Group Title: Molecular Pain 2006, 2:37
Title: Characterization of cold sensitivity and thermal preference using an operant orofacial assay
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Title: Characterization of cold sensitivity and thermal preference using an operant orofacial assay
Series Title: Molecular Pain 2006, 2:37
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Creator: Rossi HL
Vierck CJ
Caudle RM
Neubert JK
Publication Date: 39064
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Molecular Pain BioMedCentra


Characterization of cold sensitivity and thermal preference using an
operant orofacial assay
Heather L Rossi', Charles J Vierck Jr3,4, Robert M Caudle2,3,4 and
John K Neubert*1,3,4

Address: 'College of Dentistry Department of Orthodontics, University of Florida, 1600 S.W. Archer Road, P.O. Box 100444, Gainesville, FL
32610-0444, USA, 2College of Dentistry Department of Oral Surgery, University of Florida, 1600 S.W. Archer Road, P.O. Box 100416, Gainesville,
FL 32610-0444, USA, 3College of Medicine Department of Neuroscience, University of Florida, 100 Newell DR., P.O. Box 100015, Gainesville, FL
32610-0444, USA and 4Evelyn F. and William L. McKnight Brain Institute, University of Florida, 100 Newell DR., P.O. Box 100015, Gainesville,
FL 32610-0444, USA
Email: Heather L Rossi; Charles J Vierck; Robert M Caudle;
John K Neubert*
* Corresponding author

Published: 13 December 2006 Received: 23 October 2006
Molecular Pain 2006, 2:37 doi:10.1 186/1744-8069-2-37 Accepted: 13December2006
This article is available from:
2006 Rossi et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: A hallmark of many orofacial pain disorders is cold sensitivity, but relative to heat-
related pain, mechanisms of cold perception and the development of cold allodynia are not clearly
understood. Molecular mediators of cold sensation such as TRPM8 have been recently identified
and characterized using in vitro studies. In this study we characterized operant behavior with
respect to individually presented cold stimuli (24, 10, 2, and -4C) and in a thermal preference task
where rats chose between -4 and 48oC stimulation. We also evaluated the effects of menthol, a
TRPM8 agonist, on operant responses to cold stimulation (24, 10, and -4C). Male and female rats
were trained to drink sweetened milk while pressing their shaved faces against a thermode. This
presents a conflict paradigm between milk reward and thermal stimulation.
Results: We demonstrated that the cold stimulus response function was modest compared to
heat. There was a significant effect of temperature on facial (stimulus) contacts, the ratio of licking
contacts to stimulus contacts, and the stimulus duration/contact ratio. Males and females differed
only in their facial contacts at 10C. In the preference task, males preferred 48oC to -4C, despite
the fact that 48oC and -4C were equally painful as based on their reward/stimulus and duration/
contact ratios. We were able to induce hypersensitivity to cold using menthol at I 0C, but not at
24 or -4C.
Conclusion: Our results indicate a strong role for an affective component in processing of cold
stimuli, more so than for heat, which is in concordance with human psychophysical findings. The
induction of allodynia with menthol provides a model for cold allodynia. This study provides the
basis for future studies involving orofacial pain and analgesics, and is translatable to the human

Page 1 of 10
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A hallmark of many orofacial pain disorders is cold sensi-
tivity, but unlike heat-related pain, the mechanisms
underlying cold perception and the development of cold
hypersensitivity are less clearly understood. Most cold
research has focused primarily on in vitro or immunohis-
tochemical studies; in contrast to heat pain, few behavio-
ral assays relating to cold are available. The hindpaw
acetone cooling assay [1,2] has been used to evaluate cold
allodynia, but this test is difficult to quantify in terms of
the temperature delivered, and it is difficult to separate the
mechanical sensation of wetness from the onset of skin
cooling. Recently, Allchorne et al. demonstrated a stimu-
lus-response function to cold stimulation using a peltier
device and recorded hindpaw withdrawal latency as the
outcome measure in freely moving animals [3]. This
method is more quantifiable than the acetone assay and
does not involve restraint-related stress; however, this
reflex-based assay provides limited information regarding
evaluation of the central processing of pain. In contrast,
use of operant assays allows for a more complete assess-
ment of these aspects of pain processing. Mauderli et al.
[4] and Vierck et al. [5] assessed escape latency and dura-
tion in an operant assay and found that escape duration
revealed stimulus-response relationships better than
latency, and the animals were more sensitive to increasing
heat than to increasing cold for stimulation of the plantar
surfaces of the paws.

We previously developed an operant behavior system to
evaluate thermal sensitivity in the face [6]. This assay pro-
vides a useful means of evaluating animal models of
trigeminal pain in a manner that is relevant and translata-
ble to humans. The objectives of this study were to char-
acterize operant behavior with respect to cold stimuli
applied to the face and to evaluate the effects of menthol
on that behavior. Menthol is a modulator of cold sensa-
tion [7-9], and it stimulates the TRPM8 receptor [10,11].
TRPM8, a member of the transient receptor potential
(TRP) receptor family, is a molecular mediator involved in
cold sensation and is activated by temperatures below
25 C [10,11]. We hypothesized that increasingly cold
stimuli would affect behavioral outcome measures in a
manner similar to stimulation of the paws and that appli-
cation of menthol would enhance cold-induced pain.

Here we substantiated the stimulus response curve for
cold and found that it was modest relative to heat on the
face. However, the thermal preference paradigm indicated
that cold is especially aversive, compared to a level of heat
stimulation that produced comparable avoidance behav-
ior. The importance of describing a stimulus-response
function was revealed by tests following application of
menthol, which enhanced avoidance only for 10 C stim-
ulation. Using menthol in the presence of a cool stimulus

appears to provide a model of thermal allodynia analo-
gous to capsaicin-induced hypersensitivity to heat, and
taken together with the -4 C stimulus, allows for further
translatable studies regarding cold pain.

Behavioral response to cold and the effects of sex
In order to characterize normal behavioral responses of
male (n = 6) and female (n = 6) rats, we tested them in the
cold to neutral range (-4 to 37 C). During training and in
recorded sessions at 370C, there was no trend observed
for either sex to consume more milk reward or to satiate at
different rates. Table 1 shows the differences (male -
female) in raw outcome measures at each temperature
tested. For cold temperatures above freezing (24, 10 and
2 C), the males licked less often and consumed less milk
reward than females, although these differences were not
significant (Table 1). The male-female differences reached
statistical significance only for 10C stimulation, which
induced significantly more thermode contacts by males.
Also, the ratio of licks to thermode contacts was signifi-
cantly reduced for 10 C stimulation of males. Thus, par-
ticularly for mildly aversive (100C) cold stimulation,
males were hindered from completing the task, as
revealed by less licking and more stimulus contacts that
did not progress to licking.

For males and females combined, there was a significant
effect of temperature on facial (stimulus) contacts (F4,71
2.503), the reward/stimulus ratio (F4,71 = 4.078), the stim-
ulus duration/contact (F4,71 = 4.034), and intake (F4,71 =
2.590). Across the range of temperatures presented,
increasing cold gradually reduced food intake, the
number of licks per contact and the duration of individual
contacts (Fig. 1A). Each of these measures depends upon
secure contact with the thermode as the animal feeds. In
contrast, the number of thermode contacts increased with
increasing cold, as the animals more frequently withdrew
from the thermode before contacting the feeding tube
(Fig. 1B). Post-hoc analyses revealed that the stimulus
contacts were significantly increased (Fig. 1B) and both
the reward/stimulus ratio and the stimulus duration/con-
tact were significantly lower for -4 C relative to 3 7 C (Fig.
1C, D). There were no significant differences between -
4 C and 2, 10, or 24 C. Additionally, there was no signif-
icant effect of temperature on the total number of licking
events or total duration of facial contacts, and there was
no significant effect due to estrus stage of females (data not

Sensitivity to cold can change over time due to factors
such as changes in blood flow and receptor adaptation.
Therefore we looked at changes in the ratio of licks to
facial contacts over the 30-min trial. Fig. 1E shows
changes in the licks to facial contact ratio over time for 37,

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Molecular Pain 2006, 2:37

Table I: Comparison of behavioral outcomes for males and females.

Outcome Intake (grams)

Temp. CO-$ t

Licking Events

Facial contact
Events (count)



Duration (seconds)

Licks/Facial contacts ratio


Duration (s)/facial
contacts ratio


Shown are mean difference between males and females and the corresponding t statistic for the six outcome measures (intake, licking events,
stimulus contacts, duration, licks to stimulus contacts, and stimulus contact duration) across a range of temperatures (37, 24, 10, 2, and -4C). *
indicates a significant difference between males and females (P < 0.05).

24, 10, 2, and -4 C. There was a clear separation between
370C and the cool and cold temperature trials Through-
out the 30-minute period, 370C produced the greatest
licks to facial contact ratio over time, while -4 oC produced
the lowest licks to facial contact ratio over time, and the 2,
10 and 24 C produced intermediate response profiles.
Note that the animals attained their maximal rate of licks
per contact early in the 370C stimulation. After a sharp
peak of licking probably related to hunger, a high rate of
licks per contact was maintained for approximately 8 min-
utes, followed by a very gradual reduction as satiation pro-
gressed without cold stimulation. In contrast, the
maximum ratios of licking per contact during cold stimu-
lation were attained in excess of 10 minutes into the trials.
This suggests that the rate of licking per contact increased
as cold adaptation progressed within trials.

Behavioral response to thermal preference
A separate group (n = 7) of male rats was first tested under
neutral conditions (37 0C) on both sides of the place pref-
erence box (Fig. 2A). There were no significant differences
in the percent of total intake, licks, facial contacts, or dura-
tion between the left and right sides at 370C (Fig. 2B -
facial contacts shown), indicating that the rats did not have
a side preference. Rats switched sides an average of seven
times. When presented with a choice between -4 and
48 C stimulation, the percentage of licks (t3 = -2.98, *P
< 0.05), facial contacts (t13 = -6.05, P < 0.001), and dura-
tion (t13 = -3.22, P < 0.05) was greater for the hot ther-
mode than the cold, regardless of the side assignment for
each temperature (Fig. 2C -facial contacts shown). The rats
switched sides an average of six and seven times for each
of two trials; thus there wasn't a decrease in exploration
that could account for a lack of activity on one side or the
other. The licks per thermode contact and duration per
thermode contact in the thermal preference tests were also
calculated for 37, 48, and -4 C as a comparison of the rel-
ative amount of pain at each of these temperatures (Fig.
2D, E). These ratios were calculated using data obtained in

the preference condition, not in single-temperature trials.
Both of these ratios for 48 and -40 C were significantly dif-
ferent than those for 370C (licks/facial contact F2,2 =
5.40, duration/facial contact F2,20 = 4.63, P < 0.05). How-
ever, there was no significant difference between 48 and -
4 C for either ratio. Thus, the rats expressed a greater aver-
sion for -4 C cold, or conversely, a preference for 48C
heat, even though success performance at the two ther-
modes was comparable.

Behavioral response to menthol and vehicle treatment
Rats were tested at 24, 10, and -4 C fifteen minutes fol-
lowing injection of either menthol or vehicle. Each indi-
vidual rat's outcome measures were compared to the
baseline average for its sex to produce a percent increase
or decrease from baseline for the menthol and vehicle
treatment groups. At 10 C, there was a significant treat-
ment effect on total facial contacts (F2,35 = 8.582), the
ratio of licks to facial contacts (F2,35 = 5.476), and the
duration of facial contact (F2,35 = 4.640) (Fig. 3A-C),
when comparing menthol with vehicle. Following men-
thol treatment, total facial contacts increased relative to
vehicle treatment and baseline at 100 C. The ratio of licks
to facial contacts and the duration per facial contact
decreased following menthol treatment as compared to
vehicle or baseline. These changes are indicative of allody-
nia following menthol treatment. At 24 and -40C there
were no significant effects following menthol treatment,
and there were no significant differences between the
sexes following menthol treatment.

Given the significant effect of menthol at 100 C for induc-
ing cold sensitivity, we decided post-hoc to further inves-
tigate this difference by examining the change in success
ratio over time at 10 C (37 C is also shown for compari-
son). We were interested in evaluating if there was a signa-
ture temporal pattern to this behavioral outcome. Recall
that at 10 C there was a significant decrease in the success
ratio relative to 37 C (corresponding to increased failure

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Molecular Pain 2006, 2:37

A. Intake

-4 2 10
Temp (oC)

24 37

C. Licks/Facial contact
C 60

-4 2 10 24
Temp (oC)

B. Facial contacts Events
200- +


. 80

Temp (oC)

D. Duration/Facial contact
8 80

-4 2 10
Temp (oC)

E. Temporal Profile
5-- 37'C 24C 10'C 2'C -4*C
0 40

01) 10
=1 0----------------
0 200 400 600 800 10001200140016001800
Time (s)
Figure I
Behavioral response to cold stimuli. Four outcome measures are shown: intake (A), facial contact events (B), licks
(reward)/facial contact ratio (measures success) (C), and facial duration/contact (D) for cold temperatures (-4, 2, 10, and
24oC), expressed as a percent of baseline (37oC). + indicates significant increase, and indicates a significant decrease (P <
0.05). (E) represents the change in the licks/facial contact (success ratio) over the 30-minute test period for each of the tem-
peratures tested.

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Molecular Pain 2006, 2:37


A. Temperature preference apparatus

B. Side

preference at 37C


-F 40
. .
14 -
3 20

Left Right

Testing Side

D. Reward/Stimulus Ratio
S 25
8 20
i2 10
5 ~
01 I -- I-E- --

C. Stimulus temperature preference

> 80
U 40


-4 48
Temp (oC)

E. Stimulus Duration/Contact

" 4.

0 3.

I.7 1

-4 37 48 -4 37 48
Temp (C) Temp (oC)

Figure 2
Behavioral response to thermal preference. Rats were first placed in the thermal preference testing box (A) with left
and right thermodes set at 37oC to ensure that there was not a side preference (B). In two additional testing sessions, rats
chose between -4 and 48oC (C) and their reward/stimulus (D) and stimulus duration/contact ratios were found to be no differ-
ent for -4C and 48oC, but significantly lower for -4C and 48oC relative to 37oC (ratios calculated from temperature choice
trials). indicates significant decrease, P < 0.05.

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Molecular Pain 2006, 2:37

Molecular Pain 2006, 2:37

A. Facial Contacts


. 160.
" 120


- 40.
. 40

(1 4

- 4C

C. Duration/Facial Contact



t 200

- 120


D 40

I---Menthol -0-- Vehicle




B. Licks/Facial Contact


0 160

U 120

U= 80

U) 40

- 40C



D. 10C baseline and treatments

0 200 400 600 80010001200140016001800
Time (s)

Figure 3
Behavioral response to menthol and vehicle treatment. Stimulus contacts (A), reward/stimulus success ratio (B), and
stimulus duration/contact (C) at 24, 10, and -4oC in the presence of menthol (solid lines) or vehicle (dotted lines), expressed as
a percent change from untreated baseline temperatures. Data points indicate average change from baseline, + indicates signifi-
cant increase, and indicates a significant decrease. The change in reward/stimulus success ratio over time (D) is also given for
menthol (blue), vehicle (orange), baseline at 10C (green), and baseline at 37oC (pink).

to lick, which is comparable to escape) when averaged for
the entire trial (Fig. 3B). After almost 10 minutes of
enhanced licking relative to the 100C baseline, menthol
substantially reduced the rate of licks per contact during
the remainder of the 10C trial period (Fig. 3D).

While many pain patients complain of heightened cold
sensitivity, understanding the underlying mechanisms
has proved to be difficult due to current limitations in

translatable animal behavioral outcome measures. We
recently developed an operant assay that can provide
information regarding several dimensions of an animal's
evaluation of thermal stimuli that are not addressed by
traditional, reflex-based tests. Our goals in this study were
two fold: we wanted to characterize behavioral responses
to facial cold stimulation and evaluate the effects of men-
thol on cold sensitivity using an operant testing paradigm.
We previously showed a significant decrease in operant
behavioral outcome measures with increasingly hot tem-

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peratures in male rats under normal and inflammatory
conditions [6]. We found in the present study that increas-
ingly cold temperatures did not produce the dramatic
reduction in behavioral outcome measures previously
observed for heat. For facial stimulation we did not see a
significant effect on outcome measures until the tempera-
ture was reduced to -4C. Cold temperatures are not
immediately damaging to tissue as is noxious heat and
animals may be able to tolerate and overcome any initial
discomfort from cold when motivated by food reinforce-

Mauderli et al. [4] and Vierck et al. [5] have assessed escape
latency and duration to a range of cold temperatures in an
operant assay and compared them to typical reflex and
innate measures. Escape latency for a range of cold tem-
peratures (0.3-20C) was much shorter than latencies to
lick or guard, indicating that thresholds differed substan-
tially for reflex and operant responses to cold. Also the
duration of time spent off the stimulus platform (escape
duration) increased with decreasing temperature. In con-
trast, there was little effect of temperature on time spent
guarding or licking. Thus, reflex measures of cold nocice-
ption reveal different patterns of responsivity than do
operant tests. Although we did not evaluate time away
from the thermode, our findings are similar in that
changes in escape responses to increasing cold and success
ratios were modest for both operant tasks, especially when
compared with the rate of response to increasing heat.
While operant responding was not significantly hindered
by moderate cold in the present study, we observed that
the rats exhibited an increase in isolated facial grooming
following contact with these temperatures, as previously
described in models of orofacial hypersensitivity that
measure unlearned behaviors [12].

Human psychophysical studies have revealed that
responses to thermal stimulation are not merely reflective
of the physical experience but also reflect the emotional
state and views of the subject [13-15]. Cold stimulation in
particular produces a more varied qualitative response in
subjects than noxious heat, which is primarily described
as a "burning" sensation [16]. These factors contribute to
the experience of thermal pain in both normal and
injured individuals. Traditional behavioral tests in ani-
mals are often unable to evaluate affective aspects of pain
processing that are just as crucial as the physical transduc-
tion of thermal stimuli, and these procedures often intro-
duce additional stress which can confound results [17].
This is likely the reason that some potential analgesics
may seem promising in traditional animal studies, but fail
during clinical trials.

In humans, sex differences with respect to cold pain toler-
ance and ratings have been noted [18,19]. It has been sug-

gested that such difference may be due to psychosocial
factors [20], although there is some evidence that circulat-
ing female hormones may mediate adaptation to cold
pressor pain [21] and sex differences in cortisol may effect
response to repeated cold stimulation [22]. It is known
clinically that more women than men suffer from neuro-
pathic pain [23,24] and that a common symptom of indi-
viduals with such pain is increased cold sensitivity [25].
Few studies have addressed behavioral responses to cold
stimuli in animals and thus far none have looked at sex
differences in sensory processing of cold stimuli for
healthy animals in either a reflex based assay or an oper-
ant assay. In this study, we wanted to complete a general
survey of the effects of sex and cold on operant measures.
We observed significant differences between the sexes
only at 100C, with males exhibiting signs of increased
sensitivity (greater facial contacts and decreased success
ratio) as compared to females, which is in contrast to typ-
ical sex differences reported in human studies. However,
the lack of significance at all other temperatures and out-
comes indicates that the difference demonstrated here was
not robust in healthy rats. Sex differences may be more
pronounced with sustained stimulation [26]. Since ani-
mals self stimulate in this assay, the length of stimulation
may not have been sufficient to reveal sex differences. This
is certainly an area that merits further exploration.

In order to more directly compare cold and hot mediated
behaviors, we used a thermal preference paradigm. Rats
were given a choice between nociceptive cold (-4 C) and
hot (48 C) stimulation, which produced nearly identical
pain indices (reward licks/stimulus contact ratio and stim-
ulus duration/contact ratio). This indicated that the mag-
nitudes of pain sensations elicited by these stimuli were
similar (Fig. 2D, E). However, when given the option to
choose between these nociceptive hot or cold stimuli, rats
spent more time on the hot side. Interestingly, this ther-
mal preference test may reflect a greater influence of the
affective component of cold as compared to heat pain of
comparable sensory magnitude. This result is similar to
human psychophysical ratings of hot and cold stimuli.
Rainville et al. found that the relative unpleasantness,
determined as a ratio of pleasantness ratings to perceived
intensity, was greater for tonic (5 0C) cold pressor stimu-
lation than for phasic contact heat [27]. Greenspan et al.
observed that for hot stimuli the initial temperature rated
as unpleasant was within 1.4 C of the temperature rated
as painful, but for cold stimuli unpleasantness ratings
began at temperatures 5.6 C higher than the temperature
rated as painful [28]. These findings indicate that in
humans cool temperatures are perceived as more unpleas-
ant than warm temperatures of a comparable magnitude,
as we demonstrated in rats. This strong affective compo-
nent to perception of cold stimuli may make it difficult to
fully isolate the sensory component of cold perception.

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Molecular Pain 2006, 2:37

Different exposure conditions lead to different sensory
experiences, especially for cold stimuli, presumably due to
activation of different populations of receptors and the
influence of sympathetic activation on thermal sensa-
tions. In human subjects, repeated stimulation of gla-
brous skin with a cooled probe produces a deep radiating
ache over time that increases in proportion to the decrease
in superficial and deep skin temperature [29]. This effect
is presumed to be due to activation of nociceptors near
vessels. In addition, burning cold is evoked by tempera-
tures near freezing and is attributed to activation of C-fib-
ers that respond to both hot and cold stimuli [30]. It is
likely that our operant paradigm involving numerous epi-
sodes of cold stimulation elicits both sensations.

Several populations of peripheral afferents have been
identified that are responsive to cold and express different
TRP family channels. TRPM8 is a receptor expressed on
sensory neurons that has been identified as cold activated,
although there is debate about its role in the processing of
nociceptive cold stimulation [31-34]. In an attempt to
address this issue, we examined the use of the mint
derived substance, menthol, which acts as a specific ago-
nist to the TRPM8 receptor. Following treatment with
menthol, there was a significant increase in facial contacts
and significant decreases in the ratio of licks to facial con-
tacts and the duration of facial contacts at 10 C, indicat-
ing the induction of sensitization and development of

The finding that successful completion of the operant task
was hindered by menthol in the presence of 10 but not in
the presence of 240 C provides evidence that activation of
the TRPM8 receptor elicits sensations of cold pain. TRPM8
mRNA has been identified in both small and medium
diameter DRG neurons in vivo, which are presumed to cor-
respond to the cell bodies of C- and A8 fibers, respectively.
It is likely that both fiber types with TRPM8 receptors rep-
resent subpopulations of cold responsive nociceptors
[35]. However, 24 C may not be of sufficient magnitude
to activate TRPM8-containing C-fibers but could activate
cold responsive A8 fibers that block C-fiber activity and
prevent the induction of menthol-induced allodynia [36].

Within the spinal dorsal horn, cool-sensitive lamina I spi-
nothalamic (STT) cells have a specific sensitivity to tem-
peratures between 34 and 15C. The sensitivity of
polymodal nociceptive HPC (for heat, pinch, cold) cells
to noxious cold begins at about 24 C, and their response
to cold accelerates at temperatures below 15C. It has
been suggested that an increase in HPC activity beyond
that of cool-sensitive cells signals the sensation of burning
pain [37]. Even in the presence of menthol it seems 24 C
does not provide input of sufficient magnitude to increase
HPC STT cell activity beyond cool-sensitive STT cells in

lamina I and thus could explain why menthol did not
enhance sensitivity to 24 C.

We had expected that menthol would cause cold hyperal-
gesia at -4 C, but we did not observe a significant differ-
ence when comparing the different treatments. Menthol is
known to produce a burning sensation in humans at a
concentration of 40% and also increases sensitivity to
cold, presumably by its action on C-fibers [7]. It is possi-
ble that 10% menthol while sufficient to induce allodynia
at 10C, was not sufficiently concentrated to induce
hyperalgesia at -4 0 C. It is important to note that it also did
not induce an insensitivity to -40 C, ruling out the possi-
bility that TRPM8 expressing cells were desensitized to
further cold stimulation. It is also possible that sensations
produced by -4 C stimulation are mediated more so by a
population of cells expressing another putative cold trans-
ducing receptor, such as TRPA1 [31], or another as yet uni-
dentified receptor [38,39].

It seems clear that like heat, there are many molecular
players involved in peripheral transduction of cold sensa-
tions. Additionally, higher order processing of cold sensa-
tions cannot be ignored. Thus, it is important to use
behavioral tests that require engagement of higher order
processing, and synthesize information regarding multi-
ple dimensions of pain. Understanding how these varying
aspects of the pain experience interact in vivo will help
understand the regulation of molecular mechanisms of
spontaneous and induced cold allodynia in humans. This
understanding should ultimately lead to more effective
treatments for patients suffering from pain.

Male and female hairless Sprague-Dawley rats (seven
weeks old, Charles River, Raleigh, NC) were housed in
groups of three to four in enriched housing, were main-
tained in a standard 12-hour light/dark cycle and were
allowed access to food and water ad libitum when not
being tested. We chose to use both males and females as
there is evidence for sex related differences in thermal sen-
sitivity for operant testing of the hindpaw (Vierck personal
communication). Rats' weights were recorded every week to
monitor general health. Females' estrus cycles were moni-
tored by examination of vaginal lavage cytology as
described in [40]. Animal testing procedures and general
handling complied with the ethical guidelines and stand-
ards 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 (Council 1996).

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Molecular Pain 2006, 2:37

Behavioral Facial Testing
Facial testing was completed using a reward-conflict oper-
ant testing paradigm as described previously [6]. Briefly,
the rats were trained to drink sweetened condensed milk
while making facial contact with a thermode. During the
training period (approximately 2 weeks) their baseline
intake was recorded, and the rats were considered ready
for experimental testing once their average baseline intake
was 10 g or greater at 370C. The facial testing region for
each animal was depilitated under light isofluorane
anesthesia (inhalation, 2.5 %) once a week to maximize
thermal stimulus contact. The rats were fasted over night
(13-15 hrs) prior to each session and were tested at 37,
24, 10, and -4C on separate days.

Thermal preference of the animals was recorded using a
modified testing box that had thermodes and sipper tubes
situated side by side in a single compartment, with the rats
free to access either reward tube equally (Fig. 2A). The rats
(n = 7) were previously trained and tested in the single
thermode boxes at 37, -4, and 48C. Rats were initially
placed in the thermal preference apparatus with both ther-
modes set at 37 C to allow them to become accustomed
to this new task. A second such session was recorded to
ensure rats did not demonstrate a side preference. In the
third and fourth sessions one thermode was set cold (-
4C 1 C) and the other was set hot (48 0.2C). Rats
were able to move freely from one side of the compart-
ment to the other and explore both thermodes at will.

For the single thermode test, six outcome measures were
evaluated as described previously [6]: reward intake (g),
number of licking contacts, number of facial contacts with
the thermode, total duration of facial contacts, ratio of
licking contacts (reward) to facial contacts (stimuli), and
the ratio of total duration of contacts/number of facial
contacts were averaged over the 30 min trial periods. The
ratio of licks to thermode contacts was also calculated as a
function of time for each trial. For the thermal preference
task, the first four outcome measures (intake, licking con-
tacts, facial contacts, and duration of facial contacts) were
calculated for each side of the box and totaled to deter-
mine the percentage of each outcome spent on each side
and at each temperature. The ratio of licking contacts
(reward) to facial contacts (stimuli) and the ratio of total
duration of contacts/number of facial contacts were calcu-
lated for 37, -4, and 48 C. One and two-way ANOVAs
were used to compare behavioral changes across different
temperatures and treatments using SPSS (v. 14.0, SPSS,
Inc.). When significant differences were found, post-hoc
comparisons were made using Tukey's test. For the ther-
mal preference task, a paired t-test was used to compare
the percentage of time spent on the hot and the cold ther-
modes. A probability level ofP < 0.05 was considered sig-

Menthol and Vehicle Treatment
Menthol (10%) or vehicle (1.6% EtOH/0.01% Tween 80
in PBS) was delivered by subcutaneous injection (150 1)
into the cheeks of gently restrained rats. Rats were
returned to their holding cages for 15 minutes prior to
facial testing at -40C, 10C, or 24 C as described above.
A cross-over design was used for drug administration
whereby animals received both treatments at each temper-
ature on separate testing days.

Competing interests
The authors) declare that they have no competing inter-

Authors' contributions
HR was responsible for overseeing the behavioral testing,
injecting the animals, analyzing and interpreting the data,
and drafting the manuscript. CV and RC helped to draft
and revise the manuscript. JN conceived the study, partic-
ipated in its design, assisted with data analysis and inter-
pretation, and helped to draft the manuscript. All authors
read and approved the final manuscript.

Support from this research was provided bygrant # K22DE014865- IA I,
National Institute of Dental and Craniofacial Research, National Institutes
of Health, Department of Health and Human Services, Bethesda, MD, USA.
We also thank Jean Kaufman for her assistance with behavioral testing and
general care of the animals.

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