (i.c.m.) w.t. animals (Figure
6A). There was no difference in the response between
TRPV1 k.o. and RTX-treated C57BL/6J mice.

For the operant testing, we found at 48 C, the TRPV1-k.o.
mice had significantly higher licking events as compared
to the C57BL/6J mice, indicating that the k.o. mice were
insensitive to the hot stimulus that is within the activation
range of TRPV1. When treated with RTX, the C57BL/6J
w.t. mice had significantly higher licks as compared to


vehicle treated animals, on par with the TRPV1-k.o. mice
(Figure 6C). When tested at 55 C, all of the TRPV1-k.o.
mice (vehicle, RTX) had significantly lower licks as com-
pared to baseline (37C, Figure 6B). In TRPV1 k.o. mice,
no differences in licking behavior was observed at any of
the temperatures between mice treated with RTX as com-
pared to vehicle treated mice (Figure 6B). The RTX-treated
CB57BL/6J mice had significantly higher licks as com-
pared to the vehicle treated animals when tested in the
noxious heat range. Taken together these results confirm


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Hairless

* C57BL/6J


A. Reward Licking Events


B. Normalized Reward Licking Events


, 100

2 75

I 50
S'S


N.S.
-I I


S 37
37


N.S.








47


Temp (oC)
Figure 4
Operant reward licking comparison of hairless SKHI-Hrhrversus C57BL/6J wild-type mice. The hairless SKHI-Hrhr
mice had significantly greater (+P < 0.05) licking events as compared to the C57BL/6J mice at both neutral (37oC) and hot
(47oC) stimulus temperatures (A). When the data was normalized to the neutral temperature (37oC) as a percent baseline for
each strain, the differences between the two strains were not significant (N.S.). For both strains, there was a significant
decrease (*P < 0.05) in both raw and normalized licking events at 47oC as compared to 37oC, indicating an aversive response
elicited by the noxious stimulus temperature.


that RTX is specific for TRPV1 and that the effects of phar-
macological lesion are not the same as genetic removal of
TRPV1. These results indicate that another high-threshold
receptor is likely linked to TRPV1. Reduction of this other
receptor can reduce sensitivity to high thermal stimuli in
the w.t. mice, thus, producing a significantly higher lick
output. Note that naive animals in both the TRPV1-k.o.
and C57BL/6J groups performed similarly to the vehicle-
treated animals of each respected group (data not shown).

Discussion
Uncontrolled pain remains a public health epidemic, with
countless people suffering from a multitude of disorders
that cost society billions of dollars annually. The Ameri-
can Pain Society recently released a statement indicating
that translational approaches to pain relief (i.e., bench to
bedside) would have "immediate and profound benefits"
[31]. Despite significant advances made to our under-
standing of molecular pain mechanisms, few novel anal-
gesic therapies have managed to reach clinical practice
[32]. This failure to translate from bench to beside is in
part due to the use of inefficient behavioral assays in ani-
mal models of pain. Many assays typically require that the
investigator both apply the pain stimulus and evaluate a
reflexive response by the animal. This can be both time
consuming and highly subjective. The use of these assays


to model and quantify pain in animals is a major bottle-
neck in the development of new analgesics, and providing
obstacles in the validation and optimization of clinical
treatment strategies.

The use of mice as the preferred model for pain testing
provides the opportunity to utilize genetically-altered
(i.e., knock-out) strains to study specific targets related to
pain processing. The drawback of behaviorally testing
mice relates to difficulties in producing fast and reliable
results using an animal known for being skittish and
jumpy. In this study, we have overcome these traits using
our operant test paradigm by presenting a system whereby
the investigator is removed from the field and the animal
tests itself. We build on our prior work completed in rat
models of orofacial pain [17,19,27].

As part of our battery of behavioral assessment assays, we
evaluate the effects of a variety of factors (e.g., drugs) on
general activity to determine if these factors could have an
impact on the ability of an animal to perform the operant
task. For example, we previously demonstrated that doses
of morphine (> 2.5 mg/kg) produced significant reduc-
tion of rearing behavior in rats [27]. Another factor that
may affect performance relates to inherent differences
between strains of animals, as some strains may be rela-


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H


2,500

2,000

1,500

S1,000

500


0-


1251


47*


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B.


2000


1750


1500


1250


1000


750


500


250


0


2000


1750


1500


1250


1000


750


500


250


Capsaicin Capsaicin/
Morphine
Treatment


Morphine


Treatment


Figure 5
Effects of neurogenic inflammation and morphine on operant reward licking behavior in hairless SKHI-Hrhr
mice. Hairless SKHI-Hrhr mice treated with capsaicin cream (0.035%, topical, 5') had significantly lower reward licking events
(*P < 0.05) as compared to baseline (A) at 47oC. Morphine (0.5 mg/kg) given concurrently with the capsaicin-treatment pro-
vided a significant antihyperalgesic effect under similar testing conditions, as the reward licking counts were significantly higher
than the capsaicin-alone group (A). A different set of animals (N = 10) was tested with either morphine (0.5 mg/kg, s.c.) or
water (100 [Il, s.c.) at 37oC in order to assess the effects of morphine on feeding and reward behavior in the absence of a pain-
ful stimulus (B). There were no significant differences between the two treatment groups at this neutral stimulus temperature.


tively more active or inquisitive. Therefore, prior to testing
the different mice strains (SKH1-Hrhr, C57BL/6J, TRPV1
k.o.) on the operant device, we evaluated them using the
rearing assay. We found that the general activity for these
three strains was not so much a factor, as they all had sim-
ilar responses. In fact the rearing duration/event outcome
was virtually identical for each strain by the end of two
week acclimation period. We concluded that the general
activity or exploratory behavior differences between these
strains of mice would not likely influence their ability to
complete the operant task. The rearing data indicates a
potential habituation to the environment for all strains
over the two-week test schedule, with each strain display-
ing decreased events and duration. However, in contrast,
we found that the operant results improved with each ses-


sion; indicating that motivation and cognitive factors are
encouraging the animal to complete the task.

When we decided to modify the existing rat operant unit
to test mice, we searched for a mouse that was equivalent
to the hairless Sprague-Dawley (S.D.) rat that we routinely
use and found that the hairless SKH1-Hrhr mouse strain.
While the SKH1-Hrhr mouse is typically used in dermato-
logical studies, it is not the typical strain used in pain stud-
ies. Here we demonstrated that this strain indeed follows
a normal thermal stimulus response, as compared to the
wild-type C57BL/6J mice. Additionally, they respond in
the appropriate fashion following capsaicin-induced pain
with decreased operant licking behavior, and then with
morphine-rescue, with return of licking events to baseline


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A.


a)
Lu


C,)





W-
UJ


None


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A. Capsaicin Eye Wipes

60 50.2 53.5

50
TREATMENT

40] None
U 30 RTX

S20 1 Vehicle
RTX)
10
0.2 0.2
0
TRPV1 k.o. C57BL/6J
Strain

B. TRPV1 k.o. C. C57BL/6J w.t.

1600- 1600-


1200 1200-


800 800


400 400


0 0
48 55 48 55
Temp (oC) Temp (C)
Figure 6
Intracisternal (ICM) injection of RTX blocked heat pain in the face for wild-type, but not TRPVI knock-out
mice. Animals treated I-week previously with either RTX (100 ng, I Il) or vehicle (0.25% Tween 80 in PBS) were tested using
the capsaicin eye-wipe assay and using the operant orofacial device at 48 and 55oC. TRPVI k.o. mice and RTX-treated C57BL
6J mice were completely insensitive to capsaicin (0.1 %, 20 ~il, corneal application), while vehicle-treated C57BL6J had a
response similar to non-treated animals (A). TRPVI k.o. mice treated with RTX responded similarly to vehicle treated k.o.
mice at both 48 and 55oC (B). C57BL6J mice treated with RTX were not sensitive to the 48oC stimuli (B) and behaved like the
TRPVI k.o. mice. Interestingly, the RTX-treated C57BL6J mice demonstrated a significant increase in response at 55oC, with
licking events increased even above the TRPVI k.o. mice. = significantly lower (P < 0.05), compared to baseline; ** = signifi-
cantly lower compared to RTX-treated animals.



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levels in the presence of capsaicin. As demonstrated in Fig-
ure 3, they are quick to learn the task, therefore minimiz-
ing training time. We find this hairless strain to be
extremely docile and easy to handle, more convenient to
use, as we do not need to shave them in order to test them.
The video clip provides a real-time demonstration of the
ease of using these animals. Note how quickly the animals
are independently and successfully completing the task
once placed in the box. Given these traits, the SKH1-Hrhr
mice are certainly appropriate and ideal for use in future
pharmacological studies.

Trigeminal nociceptors project to the nucleus caudalis and
synapse with second-order neurons in the superficial lay-
ers, which are organized in the same way as the dorsal
horn of the spinal cord, with the nucleus caudalis being
laminated like the dorsal horn in spinal cord [33-36]. In
fact, the nucleus caudalis extends and merges with the spi-
nal dorsal horn in the cervical spinal cord [37] and has
been termed the medullary dorsal horn [38]. Neurons
within this superficial region have been shown to respond
to cooling, cold, warming, and hot stimuli [39-42] and
chemical irritants [42,43]. As it is considered a correlate to
the spinal cord dorsal horn, the trigeminal spinal nucleus
and trigeminal sensory system provides a relevant region
with respect to the understanding of pain mechanisms in
general [44,45]. Given this, we targeted this region using
the ultrapotent TRPV1 agonist, RTX, in order to assess
changes in orofacial pain.

We found that w.t. C57BL/6J mice treated with RTX had a
significant decrease in the ability to sense hot (48 C) and
very hot (55 C) stimulus temperatures. This contrasts the
response of the TRPV1 k.o. mice, as they were only signif-
icantly affected at the hottest (55 C) temperature. These
results appear to be inconsistent with the work completed
by Caterina, et al., as they demonstrated a significant dif-
ference at high temperature stimuli, such as 55 C on the
hot plate assay, when comparing the TRPV1 k.o. versus
the w.t. mice [46]. This is interesting because the thermal
assay difference between the reflex and operant tasks may
explain this discrepancy. For example, for the reflex-based
hot-plate assay, the difference between the k.o. and w.t.
groups may be a function of the wild-type mice being
more sensitive to a rapid temperature increase, so the
response may be a function of the temperature difference
detection plus the thermal pain producing the response.
In addition, when within-group effects were evaluated for
temperature, there was a decreased latency for both the
TRPV1 k.o. and w.t. mice. While these authors did not
report on this comparison, there appears to be a signifi-
cant decrease in hot plate latency in the TRPV1 k.o. group
as the stimulus moves to higher temperatures. This is con-
sistent with what we found using the operant test, with a
significant decrease in licking outcome at the 55C stim-


ulus. Collectively, these results indicate that while RTX is
specific for the TRPV1 receptor, other receptors co-
expressed (e.g., TRPV2) on the same neurons with TRPV1
may be susceptible to the RTX-lesioning effects within the
nucleus caudalis. Another possibility is that a population
of A-6 fibers expressing TRPV1 is also lesioned following
RTX-treatment in the w.t. mice.

When we evaluated the pain sensitivity of these mice
using an unlearned-reflexive measure such as the capsai-
cin eye-wipe assay, the RTX-treated C57BL/6J mice
appeared to respond identically as the TRPV1 k.o. mice.
This brings up an interesting observation that this partic-
ular type of assay is sufficient for evaluating a gross-
impairment in the nociceptive signaling pathway. How-
ever, it cannot tease out subtle differences uncovered
using the operant assay, such as the different responses of
these two groups at 55 C. This additional information
may be relevant for the development of novel analgesics.

In conclusion, we have successfully used the operant oro-
facial assay to evaluate and characterize thermal pain sen-
sitivity in mice, thus providing a revolutionary step in the
ability to model and study pain. Pain is ultimately experi-
enced as a culmination of complex information from the
periphery (including the location, intensity, quality, and
time course). While in vitro studies can provide insight
into the individual components, adequate behavioral
assessment of animal models is requisite for understand-
ing the integration of these components into the percep-
tion of pain. These findings demonstrate that operant
methods, which reflect physiological and cerebral
processing of pain, can provide insights not possible with
reflex-based testing alone. Such insight may enhance our
understanding of pain and ultimately lead to the ability to
treat uncontrollable. This replicates what occurs with
humans, whereby a person may need to choose between
tolerating pain in order to receive some reward (e.g., going
to a nice dinner with a migraine headache versus not
going to a bad restaurant with the same type of headache).
As pain spans any number of diseases, ranging from dia-
betes to cancer, providing a means of quickly identifying
new analgesic agents would provide a tremendous soci-
etal benefit, and this mouse operant system provides such
a way.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
IN conceived and participated in the design and supervi-
sion of the study, analyzed the data and drafted the man-
uscript. CK was involved with the behavioral testing and
writing portions of the manuscript. WM and JW were
involved with behavioral testing. FW, AJ, and HR were


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involved with the design of the study and behavioral test-
ing. RC was involved with the design of the study and
interpretation of the data. All authors read and approved
the final manuscript.


Additional material


Additional file 1
Movie showing a hairless SKH-Hlhr mouse testing itself on the operant
orofacial device. This movie demonstrates the ease of testing a mouse in
an investigator-independent fashion using the operant orofacial device.
Note the technician placing the mouse in the testing box at the beginning
of the video -this is the last time the technician interacts with the animal
during the trial. At 16 sec, the animal initiates the firstsuccessful contact
with the thermode and reward bottle. We pan to the computer at -31 sec
to illustrate the online recordings (top trace = reward licking signal; bot-
tom trace = stimulus thermode signal), and at -43 sec, we demonstrate
how the uncompressed, high-speed acquisition signal appears. Please note
that each peak on the top trace represents a single lick.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1744-
8069-4-43-Sl.mlv]


Acknowledgements
This work was supported by grant #5R21 DEO 16704-02, National Institute
of Dental and Craniofacial Research, and grant #1 R43DA026220-01,
National Institute on Drug Abuse, National Institutes of Health, Depart-
ment of Health and Human Services, Bethesda, MD, USA. We thank Dr.
Charles Widmer for providing the custom-written Labview software rou-
tines used in the analysis of the rearing and operant facial data.

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