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Brain Research 970 (2003) 58–72
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Research report
Contribution of the anterior cingulate cortex to laser-pain conditioning
in rats
Jen-Chuang Kung, Ning-Miao Su, Ruey-Jane Fan, Sin-Chee Chai, Bai-Chuang Shyu*
Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, ROC
Accepted 20 December 2002
Abstract
The emotional component of nociception is seldom distinguished from pain behavioral testing. The aim of the present study was to
develop a behavioral test that indicates the emotional pain responses using the classical conditioning paradigm. The role of the anterior
cingulate cortex (ACC) in the process of this pain conditioning response was also evaluated. In laser-pain conditioning, free moving rats
were trained to associate a tone (conditioned stimulus, CS) and short CO 2 laser pulsation (unconditioned stimulus, US). Monotonous tone
(800 Hz, 0.6 s) was delivered through a loud-speaker as CS. CO 2 laser pulses (5 W at 50 or 100 ms in duration) applied to the hind paw
was adopted as US. The CS–US interval was 0.5 s. Laser-pain conditioning was developed during 40 CS–US pairings. CS and US pairing
with 100-ms laser pulse stimuli was more effective in establishing conditioning responses than that of 50-ms stimuli. The conditioning
responses remained, tested by presenting CS alone, immediate to and 24 h subsequent to training. The performance of laser-pain
conditioning was significantly reduced after bilateral lesioning of the ACC. Similar results were also obtained by bilateral lesions of the
amygdala. The conditioning responses were also diminished following morphine treatment. The association between a neutral stimulus
and a noxious stimulus could be demonstrated in a Pavlovian conditioning test in free moving rats. Thus, the conditioned response may be
employed as a measure of the emotional component of the nociception. It is also suggested that the ACC may play an important role in
mediating this conditioning effect.
 2002 Elsevier Science B.V. All rights reserved.
Theme: Sensory systems
Topic: Pain modulation: anatomy and physiology
Keywords: Anterior cingulate cortex; Amygdala; Classical conditioning; Conditioned response; Laser stimulus; Morphine; Pain
1. Introduction
Several functional brain imaging studies of nociceptive
responses in human have consistently shown that the
anterior cingulate cortex (ACC) is activated during the
application of acute, noxious heat stimuli to the body
surface [3,5,9,11,12,26,48,57]. Furthermore, behavioral
and neurophysiological studies in animals have shown that
the ACC is involved in the processing of affective nociceptive information. Electrical stimulation of the ACC induces
*Corresponding author. Tel.: 1886-2-2652-3915; fax: 886-2-27829224.
E-mail addresses: [email protected] (B.-C. Shyu), http: /
/ www.ibms.sinica.edu.tw / html / PI / Bai c.html (B.-C. Shyu).
]
vocalisation that is thought to be associated with escape
responses [17,51]. Behavioral studies have also demonstrated that the ACC mediates the affective response of
tonic pain in hot-plate, formalin pain testing [15,41,60,61]
and formalin-induced conditioned place avoidance [25].
Electrophysiological evidence from our lab indicates that
there are synaptic and functional connections between
medial thalamus (MT) and ACC [23,24,30]. This finding,
together with another report on the rabbit [55], suggests
that the nociceptive information in the MT may transmit to
the ACC [24]. Nociceptive neurons in the ACC have little
or no somatotopic organisation and therefore are suited for
information processing involving affective property of
noxious stimuli [55].
The neuronal pathways that lead to the activation of
0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved.
doi:10.1016 / S0006-8993(02)04276-2
J.-C. Kung et al. / Brain Research 970 (2003) 58–72
sensory and affective components are excited in parallel by
noxious inputs. The behavioral manifestations of these two
components are not well differentiated in an overall
nocifensive behavioral response. Thus, a behavioural
model is needed to selectively assess the emotional
component of nocifensive responses and subsequently
investigate the functional role of the ACC.
Conditioning paradigms have been used to examine
emotional responses to aversive stress in rats [4,32] and
rabbits [19]. It has been shown that lesions of the cingulate
cortex disrupt active-shock avoidance learning in rats
[43,58]. A recent study, using a place-conditioning
paradigm, has demonstrated that a learned behavior induced by formalin might reflect the affective consequences
of nociceptive stimulation [25]. Furthermore, cingulectomized rabbits fail to learn an inactive avoidance learning that
involves avoidance of foot shock [19,20]. The conditioned
emotional response is an accepted animal model of emotional stress in which an animal learns to form associations
between an aversive unconditioned stimulus (US) and a
conditioning stimulus (CS) [39,40]. If a CS is presented
with no US, the physiological responses of an animal are
therefore thought to represent purely emotional responses
anticipating the aversive stimulus.
Our lab has previously developed a nocifensive behavioral model in rats evoked by a short-pulsed CO 2 laser
beam [16]. A laser pulse radiates intense and highly
focused thermal energy and has been used for noxious
stimulation in several studies [1,7,8]. Mor and Carmon
[36] used a CO 2 laser beam to stimulate human skin to
induce pain sensations and evoke a cortical potential. This
method of stimulation not only possesses the attributes of
the conventional thermal stimulation, but also allows the
spatial and temporal parameters of the stimulus to be even
more precisely controlled. Pulse stimulation is traditionally
used in electrophysiological studies because a discrete
stimulus provides a relatively uncomplicated response for
interpretation of the effect. The same stimulus could be
used to elicit both behavior reactions and cortical potentials in order to study the relationship between them. In
view of these facts, the use of CO 2 laser pulses to study
pain reactions in waking animals seems most appropriate.
Therefore we propose that a nocifensive behavior induced
by noxious laser beam can be used as an UR and that an
association of the nocifensive behavior with a neutral tone
stimuli would be useful as an indication of conditioned
response (CR) in the present conditioning experiment.
The purpose of the present study was to develop a
behavioral paradigm of pain conditioned responses by
pairing a tone (CS) with CO 2 laser stimuli (US). This
behavioral model was further employed to assess the
emotional component of nociceptive responses. In addition, morphine was administered to evaluate the analgesic
effect on the CR. Effects of lesions of the ACC on pain
conditioned responses were also examined.
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2. Materials and methods
2.1. Experimental subjects
Fifty Sprague–Dawley rats (250–300 g / body weight)
were used in present study. They were individually housed
in standard wire-mesh cages and maintained in an airconditioned room (21–23 8C, humidity 50%, 12-h light /
dark cycle starting at 06:00 h) with free access to food and
water. All experiments were carried out in accordance with
the Animal Scientific Procedures Act of 1986 and with
Institutional Ethical Committee approval. Efforts were
made to minimize animal suffering and to use a equitable
number of animals. These were randomly assigned to four
experimental groups; laser intensity testing, ACC lesioning, amygdala lesioning and morphine testing. In laser
intensity testing, laser power was set at 5 W with either 50
ms (n55) or 100 ms (n55). For ACC lesioning, animals
received lesioning prior to training (n55) or lesioning
Fig. 1. A schematic diagram of the experimental design. The rat was
placed inside a cage that was suspended by a force transducer which
registered the vertical movement of the rat either induced spontaneously
or by laser stimulation. The horizontal vibration was mostly restricted by
a pair of dampers placed in both sides of the cage. A loudspeaker (CS)
produced a auditory tone (82 dB, 800 Hz, 600 ms). The laser probe was
placed beneath the cage and could be easily moved horizontally and the
laser beam (US) positioned on the left paw of the rat. The transducer,
loudspeaker and the laser probe were connected in tandem to a PC-based
data acquisition and control unit.
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subsequent to training treatments (n55). Each lesioned
group had a sham-operated group (n55) to serve as
controls. In amygdala lesion experiments, one group of rats
(n55) received lesioning prior to training and a shamoperated group (n55) was used as control. In morphine
tested animals, each rat received 10 CS then injected with
either morphine (n55) or vehicle (n55). A double-blind
procedure was followed for these experiments. One person
was placed in charge of animal operation; the other
performed mainly behavior training and testing. Neither
person knew what treatment the animals had been subjected to.
2.2. Stimulation
The CS was an 82-dB, 800-Hz pure auditory tone
presented through a speaker mounted above the conditioning chamber. The duration of the CS was 0.6 s.
The US was short CO 2 laser pulses. The intensity of
laser power was 5 W and both 50- and 100-ms pulse
durations used in the present study. The laser pulse was
generated from a surgical CO 2 laser (Model 20 CH, Direct
Energy Inc., CA, USA), which was used to produce a laser
radiation beam in the infrared spectrum of 10.6 mm
wavelength. Its output energy has a maximum power of 20
W and pulse duration is adjustable. The device has a
build-in calibration system to measure the peak power of
laser pulses and a hand-held laser probe for directing the
beam. During the laser stimulation, the experimenter held
the laser probe and projected the laser beam to the hind
paw of the freely moving animal. The unfocused projection size of the laser beam on the animal’s paw was about
20 mm 2.
2.3. Experimental procedures
The experimental design is shown in the Fig. 1. Rats
were transferred from their home cages to a standard
conditioning chamber. The chamber was suspended on a
force transducer (Model FT 10, Grass Inc., USA). A
damper was used to restrict the horizontal movement of the
chamber, so the vertical movement of the chamber was
recorded as the rat’s movement.
During training, a CO 2 laser probe was placed under the
cage and the probe could be easily moved horizontally and
positioning the laser beam to the left hind-paw of the rat.
Following a period of adjustment the rat usually came to
remain stationary with the paws resting on the grid floor
and a stimulus could be easily aimed at the hind paw.
2.3.1. Training session
For the initial 10 trials, the rat received only the tone in
order to adapt to the sound and thus reduce orienting
responses. Subsequently, the animals perceived 40 training
trials. In each trial, the rat received paired stimuli consist-
ing of a tone and a laser pulse (CS–US). The interstimulus interval was 0.5 s. The CS was presented at an
inter-trial interval about 70 s (ranging between 60 and 80
s).
2.3.2. Test session
In the testing period, the rats were tested with 10 trials
of CS alone immediately after and at 24 h subsequent to
training session.
2.4. Conditioned and unconditioned responses
Laser pulses have been shown to induce eight types of
nocifensive responses including foot movement, foot elevation, head turning, withdrawal and licking [16]. Previous
study has also suggested that the numbers of types of
nocifensive responses and response frequency are increased with the increased laser power. In the present
behavioral paradigm, the force transducer was applied to
record rat’s movement. The amplitudes of the output
signals were increased with the laser power and corresponded to the numbers of nocifensive responses. The
output signals from the transducer therefore can be seen as
unconditioned responses (UR). In testing of CS and US
pairings or CS alone, rats at times responded to the CS,
within the first 0.5 s before the delivery of US. The
response movement can be registered by the force transducer. This pre-US movement was defined as conditioned
response (CR).
2.5. Measurement of the cr
The output signals from the transducer were connected
to a polygraph (Model RS3600, Gould Inc., USA) and a
PC-based data acquisition system. Thus the vertical displacement of the chamber due to the movement of the rats
caused pen deflections on the polygraph and these in turn
were converted to digital data and recorded in the hard
disk for off-line analysis. The transistor–transistor logic
pulses used to trigger the US and CS were delivered via
the digital input / output port and were programmed and
integrated into the data acquisition program system.
A period of 2 s of analog signal was registered in each
trial. The first 0.5 s was the pre-CS period. The following
0.5 s was during the CS period and the last 1 s consisted of
responses after US. All analog signals were rectified and
integrated. The 0.5-s data segments corresponding to the
CS period were calculated. The 10 values obtained from
the first CS alone trials were used as a control. Each value
from the individual trial during the CS1US pairing and
following exclusive CS testing was compared with the
control. If the value exceeded the mean199% confidence
interval of the control, it was regarded as CR.
A schematic diagram in the Fig. 2 illustrates the relative
J.-C. Kung et al. / Brain Research 970 (2003) 58–72
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Fig. 2. A schematic diagram of the conditioned stimuli and responses. (A) The CS, a tone with 800 Hz lasted for 600 ms. (B) The US, short laser pulses, 5
W, lasted for 100 ms. The delay between CS and US is 500 ms. (C) The US, short laser pulses, 5 W, lasted for 50 ms. The delay between CS and US is 500
ms. (D–H) Behavioral movement before, during and after the CS or CS1US. (D) An example from the 10 Pre-CS1US trial. Only a CS was presented.
(E) The responses during the paired CS1US (100 ms) stimuli. (F) The responses during the paired CS1US (50 ms) stimuli. Note that a CR occurred
between the 500 ms of the CS and US interval and was followed by large UR responses. (G) An example of CR during a CS presented immediately after
40 paired CS 1US have been given. (H) An example of CR during a CS presented 24 h after 40 paired CS 1US have been given.
time courses of the CS and US. The CR and UR are also
indicated.
2.6. ACC and amygdala lesion
At the time of lesioning each rat was anaesthetized with
the nembutal (pentobarbital sodium, 50 mg / kg, i.p.).
Supplementary doses were given if necessary. An incision
was made through the scalp and the skull was exposed.
Two rows of three small holes were drilled in the skull.
For the lesion of the ACC, holes were 0.5 mm apart in
rostrocaudal direction and extended from bregma forward
(coordinates 1.5, 2.5 and 3.5 anterior to the bregma). They
were 0.5 mm on either side of the midline. For the lesion
of the amygdala, the holes were 3 mm posterior to the
bregma and 5 mm on either side of the midline. A
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thermo-probe was positioned within the brain at designated
areas according to their anatomical coordinates [42].
Thermo-lesioning (80 8C for 20 s) was produced at the tip
of the probe (Model FRG-4A, Radionics Inc., USA). The
sham control group rats underwent the same surgical
procedure as the lesioned group except the thermal lesion
device was not activated.
2.7. Test of analgesic effect of morphine
In the beginning of each test, the laser pulse duration
was set to 5 ms. When the rat did not respond to the laser
stimulus, 5 ms were added to the duration and repeated the
laser stimulation. The procedure was repeated until the rat
responded to the laser stimulus with a licking response.
The pulse duration at which the rat responded with foot
licking was recorded and was defined as the threshold
value in that test. Three to five threshold measurements
were usually performed to obtain a control mean value.
Morphine (5 mg / kg, i.p.) or vehicle (saline) was injected
after 10 CS were delivered. Threshold testing was repeated
10–20 min after morphine injection. Forty paired CS and
US stimuli were delivered to the rat after the threshold test.
Two persons were in charge of this experiment as a
double-blind procedure. The vehicle and morphine treated
rats were also subjected to the rotarod test immediately
after the training and test session. The rotarod devise has a
revolving rod and was equipped with speed control. The
length of time that each rat stayed on the rod was counted.
2.8. Histological analysis
At the end of each assessment, rats were perfused with
saline followed by 4% paraformaldehyde (in 0.1 M sodium
phosphate-buffered, pH 7.4). The brains were cut on a
cryostat at 50 mm in thickness and the sections were
stained with cresyl violet (Sigma). The rat atlas of Paxinos
and Watson [42] was used as reference when detailed
histological structures were examined.
2.9. Data analysis
The total number of the CRs which occurred during the
40 CS–US paired trials or in each sub-trial period (1–10,
11–20, 21–30,and 31–40) and ten conditioned test trials
immediately after and 24 h subsequent to training session
were recorded and counted. The percent CR occurrence
was calculated as the number of CR counted during the
trial period divided by the number of trials. The integrated
values of the CR movement in arbitrary units of the
individual trial were also calculated. The data were analysed using Student’s t-test to statistically assess the
differences between experimental and control groups and
one-way ANOVA to examine differences between more
than two groups. Post-hoc examination of the difference
between groups was performed by Student–Newman–
Keul’s test. Probability less than 5% was considered
significantly different.
3. Results
3.1. Development of the CR
The rats moved freely in the cage during the testing
period. The CS was prepared when the rat was at rest and
allowed the laser beam to be directed to their left paw. The
pre-CS random activity was minimal throughout the trials
(Fig. 3A). Some random movements could be recorded
during the presentation of CS alone. Occasionally, a small
orienting response occurred immediately after the CS. The
rats did not show significant nocifensive behavioral responses to the CS before it was paired with the US.
Characteristic nocifensive behavior, such as licking, escaping etc., could be induced by the US. Subsequent to several
CS and US pairings, rats began to respond to the CS within
the first 0.5 s. The frequency of CR occurrence increased
concomitantly with the increase in the number of trials
(Fig. 3B). The total number of paired CS–US trials was
40. In the group of rats that were trained with 100 ms laser
stimuli (US) and 82-dB tones (CS), the CS elicited about
80% conditional response (CR) throughout the training.
However, when the tones were paired with 50-ms laser
stimuli (US), only 60% of the CS elicited CR. There was
also a significant difference exhibited in the mean CR% of
these two groups, (50 vs. 100 ms, t54.68, P,0.001) (Fig.
4A). In addition, the mean integrated CR activity observed
in the 50 ms laser stimulus group was also significantly
different from the 100-ms laser stimulus group (t55.226,
P,0.01, Fig. 2B). The data were grouped for each 10
trials. In a series of four sub-trials (1–10, 11–20, 21–30,
31–40 trials), there was significant difference between the
two groups (F(3, 28)519.403, P,0.01). The first two
sub-trials of the 100-ms group has mean CR% significantly
greater than those of the corresponding 50-ms group (t5
2.8 and 2.42, respectively, P,0.01) (Fig. 4C).
3.2. Retention of CR
The retention of the conditioning effects was found in
the present pain conditioning experiment when the 10 CS
alone trials were presented immediately or at 24 h after the
CS–US pairings. The percents of the mean CR during the
training with 50 and 100 ms US were 85.063.2 and
51.866.8%, respectively. The percents of the mean CR
immediately after training were 79.263.5 and 45.367.4%
for 50- and 100-ms group, respectively. The CR was
slightly reduced to 68.364.7 and 3965.4% 24 h after
training in these two groups. These data suggest that 10 CS
alone trials within 24 h may not be sufficient to eliminate
the CS–US association.
J.-C. Kung et al. / Brain Research 970 (2003) 58–72
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Fig. 3. Typical examples of the pre-CS random movement and the development of the integrated CR activity throughout the trials. The insets indicate the
data segments from which the integrated activities were calculated. The numbers in B are counts of the CR in each of 10 trials.
3.3. Lesions of the ACC
Five rats received bilateral lesions of the ACC 1 week
before the conditioning trainings. Another five rats re-
ceived sham ACC lesions prior to training to serve as the
control group. The same kind of lesioning was performed
in another five rats, 1 week after the conditioning training.
A control group consisted of five rats which also received
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J.-C. Kung et al. / Brain Research 970 (2003) 58–72
Fig. 4. Effect of training with 50 vs. 100 ms laser stimuli on conditioned response. (A) Effect was measured in percentage of occurrence for CR. (B) Effect
was measured in integrated activity CR. ** Denotes P,0.01. (C) Effect of training with 50 vs. 100 ms laser stimuli on conditioning response in 4
sub-trials. Note the difference was significant in 1–10 and 11–20 sub-trials group. * Denotes P,0.05.
conditioning training before sham ACC lesioning. Fig. 5A
shows the coronal sections of the ACC lesion sites. The
lesion sites covered the Cg1, Cg2, prelimbic and infralimbic subareas. To assess the effect of the ACC lesion on the
nocifensive behavior, the sensitivity and the responsiveness of the nocifensive behavior were measured in these
four groups of rats. The sensitivity was measured by the
paw withdrawal threshold as indicated by the duration of
the laser pulse and the responsiveness was measured by the
amplitude of the response movement as induced by the
suprathreshold (100 ms) laser pulse stimulation. The
measurements from the both sham control and the ACC
lesion groups are shown in Table 1. Although the threshold
of the paw withdrawal in both lesioned groups showed a
slight decrease compared to the respective sham control
groups, there was no significant difference exhibited
between groups. The nocifensive behaviors, such as licking
and escaping were also elicited by the suprathreshold laser
pulse stimulation in the lesioned groups of rats. However,
no significant differences noted in their movement amplitudes among the sham control and lesion groups.
Lesions of the ACC before or after conditioning training
J.-C. Kung et al. / Brain Research 970 (2003) 58–72
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Fig. 5. The lesion of the ACC and amygdala. Coronal sections taken every 0.5 mm between 2 and 4 mm anterior to bregma for the ACC and 3–4 mm
posterior to the bregma for the amygdala. Shaded areas represent lesioned areas.
reduced the mean CR% of the respective sham control
groups to 60 or 40%. There is a significant difference
between the ACC lesioned group and control groups in the
total mean CR%, regardless of when the lesions were
performed (F(3,16)511.009, P,0.01, Fig. 6A). Among
the four sub-trials (1–10, 11–20, 21–30, 31–40 trials), the
first three lesion sub-trials without prior training had a
mean CR% significantly less (t53.52, P,0.01, t52.39,
P,0.05 and t52.91, P,0.05, respectively) than their
corresponding sham control groups. The two sub-trials
(1–10, 21–30 trials) with prior training also had a mean
CR% which was significantly reduced subsequent to ACC
lesioning (t52.49, P,0.05 and t52.72, P,0.05, respectively, Fig. 6, B).
3.4. Lesions of amygdala
In the amygdala group, five rats received bilateral
lesions of the amygdala before conditioning (Fig. 5B). The
rats showed the same nocifensive behavior as control rats.
Fig. 7A shows that lesioning of the amygdala also
significantly reduced mean CR% to a level below 40% in
the 100-ms laser stimuli group (t57.79, P,0.01). Fig. 7B
shows the mean CR% of the four sub-groups (1–10,
Table 1
The effect of lesion treatments on the laser-induced withdrawal threshold and response movement amplitude
Groups
ACC sham control
with prior training
ACC lesion
with prior training
ACC sham control
without prior training
ACC lesion
without prior training
Amygdala sham control
Amygdala lesion
a
Laser-induced withdrawal
threshold (ms)
Laser (100 ms)-induced
response amplitude a
14.8061.33
131.0068.44
12.4062.21
143.60614.58
14.6060.93
140.40611.66
13.1761.93
15.4062.03
13.1962.76
157.30613.43
138.80617.86
146.00614.50
Arbitrary unit of integrated amplitude measured from the force transducer. Data are shown as mean6S.E.M.
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Fig. 6. Effect of training on CR in sham control vs. ACC lesion group. (A) Mean CR% change in sham control with prior training, lesion with prior
training, sham control without prior training and lesion without prior training. (B) Mean CR% changes in 1–10, 11–20, 21–30 and 31–40 sub-trials in
sham controls and lesion groups. ** Denotes P,0.01 and * denotes P,0.05.
11–20, 21–30, 31–40 trials), the mean CR% of last two
sub-groups differed significantly from those of the control
groups (t53.10, P,0.05 and t56.27, P,0.01).
3.5. Effect of morphine
Injection of morphine significantly increased thresholds
for rats to lift the feet (t53.95, P,0.05) and to lick paws
(t54.39, P,0.05) while painful laser stimuli were de-
livered (Fig. 8). In addition to the laser pain threshold test,
the morphine and vehicle treated rats have also been
subjected to rotarod test. There was no significant difference in the time spend on the rod between the vehicle and
morphine treated groups. This observation indicates that
there is no impairment in motor function induced by
morphine at a dose of 5 mg / kg in the present study. The
results were consistent with our previous findings [54] that
the morphine injection at this dosage has a specific
J.-C. Kung et al. / Brain Research 970 (2003) 58–72
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Fig. 7. Effect of training on conditioning response in sham control and amygdala lesion group. (A) Mean CR% change in sham control and amygdala
lesion group. (B) Mean CR% change in 1–10, 11–20, 21–30, 31–40 sub-trial group in control and amygdala lesion groups. ** Denotes P,0.01 and *
denotes P,0.05.
analgesic effect and without the induction of catatonic or
other motor suppressive effects. Morphine injection significantly blocked integrated CR activity (P,0.05, Fig.
9A). In a series of four sub-trials, the effect of morphine
injection was significant in three (1–10, 21–30 and 31–40)
out of four sub-trials (t52.99, 2.61,t52.80, respectively,
P,0.05, Fig. 9B) compared to the controls. However,
there was no significant difference in the integrated CR
activity among these four trials.
4. Discussion
The present study shows that neutral auditory stimuli
can form association with unlearned nocifensive responses
evoked by noxious CO 2 laser pulses stimuli. This neutral
stimulus acquires the property to elicit CR in future
occasions. It is possible that this associative learning
occurs in the supraspinal brain center and the limbic
system. The results also show that lesions of the ACC or
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Fig. 8. The effect of morphine on the threshold of the nociceptive behavioral responses. (A) The threshold for lifting the foot. (B) The threshold of licking
the paw. * Denotes P,0.05.
the amygdala block conditioned responses. This conditioned response was also blocked by injections of
morphine. This finding suggests that the pain conditioning
is mediated by the limbic system and possibly involves the
opioid system.
Learned behavioral responses involving pain stimuli
have been used extensively in psychophysiological studies
[10]. The most common method is avoidance-learning
tasks that require an animal to perform a learned response
to avoid aversive stimuli. Electric shock has been routinely
used as the source of US in these types of avoidance tasks.
Although this learning involves pain mechanisms, the
nociceptive components elicited by the foot shock have
been seldom discussed. Electrical stimulation might stimulate peripheral receptors in a non-specific way which could
elicit a wide range of activations. Thus physiological or
behavioral responses observed in most avoidance tasks
involving foot shock can involve a complicated constellation of responses rather than simple pain reactions. A short
CO 2 laser stimulation has also been used in human
psychophysiological testing [7,8]. The amplitude of evoked
cortical responses increases, when stimulus intensity is
gradually increased to achieve progressively increased
levels of pain. There is a significant correlation between
the amplitude of evoked cortical responses and weighted
subjective responses [7]. The nocifensive behaviors induced by short CO 2 laser pulses have been described in
our previous study [16]. It is assumed that the nocifensive
behaviors of rats are similar to normal subjects who
experience painful laser stimulations. Thus the short laser
pulse stimulus is a more specific noxious stimulus that can
serve as a US to form associations with neutral stimuli to
elicit CR. Therefore, it would create a more specific
conditioning process involving pain stimuli.
When the laser stimulations were delivered to the hind
paw of animals, it triggered withdrawal responses as a
result of the painful stimulations. After pairing a neutral
tone (CS) with laser stimulations (US) in the present
delayed conditioning paradigm for several trials, the
neutral tone (CS) formed association with the affective
(pain) component of laser stimulations. Therefore, the
animals withdrew their hind paw, turned their head or
simply escaped, since they predicted that the CS would be
followed by painful stimulations. The behavioral repertoire
of the CR consisted of turning the head, licking the foot,
moving and withdrawing their paw. These behaviors are
similar to behaviors induced by the US with less magnitude. In the present studies, the CR was not observed
until several trials of CS and US pairings. In the present
experiment, the CR was found immediately after the
presentation of the tone and before the onset of physical
laser pulses. It is assumed that the underlying neuronal
mechanisms of these movements in CR are different from
responses in UR which are induced by stimulating the
peripheral nociceptors. Orienting responses to tone may be
one of the components of the CR. But this factor was taken
into the consideration by using statistical estimation to
differentiate the individual CR from the pre-training
movement that may have resulted from random movement
and orienting responses. In order to further distinguish the
associated component of the CR from the reflexive component of the orienting response, in an extended study, a
second CS with different tone frequency that was not
paired with US, was introduced and analyzed.
The present paradigm differs from other conditioning
paradigms in several respects. First, the CR does not
directly measure learned avoidance behaviors, but the
consequences of conditioning may be the usual results of
avoidance behaviors. Since the interval between the CS
and US is only 0.5 s, the avoidance behavior would often
J.-C. Kung et al. / Brain Research 970 (2003) 58–72
69
Fig. 9. Effect of morphine on conditioning response. (A) The integrated CR activity decreased significantly in the morphine treated group. (B) In the four
sub-trials, trials 1–10, 21–30 and 31–40 were significantly decreased compared to the control. * Denotes P,0.05.
be observed after the US was presented. Second, the CR,
turning their head toward and licking their left paw, could
have occurred within 0.5 s after the onset of CS. In
addition to emotional arousals, other psychological functions such as attention, discrimination may also contribute
to the CR. The occurrence of CR during the paired stimuli
may indicate the acquisition phase of the CR formation.
The CR appeared immediately and 24 h after the paired
stimuli. Thus a learned CR was consolidated in the later
phase. Third, freezing responses reported in fear conditioning experiments were not measured in the present
study. It is not certain whether the lack of the CR in some
test trials is an indication of immobilization. The nature of
the noxious stimuli used in fear and pain conditioning
experiments is different. The US used in the present
experiment was restricted to the left paw. However, foot
shocks are extensive and nonspecific aversive stimuli. If
they were to be sustained for longer intervals, they would
therefore produce more stressful and aversive experiences
than the present short pulse laser stimulus, and would
result in more stressful behavioral consequences.
The present experiments suggest that the ACC is
involved in laser pain conditioning. The histological data
obtained from these animals showed that the ACC lesioned
areas include, Cg1, Cg2, prelimbic and infralimbic areas.
The Cg1 and Cg2 were the largest areas affected by the
lesion. These subareas in the ACC are parts of a complex
network that make up thalamic and other limbic structures
[63]. In rats, the ACC has reciprocal connections with the
medial thalamus that receive nociceptive inputs [23,24].
70
J.-C. Kung et al. / Brain Research 970 (2003) 58–72
With its extensive connections with cortical and subcortical structures the ACC may have involved attention-demanding task processes that is required for nocifensive
conditioning responses [13,64]. The infralimbic and prelimbic areas form the ventral part of the ACC. Several
studies have reported that the ventral cingulate cortex is
involved in conditioned autonomic responses [17,18,38].
Therefore, we can not exclude the possibility that there is a
conditioned autonomic change that occurs in parallel with
the observed motor CR. Studies in motor learning indicate
that different pathways contribute to the somatomotor and
autonomic conditioning, respectively [59].
The present experiment also showed that lesions of
amygdala block pain conditioning. This is consistent with
previous evidence which has shown that the amygdala is
involved in Pavlovian type of conditioning. The histological data suggest that the lesioned areas included the central
nucleus, lateral nucleus, basal lateral and basal medial
nuclei. There is considerable evidence that the amygdala is
involved in fear conditioning [14,28,31]. In fear conditioning with auditory stimuli, the acquisition of association between US and CS was blocked by lesions of the
lateral and central nuclei of amygdala [37]. The amygdala
has been shown to receive nociceptive inputs from posterior / suprageniculate complex and from posterior insular
complex [53]. The lateral nuclei of amygdala are the main
recipient of auditory inputs [33]. There are direct projections from the basolateral amygdaloid nucleus to the
prefrontal cortex and ACC [35], further outputs from the
ACC may send to caudate putamen. The efferents of the
central nuclei of amygdala to different brain areas may
have involved the expression of conditioned responses
which are associated with different defensive mechanisms.
Killcross et al. [29] have dissociated the functions of
central and lateral nuclei of the amygdala in fear conditioned tasks. They have shown that lesions of the central,
but not basolateral, nuclei of amygdala impaired conditioned suppression of ongoing responses by shock, and
lesions of the basolateral, but not central nuclei of
amygdala impaired the ability of a shock paired CS to
influence ongoing operant responses or choice behavior.
However, no consensus exists concerning differences in
the functions of different nuclei of amygdala in conditional
aversions [29]. A recent report suggested that the representation of the motivational aspects of pain is mediated by
an amygdala–ACC–putamen circuit [52]. It is likely that
the acquisition of the CR in the present study was related
to auditory afferent inputs that also projected to the
amygdala. The pain conditioning could be formed in the
amygdala via associating neutral auditory information with
life threatening information from brain stem. In addition,
Gabriel and colleagues [44,56] showed that electrolytic
lesions of the amygdala blocked learning and prevented the
development of training induced activity in the anterior
and medial dorsal thalamic nuclei and in related areas of
the cingulate cortex in the rabbits. Temporary inactivation
of the amygdala using the GABA receptor agonist muscimol immediately before the discriminative avoidance
conditioning permanently blocked the development of
training-induced discriminative neuronal activity in the
medial geniculate nucleus of rabbits [47]. However, intraamygdalar muscimol failed to disrupt performance of the
well-established avoidance responses [46]. These findings
suggest that the amygdala was involved in discriminative
instrumental avoidance learning, particularly the acquisition phase, and in the elaboration of cingulothalamic
learning-relevant neuronal plasticity [45].
The lesion of other limbic structures such as hippocampus may also block contextual specific fear conditioning
[22]. The hippocampus has been shown to participate in
the functional connection with the ACC and the amygdala
[62]. Thus it is likely that it may also be involved in
autonomic and emotional processes involving pain stimuli.
Lesions in the region of the medial thalamic nuclei
produce changes in supraspinal mediated nociceptive
behaviors [27,50]. However, it is not clear if pain conditioned responses are affected.
Several issues need to be addressed in order to understand the relationship between the limbic systems and CR.
First, lesions of either the ACC or amygdala may not
totally eliminate the acquisition of CR. Lesions of the
ACC or the amygdala may partially reduce the acquisition
of CR. Thus other limbic areas such as hippocampus and
orbitofrontal cortex and medial thalamus may also be
involved in the pain conditioning. Second, little is know
concerning the general physiological conditions under
which a lesion will produce a reduction of the CR. Rats
under conditions of hunger or satiated drive may alter their
performance in the avoidance response [58]. Third, lesions
of either the ACC or the amygdala appear to have no
effects on the nocifensive responses; UR. Cahill and
McGaugh [6] also showed that lesions of the amygdala
blocked conditioned response but not unconditioned response in a task. Thus, this would indicate that the ACC
and amygdala may play no essential role in the initiation of
the reflexive nocifensive behaviors.
The pain conditioning in the present study was effectively reduced by morphine injections. This suggests that a
certain analgesic effect of morphine may alter the affective
part of the pain responses. Some studies have shown that
morphine analgesia is related to the limbic system [21,49].
However, it is not certain whether an opioid system is
involved in affective responses to pain. And in addition,
there is no evidence that the effect of morphine on the CR
is involved directly in the ACC. Nonetheless, the findings
of a high content of opioid receptors in the ACC would
substantiate the assumption that morphine has a modulatory effect on the neurotransmitters involved in the process
[34,63].
Relationships between limbic areas and pain information
processing are not clear. Clinical evidence has implicated
limbic structures in the affliction of pain. Cingulotomy has
J.-C. Kung et al. / Brain Research 970 (2003) 58–72
been shown to alleviate patient’s affective responses to
noxious stimuli, such as those produced by chronic,
intractable pain [2]. This clinical evidence is consistent
with recent functional brain imaging studies of nociceptive
responses in human, showing that peripheral noxious
stimulation can activate ACC and several other limbic
areas in the brain [12,26].
The existing evidence suggests that somatic pain responses can elicit complex, negative emotional processes
through forming association between neutral stimuli and
pain stimuli. This is not a consequence of somatosensory
cortical activation but rather through association processes
in parallel with the cortical and subcortical structures.
Conditioned emotional responses are essentially sensoryaffective associations. The ACC and the amygdala appear
to be the key structures in the brain that integrate sensory
experience with emotional arousal, particularly conditioning involving negative emotional association. Through
forming associations with pain stimuli, previously neutral
stimuli become warning cues which predict danger. Organisms that can learn readily from experience have adaptive
advantages over those that cannot. Biologically pain
conditioning supports survival by fostering anticipation
and prediction of potential tissue injury. The emotional
component of pain appears to support adaptation and
survival by facilitating learning, memory and related
cognitive processes. It provides a bridge by which pain can
influence the psychological status of the individual and
their behavior tendencies.
Acknowledgements
The present study was supported by grants from the
National Science Council (Project No. NSC 89-2320-B001-032) and Academia Sinica, Taiwan.
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