Group Title: Behavioral and Brain Functions 2007, 3:49
Title: Effects of mu- and kappa-2 opioid receptor agonists on pain and rearing behaviors
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Title: Effects of mu- and kappa-2 opioid receptor agonists on pain and rearing behaviors
Series Title: Behavioral and Brain Functions 2007, 3:49
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Creator: Neubert JK
Rossi HL
Pogar J
Jenkins AC
Caudle RM
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Behavioral and Brain Functions Bioved



Research

Effects of mu- and kappa-2 opioid receptor agonists on pain and
rearing behaviors
John K Neubert*1,3,4, Heather L Rossi1,3, Jonathan Pogar', Alan C Jenkins'
and Robert M Caudle2,3,4


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 Neuroscience, College of Medicine, University of Florida, Gainesville, FL,
USA and 4Evelyn F. and William L. McKnight Brain Institute, University of Florida, Gainesville, FL, USA
Email: John K Neubert* jneubert@dental.ufl.edu; Heather L Rossi hrossi@dental.ufl.edu; Jonathan Pogar jpogar@dental.ufl.edu;
Alan C Jenkins ajenkins@dental.ufl.edu; Robert M Caudle caudle@ufl.edu
* Corresponding author



Published: 20 September 2007 Received: 19 June 2007
Behavioral and Brain Functions 2007, 3:49 doi:10.1 186/1744-9081-3-49 Accepted: 20 September 2007
This article is available from: http://www.behavioralandbrainfunctions.com/content/3/1/49
2007 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: Management of pain involves a balance between inhibition of pain and minimization
of side effects; therefore, in developing new analgesic compounds, one must consider the effects of
treatment on both pain processing and behavior. The purpose of this study was to evaluate the
effects of the mu and kappa-2 opioid receptor agonists on general and pain behavioral outcomes.
Methods: As a general behavioral assessment, we modified the cylinder rearing assay and
recorded the number and duration of rearing events. Thermal sensitivity was evaluated using either
a reflexive measure of hindpaw withdrawal latency to a radiant heat source or using an orofacial
operant thermal assay. Acetic acid-induced visceral pain and capsaicin-induced neurogenic
inflammatory pain were used as painful stimuli. The mu-opioid receptor agonist, morphine or the
kappa-2 receptor agonist GR89696 was administered 30 min prior to testing. A general linear
model repeated measures analysis was completed for baseline session comparisons and an analysis
of variance was used to evaluate the effects of treatment on each outcome measure (SPSS Inc).
When significant differences were found, post-hoc comparisons were made using the Tukey
honestly significant difference test. *P < 0.05 was considered significant in all instances.
Results: We found that morphine and GR89,696 dose-dependently decreased the number of
reaching events and rearing duration. Rearing behavior was not affected at 0.5 mg/kg for morphine,
1.25 x 10-4 mg/kg for GR89,696. Hindpaw thermal sensitivity was significantly increased only at the
highest doses for each drug. At the highest dose that did not significantly influence rearing behavior,
we found that visceral and neurogenic inflammatory pain was not affected following GR89,696
administration and morphine was only partially effective for blocking visceral pain.
Conclusion: This study demonstrated that high levels of the opioids produced significant
untoward effects and made distinguishing an analgesic versus a more general effect more difficult.
Quantification of rearing behavior in conjunction with standard analgesic assays can help in gaining
a better appreciation of true analgesic efficacy of experimental drugs.





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Behavioral and Brain Functions 2007, 3:49



I. Background
Management of pain involves a balance between inhibi-
tion and suppression of pain and minimization of unto-
ward side effects. For example, patients suffering from
intractable pain can be limited in the amount of narcotics,
such as morphine, that they can receive due to adverse
cognitive effects and sedation. Therefore in order to
develop new analgesic compounds, one must consider the
effects of the treatment on both pain processing and gen-
eral behavior. In the search for better pain relief, numer-
ous novel compounds have been investigated, including
kappa-opioid receptor agonists. Since the early work of
Attali et al [1], binding, behavioral, and in vitro physio-
logic studies have provided evidence supporting the exist-
ence of two subtypes of kappa-opioid receptors, kappa-1
and kappa-2.

Kappa-1 opioid receptors preferentially bind arylaceta-
mide-like agonists such as U69,593 [2], and have been
shown to be effective in blocking mechanical allodynia
[3,4]. However; they are relatively ineffective for reducing
visceral hypersensitivity [5] and thermal allodynia and
hyperalgesia [6,7]. While there is a debate regarding the
existence of a distinct kappa-2 receptor, there is evidence
to support that it is different from the kappa-1 receptor.
Devi and colleagues [8-11] have demonstrated that recep-
tor specificity is altered when opioid receptors form heter-
omers. Their work indicates that heteromers of the delta
opioid receptor and the kappa opioid receptor have the
receptor binding properties of the classically defined
kappa2 receptors. Additionally, the combination of kappa
and delta receptors has also been demonstrated in vivo
[12]. These data indicate that kappa/delta heteromers
(kappa-2 receptors) are a distinct class of receptor. In
addition to performing binding studies demonstrating the
presence of kappa-2 receptors, Caudle and Finegold et al
found that U69,593, a kappa-1 receptor selective agent,
has no analgesic effect when injected intrathecally in rats
[13] whereas [methyl-4-[3,4-dichlorophenyl)acetyl]-3-
[1 -pyrrolidinyl)methyl]-1-piperazinecarboxylate]
(GR89,696), a putative kappa-2 opioid receptor agonist,
has very potent antihyperalgesic actions [14,15]. The
actions of GR89,696 were also clearly distinguishable
from those of mu and delta selective agonists. Further-
more, in guinea pig hippocampal slices it has been dem-
onstrated that kappa-1 receptors primarily inhibited
glutamate release while kappa2 receptor activation sup-
pressed NMDA receptor function [16-18]. These data are
also supported by the work of Schoffelmeer et al. [19],
Ohsawa and Kamei [20], and Gu et al [21]. Kappa-2 recep-
tor agonists such as 4- [(3,4-Dichlorophenyl)acetyl]-3-(1-
pyrrolidinylmethyl)-l-piperazinecarboxylic acid methyl
ester fumarate salt (GR89,696) have been shown to effec-
tively block inflammatory and neuritis-induced pain, as
well as peripheral neuropathic pain [14,15 The anti-allo-


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dynic and -hyperalgesic effects of GR89,696 are proposed
to be a result of spinal kappa-2 opioid receptor activation
and subsequent inhibition of spinal NMDA receptors [ 14-
16].

Efficacy is normally the primary outcome in the evalua-
tion of analgesics; however, it is equally important to con-
sider that the various opioid receptor agonists are not
without unpleasant side effects. For example morphine is
considered the "gold standard" analgesic, but effective
analgesic doses can produce sedation, constipation,
dependence and addiction. Kappa-1 receptor agonists
such as U69,593 and the Mexican mint derived agent,
salvinorin A have been shown to also produce sedation
and motor incoordination [22]. Activation of the kappa-1
receptor has also been reported to affect rearing and loco-
motion activity, with lower doses increasing this activity
and higher doses decreasing it [23-25]. The negative
effects of kappa-1 receptor agonism are well-defined, but
the unpleasant effects following kappa-2 receptor activa-
tion are less known. While GR89,696 has been reported as
a possible therapeutic agent, it can produce a catatonic
and immobilized state (Caudle, personal communication,
2005). However, formal characterization of the potential
side effect profile of this drug at therapeutic doses has yet
to be completed. Clinically, kappa receptor agonists are
known to produce dysphoria and hallucinations[26,27].

In the current study, general behavior was evaluated using
a simplified rearing assay to assess sedative or systemic
effects and analgesic and antihyperalgesic effects were
evaluated using reflex and operant tests. Previous rodent
studies have assessed the influence of morphine on loco-
motor effects and have demonstrated that there are con-
trasting effects depending on dose, with both stimulant
and depressive locomotor effects being reported [28-32].
Rearing behavior has been used previously as a measure of
locomotor effects following opioid administration, with
reports that morphine decreases this rearing behavior
[33,34]. Given this, we expected that rearing behavior
would provide a sensitive method for detecting locomotor
effects in the assessment of our study drugs. It is important
to note that other factors such as sedation, decreased
motivation, and increased anxiety may lead to decreases
in this activity. In the context of this study, it was not
important to distinguish the mechanism related to a
change in rearing behavior. Our contention is that the
change in behavior is the relevant endpoint here because
any event that produced this change will likely affect the
pain outcome measures as well.

2. Methods
2. General
Male Sprague Dawley rats (200-300 g, Harlan, Houston,
TX) were housed in groups of one to two and were main-


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Behavioral and Brain Functions 2007, 3:49



trained in a standard 12-hour light/dark cycle and testing
was completed in the light portion of the cycle between
09:00-12:00. Animals were placed into the behavioral
procedure room 30 min prior to testing to acclimate.
When not in testing sessions, food and water were made
available ad libitum. Animal testing procedures complied
with the ethical guidelines and standards established by
the University of Florida's Institutional Animal Care &
Use Committee and with the Guide for Care and Use of
Laboratory Animals [35].

2.2. General behavioral assessment
A modified limb-use asymmetry test [36,37] was used to
measure rearing activity as an assessment of general
behavior. An acrylic cylinder (19.5 cm diameter x 40.5 cm
height) was constructed with aluminum sheets placed
both on the floor and 13.5-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 animals were placed into sepa-
rate cylinders and the data acquisition system was acti-
vated. When the animal reared, it would place its front
paw on the metal side of the cylinder, completing an elec-
trical circuit (Fig. 1). The closed circuit registered in the
computer and front paw contact data were collected for 15
min. Baseline testing was completed for each set of ani-
mals, consisting of 3 consecutive sessions.

2.3. Thermal testing
Response to hindpaw heat pain was determined by plac-
ing unrestrained animals on a clear glass platform under a
small plastic cage and animals habituated for 5 min. A
radiant heat source was aimed directly under the ventral
hindpaw surface and the time to paw withdrawal was
recorded as described previously [38].

Orofacial thermal sensitivity was assessed using a reward-
conflict operant paradigm, as described previously [39].
Briefly, unrestrained food fasted (12-15 hrs) animals
were placed into clear acrylic testing boxes (20.3 cm W x
20.3 cm D x 16.2 cm H). The animals were allowed access
to a standard rodent watering bottle containing a diluted
(1:2 with water) sweetened condensed milk solution
(Nestle Carnation Company, room temperature) by plac-
ing their head through an opening in the box. The open-
ing was lined with grounded metal (aluminum) tubing
that served as a stimulus thermode when connected to a
water pump (Model RTE110B, NES Laboratories, Inc.).
The reward bottle was positioned in proximity to the cage
such that the animal was allowed access to the reward bot-
tle when simultaneously contacting the thermode with its
face. The metal spout on the watering bottle was con-
nected to a DC power supply and, in series, to a multi-
channel data acquisition module (WinDaq Lite Data Acq


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DI-194, DATAQ Instruments, Inc). When the rat com-
pleted the task and drank from the water bottle, the skin
on its face contacted the grounded thermode, and the ani-
mal's tongue contacted the metal spout on the water bot-
tle, completing an electrical circuit. The closed circuit
registered in the computer and data was collected at 60 Hz
for the entire length of the experiment. Each spout contact
was recorded as a "licking" event and a separate circuit was
established from the metal thermode to the animal by
grounding the floor with an aluminum sheet for recording
of "facial contact" events. Animals were first trained to
drink milk while contacting the thermode set to a temper-
ature at 370C for baseline training (N = 5 sessions) and
animals were considered trained when their intake is > 10
g of reward milk, as described previously [39].

2.4. Pain induction
We wanted to use painful stimuli that were not going to
produce an injury or deficit in the lower limbs to mini-
mize false-negative responses, so models of acetic acid-
induced visceral pain [40] and capsaicin-induced neuro-
genic inflammatory pain were used. For visceral pain, a
0.05% acetic acid solution (1 ml) was injected into the
intraperitoneal space using a 23-gauge needle and ani-
mals were immediately placed in the testing apparatus.

Orofacial neurogenic inflammatory pain was produced by
applying capsaicin cream (0.075%, Thomson Microme-
dix, Colorado) to the facial region of unanesthetized rats
and left on for 5 min. Following capsaicin application to
the face, animals normally will try to wipe off the cream
and in the process can spread the capsaicin from their
front paws to their mouth. In order to prevent this groom-
ing and subsequent intraoral capsaicin exposure, the ani-
mals were gently restrained for the stimulus duration and
then the face was wiped clean to remove residual capsai-
cin cream. Immediately after capsaicin removal, animals
were tested at 48 C using the thermal operant system.

2.5. Opioid agonists
The mu-opioid receptor agonist, morphine (subcutane-
ous injection, 0.25 5 mg/kg, 200 gl), or the kappa-2
receptor agonist GR89696 (subcutaneous injection, 1.25
x 10-4 1.0 mg/kg, 200 gl) was administered 30 min prior
to testing. Control vehicle treatment consisted of phos-
phate buffered saline solution (subcutaneous injection,
200 1).

2.6. Data analyses
The threshold for detection of front paw contacts, facial
contacts and licking contacts was set at 1.0 V and an event
was registered when the signal went above threshold and
ended when the signal dropped below threshold. Two
rearing outcome measures were calculated: (1) cumula-
tive duration of rearing events; (2) total number of reach-


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Behavioral and Brain Functions 2007, 3:49



A. Resting


C. Rearing Contacts







0 3


D. Number
140-
,2331 (2
120 -
Q100
0 80
S60
<40
20
0 3


4,1


LI


1 2 3
Session


4,


I I I I
6 9 12 11
Time (sec)
E. Duration


(186s) (179s) (173

m rh +


U I I I I
1 2 3
Session


Figure I
Illustrated is an animal that is at rest (A) and rearing (B) in the rearing assay testing device. An example of the trace recording
is illustrated in panel C with the arrows denoting distinct rearing events. Animals (N = 12) tested on three consecutive days
displayed a significant decrease (*P < 0.05) in the number of reaches (D), but not in the total rearing duration (E). The raw
number of events or time (s) is denoted in the parentheses.


ing events. For orofacial thermal sensitivity, six outcome
measures were evaluated: (1) reward intake; (2) total
number of licking events; (3) total number facial contacts;
(4) cumulative facial contact duration; (5) ratio of
reward/facial contacts; (6) duration per contact for the
facial contacts. Data analyses were completed using cus-
tom-written routines in LabView Express (National Instru-
ments Corporation) and Excel (Microsoft).

2.7. Statistical analyses
A general linear model repeat measures analysis was com-
pleted for baseline session comparisons and an analysis of


variance was used to evaluate the effects of treatment on
each outcome measure (SPSS Inc). When significant dif-
ferences were found, post-hoc comparisons were made
using the Tukey honestly significant difference test. P <
0.05 was considered significant in all instances.

3. Results
3. Rearing behavior
We found that different groups of animals reared a varying
amount; therefore in order to normalize differences
between groups, we computed and compared data as a
percentage of the baseline sessions. An example of a typi-


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B. Rearing


I,







Behavioral and Brain Functions 2007, 3:49


cal baseline session set is presented in Fig. 1D, E. There
was an overall significant decrease (F2,22 = 7.35, *P <
0.005) in the number of contacts across the three baseline
testing sessions for this group of animals (N = 12). This
decrease occurred with other groups and is likely due to
normal habituation to the closed testing field with repeat
exposures. In contrast, the rearing activity duration
remained constant and was not significantly (F2,22 = 0.26,
P = 0.77) different across baseline testing sessions and this
was consistent within each group.

3.2 Effects of opioid agonists on rearing behavior
3.2.1 Reaching number (Fig. 2A)
There was a significant dose effect produced by both
GR89,696 (F4, = 10.71, *P < 0.001) and morphine (F4,91
= 9.41, *P < 0.001) on the number of reaching contacts.
GR89,696 at doses 2 1.25 x 10-3 mg/kg, produced a signif-
icant decrease in reaching number, compared to baseline.
The 1.25 x 10-4 mg/kg dose was the highest dose tested
that did not significantly differ from baseline. Similarly,
morphine produced a significant decrease in reaching
contacts at doses 2 2.5 mg/kg.

3.2.2 Rearing duration (Fig. 2B)
There was a significant dose effect on rearing duration
when either GR89,696 (F4,29 = 10.74, P < 0.001) or mor-
phine (F4,91 = 12.16, *P < 0.001) was administered. Simi-


A. Number

35' GR89,696 125 Morphine
100] 100
I 75 75
W0 5 5
B B25
00 i
0 1.25 125 1.25 1.0 0 25 05 25 5.0
x10' xl0' x10, DOSE (mg/kg)
B. Duration
150 GR89,696 125 Morphine
125 00
S75
m: *
G 50* *
25- 25

0 1.25 1.25 1.25 1.0 0 025 05 2.5 5.0
x10' x10' x10 DOSE (mg/kg)

Figure 2
We evaluated the effects of GR89,696 and morphine on
reaching activity and cumulative rearing duration. There was
a significant dose-related decrease (*P < 0.05) in the number
of reaching events (A) and total time spent rearing (B) fol-
lowing morphine (N = 8-12, right insets) or GR89,696 (N =
6, left insets) administration, compared to baseline values.


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lar to the effects on the number of reaches, GR89,696
produced a significant reduction in the rearing duration at
doses 2 1.25 x 10-3 mg/kg. Additionally, morphine doses
> 2.5 mg/kg suppressed exploratory behavior, as evident
by a significant decrease in duration. Clinically relevant
doses (< 0.5 mg/kg) of morphine did not significantly
affect this outcome measure as compared to baseline.

3.3 Effects of opioid agonists on thermal sensitivity
3.3.1 Hindpaw withdrawal latency (Fig. 3)
There was a significant dose effect (F4,57 = 5.53, P < 0.05)
of GR89,696 on hindpaw withdrawal latency following
thermal stimulation. Only the highest dose tested, 1.0
mg/kg, produced a significantly higher latency compared
to baseline. There was additionally a significant dose
effect (F2,35 = 15.4, P < 0.001) of morphine, with only the
highest dose of 5.0 mg/kg sufficient to suppress normal
thermal transmission. The 0.5 mg/kg dose did not alter
withdrawal latency as compared to baseline.

3.3.2 Thermal operant assessment
Previously, we demonstrated that a 0.5 mg/kg dose of
morphine could completely reverse inflammatory [39]
and neurogenic inflammatory heat allodynia and hyperal-


A. GR89,696
200-

W 150-
z

aU

50-

0-


0 1.25 1.25 1.25
x10" x10 x102
B. Morphine
2001


S150-
Z
-j
w
n 100-
m
S50-


0 0.5 5.0
Dose (mg/kg)


1.0


Figure 3
The effects of GR89,696 and morphine on normal thermal
sensitivity were assessed and demonstrated that hindpaw
withdrawal latency to thermal stimulation was significantly
increased only at the highest doses tested for GR89,696 (N =
6) and morphine (N = 6).


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nn








Behavioral and Brain Functions 2007, 3:49




gesia[41] in the orofacial region. Specifically, reward
intake, licking contacts, facial contacts, facial contact
duration, ratio of reward/stimulus contacts, ratio of facial
contact duration/event all returned to baseline levels
when animals were pretreated with morphine 30 min
prior to either carrageenan or capsaicin application.

In this study, as a comparison, GR89,696 was adminis-
tered 30 min prior to topical capsaicin application. The
dose tested (1.25 x 10-4 mg/kg, subcutaneous) was the
maximum dose that did not produce a significant effect
on rearing behaviors. Contrary to the morphine results
from those previous studies, GR89,696 was not capable of


A. Reward Intake
1201
100


vehlcap


Treatment
C. Licking Contact Events
120 (Reward)
100
_ 80
S60
40
20

baseline GR89696 vehcap
/cap
Treatment

E. RewardlAttempts
1201
100 r-A


- 80
0 60
40
20
0


*

baseline GR89696 veh/cap
/cap
Treatment


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blocking the heat hyperalgesia associated with capsaicin
treatment (Fig. 4). There were no significant differences in
reward intake, licking contacts, facial contacts, facial con-
tact duration, ratio of reward/stimulus contacts, and ratio
of facial contact duration/event when comparing active
drug compared to vehicle treated animals. Both GR89,696
and vehicle-treated animals displayed significantly lower
responses for these operant outcomes, compared to base-
line values, indicating that the capsaicin produced a pain
behavior that was unaffected by either GR89,696 or vehi-
cle treatment.




B. Facial Contact Duration
1201
i'0 I


80
460
S40,


baseline GR89696 veh/cap
/cap
Treatment


D. Facial Contact Events (Stimuli)


baseline GR89696 veh/cap
/cap
Treatment


F. Facial Duration/Contact
160
140
!120
* 100 --
S80
[ 60
40
20
0
baseline GR89696 veh/cap
/cap
Treatment


Figure 4
We found that GR89,696 does not affect operant thermal outcome measures. Animals treated with either GR89,696 (1.25 x
10-4 mg/kg, subcutaneous) or vehicle (PBS, subcutaneous) prior to facial capsaicin application did not significantly differ from
each other when evaluated using the six thermal operant outcome measures (N = 10). Both groups were significantly lower
(*P < 0.05) than baseline values in all instances except for GR89696 in Facial Contact Duration (B) and for both GR89696 and
vehicle for Facial Duration/Contact (F). Overall, this indicates that the neurogenic inflammatory pain was not inhibited follow-
ing capsaicin application.




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3.4 Effects of visceral pain on rearing behavior (Fig. 5)
The induction of visceral pain using acetic acid provides a
standard pain assay used in many analgesic assessment
studies. Typically, investigators will count the number of
writhing (stretching) events following intraperitoneal
injection of acetic acid and compare this response in the
presence of an analgesic substance. We were interested in
evaluating the effects of visceral pain on exploratory
behavior and we wanted to evaluate the effects of mor-
phine and GR89,696 at doses that did not produce a sig-
nificant suppression of rearing behavior. Immediately
following acetic acid injection, animals displayed a signif-
icant decrease in activity as demonstrated by a decrease in
the number of contacts (F2,31 = 26.96, *P < 0.001) and
rearing duration (F2,31 = 13.79, P< 0.001). These animals
displayed normal behavior when tested again 24 h post-
injection (upper panels). There was a significant treatment
effect on reaching contacts (F3,41 = 13.11, P < 0.001) and
rearing duration (F3,41 = 14.45, P < 0.001) following acetic
acid injection when GR89,696 (1.25 x 10-4 mg/kg) and
morphine (0.5 mg/kg) were compared to untreated ani-
mals (lower panels). Post-hoc analysis revealed that the


A. Number


B. Duration


C. Number TIME D. Duration TIME


ta ., ht.'..: j-. e.1.t,.r. Na... -.s.. CR n-..r
Acid 89.696 Acid 89,696
TREATMENT TREATMENT


Figure 5
The effects of morphine and GR89,696 on reaching activity
and cumulative rearing duration following visceral pain
induction was determined. Acetic acid was injected (intra-
peritoneal) as an acute painful stimulus and produced a sig-
nificant decrease (*P < 0.05, compared to baseline) in both
reaching number (A) and rearing duration (B) during the
first 15 min post-injection. Animals were fully recovered by
24 h, as their rearing behavior returned to baseline levels.
GR89,696 (N = 8, 1.25 x 10-4 mg/kg, subcutaneous) was
ineffective and morphine (N = 8, 0.5 mg/kg) was only par-
tially effective for blocking this visceral pain induction, as
demonstrated by a decrease in reaching number (C) and
rearing duration (D). For (C, D), *P < 0.05 as compared to
naive animals.


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untreated and GR89,696 groups were significantly lower
than the baseline values, while the morphine treated
group was not significantly different compared to base-
line.

4. Discussion
The use of pharmacological agents as a tool for character-
ization of nociceptive pathways remains a cornerstone of
pain research. Related to this is the ability to assess the
general behavioral consequences of experimental inter-
ventions, as this is a necessary and important step in char-
acterizing specific and non-specific effects of each
treatment. When assessing behavioral outcome measures
for evaluation of analgesics, there is an underlying
assumption that the drug is having an effect only on the
relevant portion of the pain circuitry that it is targeting.
However, if an experimental compound produces a severe
cognitive or sedative effect, then these measures could be
altered or suppressed to give false-negative results. There-
fore it is important to be able to evaluate and distinguish
between analgesic effects versus central incapacitating or
sedative effects. In this paper, we compared the effects of
opioid agonists on pain and general behavioral outcomes
in order to characterize the optimal dose required for
reducing pain in the absence of cognitive impairment.

We used a modified cylinder test as a non-invasive, repeat-
able assay to quickly screen for general behavioral effects
(i.e., sedation) produced by varying doses of different opi-
oid agonists. The cylinder test is also known as the limb-
use asymmetry test and is primarily used for determining
the effects of neurological damage on sensory motor func-
tion skills [36,37]; however, the use of this system differs
slightly in the context of pain research. For example, we
were not necessarily interested in functional handedness
(right vs. left) for reaching; rather we wanted to assess the
exploratory activity as a general behavioral outcome
measure. Rearing events and number of reaching contacts
were the outcome measures evaluated using this test. The
advantage of the cylinder test is that repeated testing does
not influence outcomes and rats do not develop compen-
satory strategies. We modified the system by utilizing
automated computer data acquisition that provides an
easy and fast method for evaluation of behavior following
injury or drug administration. This can allow for an inves-
tigator to quickly screen for systemic and cognitive effects
of new therapeutics, which is particularly valuable for the
assessment of pain, or pain relief following a therapeutic
intervention. In this system, we did note that a certain
level of habituation occurs, with the number of reaches
significantly decreasing with consecutive trials. However,
the duration data remained constant and does not appear
to be susceptible to this habituation phenomenon, and
thus provides the more stable measure. This assay pro-
vides a quantitative measure that is neither stimulus-


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Behavioral and Brain Functions 2007, 3:49



evoked nor investigator-initiated and therefore can reduce
inherent variability issues associated with animal testing.

Morphine remains the "gold standard" for controlling
clinical pain and has been used extensively in animal pain
models. There is evidence in rodents supporting the idea
that morphine produces a biphasic response in locomo-
tion following morphine administration, resulting in
both decreased and increased motor activity, depending
on: the activity evaluated (e.g., locomotion, rearing),
dose, and time after administration [28,34,42-44]. In
these studies, increased locomotion has been reported at
doses of morphine 2 10 mg/kg, well above the doses used
in our study. Additionally, other investigators have evalu-
ated the effects of morphine on rearing behavior in mice
and similarly found that doses of morphine 2 5-40 mg/kg
produces a reduction in rearing events [34,45]. Indeed,
when we evaluated the effects of morphine on rearing
behavior, we found that there was a significant dose-
related decrease in both rearing events and in the number
of reaching contacts, with suppression of the exploratory
behavior occurring at doses 2 2.5 mg/kg (Fig. 2). These
data support that systemic effects can occur at these doses,
including impairment of motor and motivational
responses, as reported previously [46-48]. We would
expect that other drugs producing sedative effects would
also produce a significant decrease in rearing contacts and
duration.

In regards to analgesic efficacy, these impairments can
compromise interpretation of analgesic results, as an ani-
mal may be immobilized or unresponsive when tested at
these doses. This may be problematic in that high doses of
morphine (> 3 mg/kg) are required to suppress reflexive
assays of pain measurement [49-51] and are relatively
higher as compared to doses in humans (< 0.15 mg/kg)
that are effective for controlling clinical pain [52]. While
it is possible that a differential expression rate of opioid
receptors between species may explain this discrepancy in
dose, the non-analgesic effects including sedation and
suppression of reflex responses cannot be ignored. In fact,
we were able to use operant outcome measures to demon-
strate that this discrepancy may be due to a lack of sensi-
tivity of current reflex-based analgesic assays for detecting
analgesic effects at the lower doses of morphine. Our
group has shown that 0.5 mg/kg of morphine was suffi-
cient to suppress nociceptive activity [39,41]. In contrast,
when we completed analgesic assessment using hindpaw
withdrawal time as the outcome, a 10-fold higher dose
was required to block thermal pain.

Recently, the kappa-2 opioid receptor has become a target
for pain control, with drugs such as GR89,696 being con-
sidered. In contrast to mu-receptor agonism, kappa-2
receptor binding produces an antihyperalgesic effect, but


http://www.behavioralandbrainfunctions.com/content/3/1/49



is not considered an analgesic, as sensitivity to normal
nociceptive stimuli is not affected. The effective dose of
GR89,696 for reducing pain behaviors has been reported
to be ~0.01 mg/kg when given intrathecally [14,15]. For
subcutaneous administration, we initially chose a dose
that was 100 fold higher than this dose, as this has been
reported to produce a catatonic-like state (Caudle RM, per-
sonal communication). Subsequent doses were decreased by
~10 fold dilution until normal behavior was exhibited. In
this study, GR89,696 doses 2 1.25 x 10-3 mg/kg produced
behavioral impairment (Fig. 2A, B), as demonstrated by
the significant decrease in both rearing number and dura-
tion.

Pain associated with normal heat transduction can be
reduced with high doses of morphine (5 mg/kg) or
GR89,696 (1 mg/kg), as seen by an increase in hindpaw
withdrawal latency. However at these doses there is a sig-
nificant reduction in rearing behavior, therefore, one can-
not necessarily state that these doses are analgesic, rather
that they were sufficient to significantly change the reflex
outcome. For subsequent analgesic assessment against
visceral and orofacial neurogenic inflammatory pain, we
selected 1.25 x 10-4 mg/kg for GR89,696 and 0.5 mg/kg
for morphine, respectively, as the relevant test doses that
did not produce a significant behavioral rearing effect as
compared to baseline (Fig. 3). Also there was no effect on
normal thermal transduction following GR89,696 (1.25 x
10-4 mg/kg) or morphine (0.5 mg/kg) at these low doses,
as hindpaw withdrawal latency was not significantly
increased.

Visceral pain following intraperitoneal injection of acetic
acid has been shown to produce a significant increase in
writhing behavior for approximately 15 min [40,53]. Not
surprisingly, visceral discomfort had a significant effect on
exploratory behavior and this effect was detected by the
rearing assay in a time-appropriate fashion with the
behavioral response duration corresponding to the dura-
tion of the stimulus. There was a significant decrease at the
acute (15 min) session, but the behavior returned to nor-
mal at 24 hrs. When animals were pre-treated with mor-
phine (0.5 mg/kg) or GR89,696 (1.25 x 10-4 mg/kg), there
was a partial analgesic effect on the acetic acid/rearing
assessment produced by morphine, as the reaching and
duration values were lower than baseline, but higher than
no treatment or GR89696. This dose of morphine was not
effective in blocking normal heat sensitivity (Fig. 3B),
which is consistent with the lack of antinociceptive effect
demonstrated using the rearing assay.

As a second pain model, we applied a low dose of capsai-
cin cream to the facial region to produce neurogenic
inflammatory pain. Previously, we demonstrated that
morphine at doses < 0.5 mg/kg produced a significant


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Behavioral and Brain Functions 2007, 3:49




analgesic effect on operant thermal outcomes, with com-
plete inhibition of thermal hyperalgesia [41]. In contrast,
GR89,696 was not effective for reducing this pain at the
highest non-sedative dose when evaluated on the thermal
operant assay.

5. Conclusion
We demonstrated that high levels of the opioids produced
significant untoward effects and made distinguishing an
analgesic versus a more general effect more difficult.
While these side effects are well known for morphine, to
our knowledge, this is the first report quantifying the
effects on general behavior following agonism of the
kappa-2 opioid receptor. The balance between general
impairment and analgesia, especially in the context of
drugs that activate the kappa-2 opioid receptors, needs to
be identified. In this regard, we found that measuring rear-
ing behavior can provide a relevant endpoint for assess-
ment of these factors and can be useful in the assessment
of analgesic efficacy of experimental drugs.

List of abbreviations
see text.

Competing interests
Financial competing interests. The University of Florida has
filed a patent (U.S. Patent Application No. 11/201,452,
UF#-11521) with Drs. Neubert and Caudle regarding the
operant facial testing apparatus. Non-financial competing
interests. None.

Authors' contributions
IN contributed to the conception and design of the study
and was primarily responsible for the interpretation of the
data and writing of the manuscript. HR contributed to the
design of the study and data analysis. IP contributed to
data acquisition.

AJ contributed to the study design and data acquisition.
RM contributed to the conception and design of the study
and revision of the final manuscript. All authors have read
and accepted the final manuscript.

Acknowledgements
This work was supported by Grant #5R21 DE 16704-02, National Institute
of Dental and Craniofacial Research, 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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