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Effects of mu- and kappa-2 opioid receptor agonists on pain and rearing behaviors

Effects of mu- and kappa-2 opioid receptor agonists on pain and rearing behaviors 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, -4 1.25 × 10 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. Page 1 of 10 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:49 http://www.behavioralandbrainfunctions.com/content/3/1/49 dynic and -hyperalgesic effects of GR89,696 are proposed 1. Background Management of pain involves a balance between inhibi- to be a result of spinal kappa-2 opioid receptor activation tion and suppression of pain and minimization of unto- and subsequent inhibition of spinal NMDA receptors [14- ward side effects. For example, patients suffering from 16]. intractable pain can be limited in the amount of narcotics, such as morphine, that they can receive due to adverse Efficacy is normally the primary outcome in the evalua- cognitive effects and sedation. Therefore in order to tion of analgesics; however, it is equally important to con- develop new analgesic compounds, one must consider the sider that the various opioid receptor agonists are not effects of the treatment on both pain processing and gen- without unpleasant side effects. For example morphine is eral behavior. In the search for better pain relief, numer- considered the "gold standard" analgesic, but effective ous novel compounds have been investigated, including analgesic doses can produce sedation, constipation, kappa-opioid receptor agonists. Since the early work of dependence and addiction. Kappa-1 receptor agonists Attali et al [1], binding, behavioral, and in vitro physio- such as U69,593 and the Mexican mint derived agent, logic studies have provided evidence supporting the exist- salvinorin A have been shown to also produce sedation ence of two subtypes of kappa-opioid receptors, kappa-1 and motor incoordination [22]. Activation of the kappa-1 and kappa-2. receptor has also been reported to affect rearing and loco- motion activity, with lower doses increasing this activity Kappa-1 opioid receptors preferentially bind arylaceta- and higher doses decreasing it [23-25]. The negative mide-like agonists such as U69,593 [2], and have been effects of kappa-1 receptor agonism are well-defined, but shown to be effective in blocking mechanical allodynia the unpleasant effects following kappa-2 receptor activa- [3,4]. However; they are relatively ineffective for reducing tion are less known. While GR89,696 has been reported as visceral hypersensitivity [5] and thermal allodynia and a possible therapeutic agent, it can produce a catatonic hyperalgesia [6,7]. While there is a debate regarding the and immobilized state (Caudle, personal communication, existence of a distinct kappa-2 receptor, there is evidence 2005). However, formal characterization of the potential to support that it is different from the kappa-1 receptor. side effect profile of this drug at therapeutic doses has yet Devi and colleagues [8-11] have demonstrated that recep- to be completed. Clinically, kappa receptor agonists are tor specificity is altered when opioid receptors form heter- known to produce dysphoria and hallucinations[26,27]. omers. Their work indicates that heteromers of the delta opioid receptor and the kappa opioid receptor have the In the current study, general behavior was evaluated using receptor binding properties of the classically defined a simplified rearing assay to assess sedative or systemic kappa2 receptors. Additionally, the combination of kappa effects and analgesic and antihyperalgesic effects were and delta receptors has also been demonstrated in vivo evaluated using reflex and operant tests. Previous rodent [12]. These data indicate that kappa/delta heteromers studies have assessed the influence of morphine on loco- (kappa-2 receptors) are a distinct class of receptor. In motor effects and have demonstrated that there are con- addition to performing binding studies demonstrating the trasting effects depending on dose, with both stimulant presence of kappa-2 receptors, Caudle and Finegold et al and depressive locomotor effects being reported [28-32]. found that U69,593, a kappa-1 receptor selective agent, Rearing behavior has been used previously as a measure of has no analgesic effect when injected intrathecally in rats locomotor effects following opioid administration, with [13] whereas [methyl-4-[3,4-dichlorophenyl)acetyl]-3- reports that morphine decreases this rearing behavior [1-pyrrolidinyl)methyl]-1-piperazinecarboxylate] [33,34]. Given this, we expected that rearing behavior (GR89,696), a putative kappa-2 opioid receptor agonist, would provide a sensitive method for detecting locomotor has very potent antihyperalgesic actions [14,15]. The effects in the assessment of our study drugs. It is important actions of GR89,696 were also clearly distinguishable to note that other factors such as sedation, decreased from those of mu and delta selective agonists. Further- motivation, and increased anxiety may lead to decreases more, in guinea pig hippocampal slices it has been dem- in this activity. In the context of this study, it was not onstrated that kappa-1 receptors primarily inhibited important to distinguish the mechanism related to a glutamate release while kappa2 receptor activation sup- change in rearing behavior. Our contention is that the pressed NMDA receptor function [16-18]. These data are change in behavior is the relevant endpoint here because also supported by the work of Schoffelmeer et al. [19], any event that produced this change will likely affect the Ohsawa and Kamei [20], and Gu et al [21]. Kappa-2 recep- pain outcome measures as well. tor agonists such as 4- [(3,4-Dichlorophenyl)acetyl]-3-(1- pyrrolidinylmethyl)-1-piperazinecarboxylic acid methyl 2. Methods ester fumarate salt (GR89,696) have been shown to effec- 2.1. General tively block inflammatory and neuritis-induced pain, as Male Sprague Dawley rats (200–300 g, Harlan, Houston, well as peripheral neuropathic pain [14,15]. The anti-allo- TX) were housed in groups of one to two and were main- Page 2 of 10 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:49 http://www.behavioralandbrainfunctions.com/content/3/1/49 tained in a standard 12-hour light/dark cycle and testing DI-194, DATAQ Instruments, Inc). When the rat com- was completed in the light portion of the cycle between pleted the task and drank from the water bottle, the skin 09:00–12:00. Animals were placed into the behavioral on its face contacted the grounded thermode, and the ani- procedure room 30 min prior to testing to acclimate. mal's tongue contacted the metal spout on the water bot- When not in testing sessions, food and water were made tle, completing an electrical circuit. The closed circuit available ad libitum. Animal testing procedures complied registered in the computer and data was collected at 60 Hz with the ethical guidelines and standards established by for the entire length of the experiment. Each spout contact the University of Florida's Institutional Animal Care & was recorded as a "licking" event and a separate circuit was Use Committee and with the Guide for Care and Use of established from the metal thermode to the animal by Laboratory Animals [35]. grounding the floor with an aluminum sheet for recording of "facial contact" events. Animals were first trained to 2.2. General behavioral assessment drink milk while contacting the thermode set to a temper- A modified limb-use asymmetry test [36,37] was used to ature at 37°C for baseline training (N = 5 sessions) and measure rearing activity as an assessment of general animals were considered trained when their intake is ≥ 10 behavior. An acrylic cylinder (19.5 cm diameter × 40.5 cm g of reward milk, as described previously [39]. height) was constructed with aluminum sheets placed both on the floor and 13.5-cm above the floor. The metal 2.4. Pain induction siding was connected to a DC power supply and, in series, We wanted to use painful stimuli that were not going to to a multi-channel data acquisition module (DATAQ produce an injury or deficit in the lower limbs to mini- Instruments, Inc) and the floor served as the ground for mize false-negative responses, so models of acetic acid- the circuit. Unrestrained animals were placed into sepa- induced visceral pain [40] and capsaicin-induced neuro- rate cylinders and the data acquisition system was acti- genic inflammatory pain were used. For visceral pain, a vated. When the animal reared, it would place its front 0.05% acetic acid solution (1 ml) was injected into the paw on the metal side of the cylinder, completing an elec- intraperitoneal space using a 23-gauge needle and ani- trical circuit (Fig. 1). The closed circuit registered in the mals were immediately placed in the testing apparatus. computer and front paw contact data were collected for 15 min. Baseline testing was completed for each set of ani- Orofacial neurogenic inflammatory pain was produced by mals, consisting of 3 consecutive sessions. applying capsaicin cream (0.075%, Thomson Microme- dix, Colorado) to the facial region of unanesthetized rats 2.3. Thermal testing and left on for 5 min. Following capsaicin application to Response to hindpaw heat pain was determined by plac- the face, animals normally will try to wipe off the cream ing unrestrained animals on a clear glass platform under a and in the process can spread the capsaicin from their small plastic cage and animals habituated for 5 min. A front paws to their mouth. In order to prevent this groom- radiant heat source was aimed directly under the ventral ing and subsequent intraoral capsaicin exposure, the ani- hindpaw surface and the time to paw withdrawal was mals were gently restrained for the stimulus duration and recorded as described previously [38]. then the face was wiped clean to remove residual capsai- cin cream. Immediately after capsaicin removal, animals Orofacial thermal sensitivity was assessed using a reward- were tested at 48°C using the thermal operant system. conflict operant paradigm, as described previously [39]. Briefly, unrestrained food fasted (12–15 hrs) animals 2.5. Opioid agonists were placed into clear acrylic testing boxes (20.3 cm W × The mu-opioid receptor agonist, morphine (subcutane- 20.3 cm D × 16.2 cm H). The animals were allowed access ous injection, 0.25 – 5 mg/kg, 200 µl), or the kappa-2 to a standard rodent watering bottle containing a diluted receptor agonist GR89696 (subcutaneous injection, 1.25 -4 (1:2 with water) sweetened condensed milk solution × 10 – 1.0 mg/kg, 200 µl) was administered 30 min prior (Nestle Carnation Company, room temperature) by plac- to testing. Control vehicle treatment consisted of phos- ing their head through an opening in the box. The open- phate buffered saline solution (subcutaneous injection, ing was lined with grounded metal (aluminum) tubing 200 µl). that served as a stimulus thermode when connected to a water pump (Model RTE110B, NES Laboratories, Inc.). 2.6. Data analyses The reward bottle was positioned in proximity to the cage The threshold for detection of front paw contacts, facial such that the animal was allowed access to the reward bot- contacts and licking contacts was set at 1.0 V and an event tle when simultaneously contacting the thermode with its was registered when the signal went above threshold and face. The metal spout on the watering bottle was con- ended when the signal dropped below threshold. Two nected to a DC power supply and, in series, to a multi- rearing outcome measures were calculated: (1) cumula- channel data acquisition module (WinDaq Lite Data Acq tive duration of rearing events; (2) total number of reach- Page 3 of 10 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:49 http://www.behavioralandbrainfunctions.com/content/3/1/49 Illustrated is an a Figure 1 nimal that is at rest (A) and rearing (B) in the rearing assay testing device 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 variance was used to evaluate the effects of treatment on measures were evaluated: (1) reward intake; (2) total each outcome measure (SPSS Inc). When significant dif- number of licking events; (3) total number facial contacts; ferences were found, post-hoc comparisons were made (4) cumulative facial contact duration; (5) ratio of using the Tukey honestly significant difference test. *P < reward/facial contacts; (6) duration per contact for the 0.05 was considered significant in all instances. facial contacts. Data analyses were completed using cus- tom-written routines in LabView Express (National Instru- 3. Results 3.1. Rearing behavior ments Corporation) and Excel (Microsoft). We found that different groups of animals reared a varying 2.7. Statistical analyses amount; therefore in order to normalize differences A general linear model repeat measures analysis was com- between groups, we computed and compared data as a pleted for baseline session comparisons and an analysis of percentage of the baseline sessions. An example of a typi- Page 4 of 10 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:49 http://www.behavioralandbrainfunctions.com/content/3/1/49 cal baseline session set is presented in Fig. 1D, E. There lar to the effects on the number of reaches, GR89,696 was an overall significant decrease (F = 7.35, *P < produced a significant reduction in the rearing duration at 2,22 -3 mg/kg. Additionally, morphine doses 0.005) in the number of contacts across the three baseline doses ≥ 1.25 × 10 testing sessions for this group of animals (N = 12). This ≥ 2.5 mg/kg suppressed exploratory behavior, as evident decrease occurred with other groups and is likely due to by a significant decrease in duration. Clinically relevant normal habituation to the closed testing field with repeat doses (≤ 0.5 mg/kg) of morphine did not significantly exposures. In contrast, the rearing activity duration affect this outcome measure as compared to baseline. remained constant and was not significantly (F = 0.26, 2,22 P = 0.77) different across baseline testing sessions and this 3.3 Effects of opioid agonists on thermal sensitivity was consistent within each group. 3.3.1 Hindpaw withdrawal latency (Fig. 3) There was a significant dose effect (F = 5.53, P < 0.05) 4,57 3.2 Effects of opioid agonists on rearing behavior of GR89,696 on hindpaw withdrawal latency following 3.2.1 Reaching number (Fig. 2A) thermal stimulation. Only the highest dose tested, 1.0 There was a significant dose effect produced by both mg/kg, produced a significantly higher latency compared = 10.71, *P < 0.001) and morphine (F GR89,696 (F to baseline. There was additionally a significant dose 4,29 4,91 = 9.41, *P < 0.001) on the number of reaching contacts. effect (F = 15.4, P < 0.001) of morphine, with only the 2,35 -3 GR89,696 at doses ≥ 1.25 × 10 mg/kg, produced a signif- highest dose of 5.0 mg/kg sufficient to suppress normal icant decrease in reaching number, compared to baseline. thermal transmission. The 0.5 mg/kg dose did not alter -4 The 1.25 × 10 mg/kg dose was the highest dose tested withdrawal latency as compared to baseline. that did not significantly differ from baseline. Similarly, morphine produced a significant decrease in reaching 3.3.2 Thermal operant assessment contacts at doses ≥ 2.5 mg/kg. Previously, we demonstrated that a 0.5 mg/kg dose of morphine could completely reverse inflammatory [39] 3.2.2 Rearing duration (Fig. 2B) and neurogenic inflammatory heat allodynia and hyperal- There was a significant dose effect on rearing duration when either GR89,696 (F = 10.74, P < 0.001) or mor- 4,29 phine (F = 12.16, *P < 0.001) was administered. Simi- 4,91 We evaluated the effects of G reachin Figure 2 g activity and cumulative rearing duration R89,696 and morphine on We evaluated the effects of GR89,696 and morphine on The effects of GR89 sens withdrawal latency to thermal inc 6) a Figure 3 rn eased on iti d mor vity were as phin ly at e (N = the sess ,696 highest doses tested for GR8 ed a 6) and morphine nd demonstr stimulatio ated that hindpaw on n n was s orma ignif l9 th ,696 icantly erm (N = al reaching activity and cumulative rearing duration. There was The effects of GR89,696 and morphine on normal thermal a significant dose-related decrease (*P < 0.05) in the number sensitivity were assessed and demonstrated that hindpaw of reaching events (A) and total time spent rearing (B) fol- withdrawal latency to thermal stimulation was significantly lowing morphine (N = 8–12, right insets) or GR89,696 (N = increased only at the highest doses tested for GR89,696 (N = 6, left insets) administration, compared to baseline values. 6) and morphine (N = 6). Page 5 of 10 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:49 http://www.behavioralandbrainfunctions.com/content/3/1/49 gesia[41] in the orofacial region. Specifically, reward blocking the heat hyperalgesia associated with capsaicin intake, licking contacts, facial contacts, facial contact treatment (Fig. 4). There were no significant differences in duration, ratio of reward/stimulus contacts, ratio of facial reward intake, licking contacts, facial contacts, facial con- contact duration/event all returned to baseline levels tact duration, ratio of reward/stimulus contacts, and ratio when animals were pretreated with morphine 30 min of facial contact duration/event when comparing active prior to either carrageenan or capsaicin application. drug compared to vehicle treated animals. Both GR89,696 and vehicle-treated animals displayed significantly lower In this study, as a comparison, GR89,696 was adminis- responses for these operant outcomes, compared to base- tered 30 min prior to topical capsaicin application. The line values, indicating that the capsaicin produced a pain -4 dose tested (1.25 × 10 mg/kg, subcutaneous) was the behavior that was unaffected by either GR89,696 or vehi- maximum dose that did not produce a significant effect cle treatment. on rearing behaviors. Contrary to the morphine results from those previous studies, GR89,696 was not capable of We fou Figure 4 nd that GR89,696 does not affect operant thermal outcome measures We found that GR89,696 does not affect operant thermal outcome measures. Animals treated with either GR89,696 (1.25 × -4 10 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. Page 6 of 10 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:49 http://www.behavioralandbrainfunctions.com/content/3/1/49 3.4 Effects of visceral pain on rearing behavior (Fig. 5) untreated and GR89,696 groups were significantly lower The induction of visceral pain using acetic acid provides a than the baseline values, while the morphine treated standard pain assay used in many analgesic assessment group was not significantly different compared to base- studies. Typically, investigators will count the number of line. writhing (stretching) events following intraperitoneal injection of acetic acid and compare this response in the 4. Discussion presence of an analgesic substance. We were interested in The use of pharmacological agents as a tool for character- evaluating the effects of visceral pain on exploratory ization of nociceptive pathways remains a cornerstone of behavior and we wanted to evaluate the effects of mor- pain research. Related to this is the ability to assess the phine and GR89,696 at doses that did not produce a sig- general behavioral consequences of experimental inter- nificant suppression of rearing behavior. Immediately ventions, as this is a necessary and important step in char- following acetic acid injection, animals displayed a signif- acterizing specific and non-specific effects of each icant decrease in activity as demonstrated by a decrease in treatment. When assessing behavioral outcome measures the number of contacts (F = 26.96, *P < 0.001) and for evaluation of analgesics, there is an underlying 2,31 = 13.79, *P < 0.001). These animals rearing duration (F assumption that the drug is having an effect only on the 2,31 displayed normal behavior when tested again 24 h post- relevant portion of the pain circuitry that it is targeting. injection (upper panels). There was a significant treatment However, if an experimental compound produces a severe effect on reaching contacts (F = 13.11, P < 0.001) and cognitive or sedative effect, then these measures could be 3,41 rearing duration (F = 14.45, P < 0.001) following acetic altered or suppressed to give false-negative results. There- 3,41 -4 acid injection when GR89,696 (1.25 × 10 mg/kg) and fore it is important to be able to evaluate and distinguish morphine (0.5 mg/kg) were compared to untreated ani- between analgesic effects versus central incapacitating or mals (lower panels). Post-hoc analysis revealed that the 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 Th and cumulative rearin induction was determ Figure 5 e effects of morphine g durati ined and GR89,696 on reaching on following visceral pai activity n not influence outcomes and rats do not develop compen- The effects of morphine and GR89,696 on reaching activity satory strategies. We modified the system by utilizing and cumulative rearing duration following visceral pain automated computer data acquisition that provides an induction was determined. Acetic acid was injected (intra- easy and fast method for evaluation of behavior following peritoneal) as an acute painful stimulus and produced a sig- injury or drug administration. This can allow for an inves- nificant decrease (*P < 0.05, compared to baseline) in both tigator to quickly screen for systemic and cognitive effects reaching number (A) and rearing duration (B) during the of new therapeutics, which is particularly valuable for the first 15 min post-injection. Animals were fully recovered by assessment of pain, or pain relief following a therapeutic 24 h, as their rearing behavior returned to baseline levels. -4 intervention. In this system, we did note that a certain GR89,696 (N = 8, 1.25 × 10 mg/kg, subcutaneous) was ineffective and morphine (N = 8, 0.5 mg/kg) was only par- level of habituation occurs, with the number of reaches tially effective for blocking this visceral pain induction, as significantly decreasing with consecutive trials. However, demonstrated by a decrease in reaching number (C) and the duration data remained constant and does not appear rearing duration (D). For (C, D), *P < 0.05 as compared to to be susceptible to this habituation phenomenon, and naïve animals. thus provides the more stable measure. This assay pro- vides a quantitative measure that is neither stimulus- Page 7 of 10 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:49 http://www.behavioralandbrainfunctions.com/content/3/1/49 evoked nor investigator-initiated and therefore can reduce is not considered an analgesic, as sensitivity to normal inherent variability issues associated with animal testing. nociceptive stimuli is not affected. The effective dose of GR89,696 for reducing pain behaviors has been reported Morphine remains the "gold standard" for controlling to be ~0.01 mg/kg when given intrathecally [14,15]. For clinical pain and has been used extensively in animal pain subcutaneous administration, we initially chose a dose models. There is evidence in rodents supporting the idea that was 100 fold higher than this dose, as this has been that morphine produces a biphasic response in locomo- reported to produce a catatonic-like state (Caudle RM, per- tion following morphine administration, resulting in sonal communication). Subsequent doses were decreased by both decreased and increased motor activity, depending ~10 fold dilution until normal behavior was exhibited. In -3 on: the activity evaluated (e.g., locomotion, rearing), this study, GR89,696 doses ≥ 1.25 × 10 mg/kg produced dose, and time after administration [28,34,42-44]. In behavioral impairment (Fig. 2A, B), as demonstrated by these studies, increased locomotion has been reported at the significant decrease in both rearing number and dura- doses of morphine ≥ 10 mg/kg, well above the doses used tion. in our study. Additionally, other investigators have evalu- ated the effects of morphine on rearing behavior in mice Pain associated with normal heat transduction can be and similarly found that doses of morphine ≥ 5–40 mg/kg reduced with high doses of morphine (5 mg/kg) or produces a reduction in rearing events [34,45]. Indeed, GR89,696 (1 mg/kg), as seen by an increase in hindpaw when we evaluated the effects of morphine on rearing withdrawal latency. However at these doses there is a sig- behavior, we found that there was a significant dose- nificant reduction in rearing behavior, therefore, one can- related decrease in both rearing events and in the number not necessarily state that these doses are analgesic, rather of reaching contacts, with suppression of the exploratory that they were sufficient to significantly change the reflex behavior occurring at doses ≥ 2.5 mg/kg (Fig. 2). These outcome. For subsequent analgesic assessment against data support that systemic effects can occur at these doses, visceral and orofacial neurogenic inflammatory pain, we -4 including impairment of motor and motivational selected 1.25 × 10 mg/kg for GR89,696 and 0.5 mg/kg responses, as reported previously [46-48]. We would for morphine, respectively, as the relevant test doses that expect that other drugs producing sedative effects would did not produce a significant behavioral rearing effect as also produce a significant decrease in rearing contacts and compared to baseline (Fig. 3). Also there was no effect on duration. normal thermal transduction following GR89,696 (1.25 × -4 10 mg/kg) or morphine (0.5 mg/kg) at these low doses, In regards to analgesic efficacy, these impairments can as hindpaw withdrawal latency was not significantly compromise interpretation of analgesic results, as an ani- increased. mal may be immobilized or unresponsive when tested at these doses. This may be problematic in that high doses of Visceral pain following intraperitoneal injection of acetic morphine (> 3 mg/kg) are required to suppress reflexive acid has been shown to produce a significant increase in assays of pain measurement [49-51] and are relatively writhing behavior for approximately 15 min [40,53]. Not higher as compared to doses in humans (< 0.15 mg/kg) surprisingly, visceral discomfort had a significant effect on that are effective for controlling clinical pain [52]. While exploratory behavior and this effect was detected by the it is possible that a differential expression rate of opioid rearing assay in a time-appropriate fashion with the receptors between species may explain this discrepancy in behavioral response duration corresponding to the dura- dose, the non-analgesic effects including sedation and tion of the stimulus. There was a significant decrease at the suppression of reflex responses cannot be ignored. In fact, acute (15 min) session, but the behavior returned to nor- we were able to use operant outcome measures to demon- mal at 24 hrs. When animals were pre-treated with mor- -4 strate that this discrepancy may be due to a lack of sensi- phine (0.5 mg/kg) or GR89,696 (1.25 × 10 mg/kg), there tivity of current reflex-based analgesic assays for detecting was a partial analgesic effect on the acetic acid/rearing analgesic effects at the lower doses of morphine. Our assessment produced by morphine, as the reaching and group has shown that 0.5 mg/kg of morphine was suffi- duration values were lower than baseline, but higher than cient to suppress nociceptive activity [39,41]. In contrast, no treatment or GR89696. This dose of morphine was not when we completed analgesic assessment using hindpaw effective in blocking normal heat sensitivity (Fig. 3B), withdrawal time as the outcome, a 10-fold higher dose which is consistent with the lack of antinociceptive effect was required to block thermal pain. demonstrated using the rearing assay. Recently, the kappa-2 opioid receptor has become a target As a second pain model, we applied a low dose of capsai- for pain control, with drugs such as GR89,696 being con- cin cream to the facial region to produce neurogenic sidered. In contrast to mu-receptor agonism, kappa-2 inflammatory pain. Previously, we demonstrated that receptor binding produces an antihyperalgesic effect, but morphine at doses ≤ 0.5 mg/kg produced a significant Page 8 of 10 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:49 http://www.behavioralandbrainfunctions.com/content/3/1/49 3. 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Neurosci Lett 2001, 298:151-154. 13. Caudle RM, Finegold AA, Mannes AJ, Tobias MD, Kenshalo DR Jr., Competing interests Iadarola MJ: Spinal kappa1 and kappa2 opioid binding sites in Finanacial competing interests. The University of Florida has rats, guinea pigs, monkeys and humans. Neuroreport 1998, filed a patent (U.S. Patent Application No. 11/201,452, 9:2523-2525. 14. Eliav E, Herzberg U, Caudle RM: The kappa opioid agonist UF#-11521) with Drs. Neubert and Caudle regarding the GR89,696 blocks hyperalgesia and allodynia in rat models of operant facial testing apparatus. Non-financial competing peripheral neuritis and neuropathy. Pain 1999, 79:255-264. 15. Ho J, Mannes AJ, Dubner R, Caudle RM: Putative kappa-2 opioid interests. None. agonists are antihyperalgesic in a rat model of inflammation. J Pharmacol Exp Ther 1997, 281:136-141. 16. 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Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 10 of 10 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Behavioral and Brain Functions Springer Journals

Effects of mu- and kappa-2 opioid receptor agonists on pain and rearing behaviors

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Springer Journals
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Copyright © 2007 by Neubert et al; licensee BioMed Central Ltd.
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Biomedicine; Neurosciences; Neurology; Behavioral Therapy; Psychiatry
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10.1186/1744-9081-3-49
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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, -4 1.25 × 10 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. Page 1 of 10 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:49 http://www.behavioralandbrainfunctions.com/content/3/1/49 dynic and -hyperalgesic effects of GR89,696 are proposed 1. Background Management of pain involves a balance between inhibi- to be a result of spinal kappa-2 opioid receptor activation tion and suppression of pain and minimization of unto- and subsequent inhibition of spinal NMDA receptors [14- ward side effects. For example, patients suffering from 16]. intractable pain can be limited in the amount of narcotics, such as morphine, that they can receive due to adverse Efficacy is normally the primary outcome in the evalua- cognitive effects and sedation. Therefore in order to tion of analgesics; however, it is equally important to con- develop new analgesic compounds, one must consider the sider that the various opioid receptor agonists are not effects of the treatment on both pain processing and gen- without unpleasant side effects. For example morphine is eral behavior. In the search for better pain relief, numer- considered the "gold standard" analgesic, but effective ous novel compounds have been investigated, including analgesic doses can produce sedation, constipation, kappa-opioid receptor agonists. Since the early work of dependence and addiction. Kappa-1 receptor agonists Attali et al [1], binding, behavioral, and in vitro physio- such as U69,593 and the Mexican mint derived agent, logic studies have provided evidence supporting the exist- salvinorin A have been shown to also produce sedation ence of two subtypes of kappa-opioid receptors, kappa-1 and motor incoordination [22]. Activation of the kappa-1 and kappa-2. receptor has also been reported to affect rearing and loco- motion activity, with lower doses increasing this activity Kappa-1 opioid receptors preferentially bind arylaceta- and higher doses decreasing it [23-25]. The negative mide-like agonists such as U69,593 [2], and have been effects of kappa-1 receptor agonism are well-defined, but shown to be effective in blocking mechanical allodynia the unpleasant effects following kappa-2 receptor activa- [3,4]. However; they are relatively ineffective for reducing tion are less known. While GR89,696 has been reported as visceral hypersensitivity [5] and thermal allodynia and a possible therapeutic agent, it can produce a catatonic hyperalgesia [6,7]. While there is a debate regarding the and immobilized state (Caudle, personal communication, existence of a distinct kappa-2 receptor, there is evidence 2005). However, formal characterization of the potential to support that it is different from the kappa-1 receptor. side effect profile of this drug at therapeutic doses has yet Devi and colleagues [8-11] have demonstrated that recep- to be completed. Clinically, kappa receptor agonists are tor specificity is altered when opioid receptors form heter- known to produce dysphoria and hallucinations[26,27]. omers. Their work indicates that heteromers of the delta opioid receptor and the kappa opioid receptor have the In the current study, general behavior was evaluated using receptor binding properties of the classically defined a simplified rearing assay to assess sedative or systemic kappa2 receptors. Additionally, the combination of kappa effects and analgesic and antihyperalgesic effects were and delta receptors has also been demonstrated in vivo evaluated using reflex and operant tests. Previous rodent [12]. These data indicate that kappa/delta heteromers studies have assessed the influence of morphine on loco- (kappa-2 receptors) are a distinct class of receptor. In motor effects and have demonstrated that there are con- addition to performing binding studies demonstrating the trasting effects depending on dose, with both stimulant presence of kappa-2 receptors, Caudle and Finegold et al and depressive locomotor effects being reported [28-32]. found that U69,593, a kappa-1 receptor selective agent, Rearing behavior has been used previously as a measure of has no analgesic effect when injected intrathecally in rats locomotor effects following opioid administration, with [13] whereas [methyl-4-[3,4-dichlorophenyl)acetyl]-3- reports that morphine decreases this rearing behavior [1-pyrrolidinyl)methyl]-1-piperazinecarboxylate] [33,34]. Given this, we expected that rearing behavior (GR89,696), a putative kappa-2 opioid receptor agonist, would provide a sensitive method for detecting locomotor has very potent antihyperalgesic actions [14,15]. The effects in the assessment of our study drugs. It is important actions of GR89,696 were also clearly distinguishable to note that other factors such as sedation, decreased from those of mu and delta selective agonists. Further- motivation, and increased anxiety may lead to decreases more, in guinea pig hippocampal slices it has been dem- in this activity. In the context of this study, it was not onstrated that kappa-1 receptors primarily inhibited important to distinguish the mechanism related to a glutamate release while kappa2 receptor activation sup- change in rearing behavior. Our contention is that the pressed NMDA receptor function [16-18]. These data are change in behavior is the relevant endpoint here because also supported by the work of Schoffelmeer et al. [19], any event that produced this change will likely affect the Ohsawa and Kamei [20], and Gu et al [21]. Kappa-2 recep- pain outcome measures as well. tor agonists such as 4- [(3,4-Dichlorophenyl)acetyl]-3-(1- pyrrolidinylmethyl)-1-piperazinecarboxylic acid methyl 2. Methods ester fumarate salt (GR89,696) have been shown to effec- 2.1. General tively block inflammatory and neuritis-induced pain, as Male Sprague Dawley rats (200–300 g, Harlan, Houston, well as peripheral neuropathic pain [14,15]. The anti-allo- TX) were housed in groups of one to two and were main- Page 2 of 10 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:49 http://www.behavioralandbrainfunctions.com/content/3/1/49 tained in a standard 12-hour light/dark cycle and testing DI-194, DATAQ Instruments, Inc). When the rat com- was completed in the light portion of the cycle between pleted the task and drank from the water bottle, the skin 09:00–12:00. Animals were placed into the behavioral on its face contacted the grounded thermode, and the ani- procedure room 30 min prior to testing to acclimate. mal's tongue contacted the metal spout on the water bot- When not in testing sessions, food and water were made tle, completing an electrical circuit. The closed circuit available ad libitum. Animal testing procedures complied registered in the computer and data was collected at 60 Hz with the ethical guidelines and standards established by for the entire length of the experiment. Each spout contact the University of Florida's Institutional Animal Care & was recorded as a "licking" event and a separate circuit was Use Committee and with the Guide for Care and Use of established from the metal thermode to the animal by Laboratory Animals [35]. grounding the floor with an aluminum sheet for recording of "facial contact" events. Animals were first trained to 2.2. General behavioral assessment drink milk while contacting the thermode set to a temper- A modified limb-use asymmetry test [36,37] was used to ature at 37°C for baseline training (N = 5 sessions) and measure rearing activity as an assessment of general animals were considered trained when their intake is ≥ 10 behavior. An acrylic cylinder (19.5 cm diameter × 40.5 cm g of reward milk, as described previously [39]. height) was constructed with aluminum sheets placed both on the floor and 13.5-cm above the floor. The metal 2.4. Pain induction siding was connected to a DC power supply and, in series, We wanted to use painful stimuli that were not going to to a multi-channel data acquisition module (DATAQ produce an injury or deficit in the lower limbs to mini- Instruments, Inc) and the floor served as the ground for mize false-negative responses, so models of acetic acid- the circuit. Unrestrained animals were placed into sepa- induced visceral pain [40] and capsaicin-induced neuro- rate cylinders and the data acquisition system was acti- genic inflammatory pain were used. For visceral pain, a vated. When the animal reared, it would place its front 0.05% acetic acid solution (1 ml) was injected into the paw on the metal side of the cylinder, completing an elec- intraperitoneal space using a 23-gauge needle and ani- trical circuit (Fig. 1). The closed circuit registered in the mals were immediately placed in the testing apparatus. computer and front paw contact data were collected for 15 min. Baseline testing was completed for each set of ani- Orofacial neurogenic inflammatory pain was produced by mals, consisting of 3 consecutive sessions. applying capsaicin cream (0.075%, Thomson Microme- dix, Colorado) to the facial region of unanesthetized rats 2.3. Thermal testing and left on for 5 min. Following capsaicin application to Response to hindpaw heat pain was determined by plac- the face, animals normally will try to wipe off the cream ing unrestrained animals on a clear glass platform under a and in the process can spread the capsaicin from their small plastic cage and animals habituated for 5 min. A front paws to their mouth. In order to prevent this groom- radiant heat source was aimed directly under the ventral ing and subsequent intraoral capsaicin exposure, the ani- hindpaw surface and the time to paw withdrawal was mals were gently restrained for the stimulus duration and recorded as described previously [38]. then the face was wiped clean to remove residual capsai- cin cream. Immediately after capsaicin removal, animals Orofacial thermal sensitivity was assessed using a reward- were tested at 48°C using the thermal operant system. conflict operant paradigm, as described previously [39]. Briefly, unrestrained food fasted (12–15 hrs) animals 2.5. Opioid agonists were placed into clear acrylic testing boxes (20.3 cm W × The mu-opioid receptor agonist, morphine (subcutane- 20.3 cm D × 16.2 cm H). The animals were allowed access ous injection, 0.25 – 5 mg/kg, 200 µl), or the kappa-2 to a standard rodent watering bottle containing a diluted receptor agonist GR89696 (subcutaneous injection, 1.25 -4 (1:2 with water) sweetened condensed milk solution × 10 – 1.0 mg/kg, 200 µl) was administered 30 min prior (Nestle Carnation Company, room temperature) by plac- to testing. Control vehicle treatment consisted of phos- ing their head through an opening in the box. The open- phate buffered saline solution (subcutaneous injection, ing was lined with grounded metal (aluminum) tubing 200 µl). that served as a stimulus thermode when connected to a water pump (Model RTE110B, NES Laboratories, Inc.). 2.6. Data analyses The reward bottle was positioned in proximity to the cage The threshold for detection of front paw contacts, facial such that the animal was allowed access to the reward bot- contacts and licking contacts was set at 1.0 V and an event tle when simultaneously contacting the thermode with its was registered when the signal went above threshold and face. The metal spout on the watering bottle was con- ended when the signal dropped below threshold. Two nected to a DC power supply and, in series, to a multi- rearing outcome measures were calculated: (1) cumula- channel data acquisition module (WinDaq Lite Data Acq tive duration of rearing events; (2) total number of reach- Page 3 of 10 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:49 http://www.behavioralandbrainfunctions.com/content/3/1/49 Illustrated is an a Figure 1 nimal that is at rest (A) and rearing (B) in the rearing assay testing device 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 variance was used to evaluate the effects of treatment on measures were evaluated: (1) reward intake; (2) total each outcome measure (SPSS Inc). When significant dif- number of licking events; (3) total number facial contacts; ferences were found, post-hoc comparisons were made (4) cumulative facial contact duration; (5) ratio of using the Tukey honestly significant difference test. *P < reward/facial contacts; (6) duration per contact for the 0.05 was considered significant in all instances. facial contacts. Data analyses were completed using cus- tom-written routines in LabView Express (National Instru- 3. Results 3.1. Rearing behavior ments Corporation) and Excel (Microsoft). We found that different groups of animals reared a varying 2.7. Statistical analyses amount; therefore in order to normalize differences A general linear model repeat measures analysis was com- between groups, we computed and compared data as a pleted for baseline session comparisons and an analysis of percentage of the baseline sessions. An example of a typi- Page 4 of 10 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:49 http://www.behavioralandbrainfunctions.com/content/3/1/49 cal baseline session set is presented in Fig. 1D, E. There lar to the effects on the number of reaches, GR89,696 was an overall significant decrease (F = 7.35, *P < produced a significant reduction in the rearing duration at 2,22 -3 mg/kg. Additionally, morphine doses 0.005) in the number of contacts across the three baseline doses ≥ 1.25 × 10 testing sessions for this group of animals (N = 12). This ≥ 2.5 mg/kg suppressed exploratory behavior, as evident decrease occurred with other groups and is likely due to by a significant decrease in duration. Clinically relevant normal habituation to the closed testing field with repeat doses (≤ 0.5 mg/kg) of morphine did not significantly exposures. In contrast, the rearing activity duration affect this outcome measure as compared to baseline. remained constant and was not significantly (F = 0.26, 2,22 P = 0.77) different across baseline testing sessions and this 3.3 Effects of opioid agonists on thermal sensitivity was consistent within each group. 3.3.1 Hindpaw withdrawal latency (Fig. 3) There was a significant dose effect (F = 5.53, P < 0.05) 4,57 3.2 Effects of opioid agonists on rearing behavior of GR89,696 on hindpaw withdrawal latency following 3.2.1 Reaching number (Fig. 2A) thermal stimulation. Only the highest dose tested, 1.0 There was a significant dose effect produced by both mg/kg, produced a significantly higher latency compared = 10.71, *P < 0.001) and morphine (F GR89,696 (F to baseline. There was additionally a significant dose 4,29 4,91 = 9.41, *P < 0.001) on the number of reaching contacts. effect (F = 15.4, P < 0.001) of morphine, with only the 2,35 -3 GR89,696 at doses ≥ 1.25 × 10 mg/kg, produced a signif- highest dose of 5.0 mg/kg sufficient to suppress normal icant decrease in reaching number, compared to baseline. thermal transmission. The 0.5 mg/kg dose did not alter -4 The 1.25 × 10 mg/kg dose was the highest dose tested withdrawal latency as compared to baseline. that did not significantly differ from baseline. Similarly, morphine produced a significant decrease in reaching 3.3.2 Thermal operant assessment contacts at doses ≥ 2.5 mg/kg. Previously, we demonstrated that a 0.5 mg/kg dose of morphine could completely reverse inflammatory [39] 3.2.2 Rearing duration (Fig. 2B) and neurogenic inflammatory heat allodynia and hyperal- There was a significant dose effect on rearing duration when either GR89,696 (F = 10.74, P < 0.001) or mor- 4,29 phine (F = 12.16, *P < 0.001) was administered. Simi- 4,91 We evaluated the effects of G reachin Figure 2 g activity and cumulative rearing duration R89,696 and morphine on We evaluated the effects of GR89,696 and morphine on The effects of GR89 sens withdrawal latency to thermal inc 6) a Figure 3 rn eased on iti d mor vity were as phin ly at e (N = the sess ,696 highest doses tested for GR8 ed a 6) and morphine nd demonstr stimulatio ated that hindpaw on n n was s orma ignif l9 th ,696 icantly erm (N = al reaching activity and cumulative rearing duration. There was The effects of GR89,696 and morphine on normal thermal a significant dose-related decrease (*P < 0.05) in the number sensitivity were assessed and demonstrated that hindpaw of reaching events (A) and total time spent rearing (B) fol- withdrawal latency to thermal stimulation was significantly lowing morphine (N = 8–12, right insets) or GR89,696 (N = increased only at the highest doses tested for GR89,696 (N = 6, left insets) administration, compared to baseline values. 6) and morphine (N = 6). Page 5 of 10 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:49 http://www.behavioralandbrainfunctions.com/content/3/1/49 gesia[41] in the orofacial region. Specifically, reward blocking the heat hyperalgesia associated with capsaicin intake, licking contacts, facial contacts, facial contact treatment (Fig. 4). There were no significant differences in duration, ratio of reward/stimulus contacts, ratio of facial reward intake, licking contacts, facial contacts, facial con- contact duration/event all returned to baseline levels tact duration, ratio of reward/stimulus contacts, and ratio when animals were pretreated with morphine 30 min of facial contact duration/event when comparing active prior to either carrageenan or capsaicin application. drug compared to vehicle treated animals. Both GR89,696 and vehicle-treated animals displayed significantly lower In this study, as a comparison, GR89,696 was adminis- responses for these operant outcomes, compared to base- tered 30 min prior to topical capsaicin application. The line values, indicating that the capsaicin produced a pain -4 dose tested (1.25 × 10 mg/kg, subcutaneous) was the behavior that was unaffected by either GR89,696 or vehi- maximum dose that did not produce a significant effect cle treatment. on rearing behaviors. Contrary to the morphine results from those previous studies, GR89,696 was not capable of We fou Figure 4 nd that GR89,696 does not affect operant thermal outcome measures We found that GR89,696 does not affect operant thermal outcome measures. Animals treated with either GR89,696 (1.25 × -4 10 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. Page 6 of 10 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:49 http://www.behavioralandbrainfunctions.com/content/3/1/49 3.4 Effects of visceral pain on rearing behavior (Fig. 5) untreated and GR89,696 groups were significantly lower The induction of visceral pain using acetic acid provides a than the baseline values, while the morphine treated standard pain assay used in many analgesic assessment group was not significantly different compared to base- studies. Typically, investigators will count the number of line. writhing (stretching) events following intraperitoneal injection of acetic acid and compare this response in the 4. Discussion presence of an analgesic substance. We were interested in The use of pharmacological agents as a tool for character- evaluating the effects of visceral pain on exploratory ization of nociceptive pathways remains a cornerstone of behavior and we wanted to evaluate the effects of mor- pain research. Related to this is the ability to assess the phine and GR89,696 at doses that did not produce a sig- general behavioral consequences of experimental inter- nificant suppression of rearing behavior. Immediately ventions, as this is a necessary and important step in char- following acetic acid injection, animals displayed a signif- acterizing specific and non-specific effects of each icant decrease in activity as demonstrated by a decrease in treatment. When assessing behavioral outcome measures the number of contacts (F = 26.96, *P < 0.001) and for evaluation of analgesics, there is an underlying 2,31 = 13.79, *P < 0.001). These animals rearing duration (F assumption that the drug is having an effect only on the 2,31 displayed normal behavior when tested again 24 h post- relevant portion of the pain circuitry that it is targeting. injection (upper panels). There was a significant treatment However, if an experimental compound produces a severe effect on reaching contacts (F = 13.11, P < 0.001) and cognitive or sedative effect, then these measures could be 3,41 rearing duration (F = 14.45, P < 0.001) following acetic altered or suppressed to give false-negative results. There- 3,41 -4 acid injection when GR89,696 (1.25 × 10 mg/kg) and fore it is important to be able to evaluate and distinguish morphine (0.5 mg/kg) were compared to untreated ani- between analgesic effects versus central incapacitating or mals (lower panels). Post-hoc analysis revealed that the 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 Th and cumulative rearin induction was determ Figure 5 e effects of morphine g durati ined and GR89,696 on reaching on following visceral pai activity n not influence outcomes and rats do not develop compen- The effects of morphine and GR89,696 on reaching activity satory strategies. We modified the system by utilizing and cumulative rearing duration following visceral pain automated computer data acquisition that provides an induction was determined. Acetic acid was injected (intra- easy and fast method for evaluation of behavior following peritoneal) as an acute painful stimulus and produced a sig- injury or drug administration. This can allow for an inves- nificant decrease (*P < 0.05, compared to baseline) in both tigator to quickly screen for systemic and cognitive effects reaching number (A) and rearing duration (B) during the of new therapeutics, which is particularly valuable for the first 15 min post-injection. Animals were fully recovered by assessment of pain, or pain relief following a therapeutic 24 h, as their rearing behavior returned to baseline levels. -4 intervention. In this system, we did note that a certain GR89,696 (N = 8, 1.25 × 10 mg/kg, subcutaneous) was ineffective and morphine (N = 8, 0.5 mg/kg) was only par- level of habituation occurs, with the number of reaches tially effective for blocking this visceral pain induction, as significantly decreasing with consecutive trials. However, demonstrated by a decrease in reaching number (C) and the duration data remained constant and does not appear rearing duration (D). For (C, D), *P < 0.05 as compared to to be susceptible to this habituation phenomenon, and naïve animals. thus provides the more stable measure. This assay pro- vides a quantitative measure that is neither stimulus- Page 7 of 10 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:49 http://www.behavioralandbrainfunctions.com/content/3/1/49 evoked nor investigator-initiated and therefore can reduce is not considered an analgesic, as sensitivity to normal inherent variability issues associated with animal testing. nociceptive stimuli is not affected. The effective dose of GR89,696 for reducing pain behaviors has been reported Morphine remains the "gold standard" for controlling to be ~0.01 mg/kg when given intrathecally [14,15]. For clinical pain and has been used extensively in animal pain subcutaneous administration, we initially chose a dose models. There is evidence in rodents supporting the idea that was 100 fold higher than this dose, as this has been that morphine produces a biphasic response in locomo- reported to produce a catatonic-like state (Caudle RM, per- tion following morphine administration, resulting in sonal communication). Subsequent doses were decreased by both decreased and increased motor activity, depending ~10 fold dilution until normal behavior was exhibited. In -3 on: the activity evaluated (e.g., locomotion, rearing), this study, GR89,696 doses ≥ 1.25 × 10 mg/kg produced dose, and time after administration [28,34,42-44]. In behavioral impairment (Fig. 2A, B), as demonstrated by these studies, increased locomotion has been reported at the significant decrease in both rearing number and dura- doses of morphine ≥ 10 mg/kg, well above the doses used tion. in our study. Additionally, other investigators have evalu- ated the effects of morphine on rearing behavior in mice Pain associated with normal heat transduction can be and similarly found that doses of morphine ≥ 5–40 mg/kg reduced with high doses of morphine (5 mg/kg) or produces a reduction in rearing events [34,45]. Indeed, GR89,696 (1 mg/kg), as seen by an increase in hindpaw when we evaluated the effects of morphine on rearing withdrawal latency. However at these doses there is a sig- behavior, we found that there was a significant dose- nificant reduction in rearing behavior, therefore, one can- related decrease in both rearing events and in the number not necessarily state that these doses are analgesic, rather of reaching contacts, with suppression of the exploratory that they were sufficient to significantly change the reflex behavior occurring at doses ≥ 2.5 mg/kg (Fig. 2). These outcome. For subsequent analgesic assessment against data support that systemic effects can occur at these doses, visceral and orofacial neurogenic inflammatory pain, we -4 including impairment of motor and motivational selected 1.25 × 10 mg/kg for GR89,696 and 0.5 mg/kg responses, as reported previously [46-48]. We would for morphine, respectively, as the relevant test doses that expect that other drugs producing sedative effects would did not produce a significant behavioral rearing effect as also produce a significant decrease in rearing contacts and compared to baseline (Fig. 3). Also there was no effect on duration. normal thermal transduction following GR89,696 (1.25 × -4 10 mg/kg) or morphine (0.5 mg/kg) at these low doses, In regards to analgesic efficacy, these impairments can as hindpaw withdrawal latency was not significantly compromise interpretation of analgesic results, as an ani- increased. mal may be immobilized or unresponsive when tested at these doses. This may be problematic in that high doses of Visceral pain following intraperitoneal injection of acetic morphine (> 3 mg/kg) are required to suppress reflexive acid has been shown to produce a significant increase in assays of pain measurement [49-51] and are relatively writhing behavior for approximately 15 min [40,53]. Not higher as compared to doses in humans (< 0.15 mg/kg) surprisingly, visceral discomfort had a significant effect on that are effective for controlling clinical pain [52]. While exploratory behavior and this effect was detected by the it is possible that a differential expression rate of opioid rearing assay in a time-appropriate fashion with the receptors between species may explain this discrepancy in behavioral response duration corresponding to the dura- dose, the non-analgesic effects including sedation and tion of the stimulus. There was a significant decrease at the suppression of reflex responses cannot be ignored. In fact, acute (15 min) session, but the behavior returned to nor- we were able to use operant outcome measures to demon- mal at 24 hrs. When animals were pre-treated with mor- -4 strate that this discrepancy may be due to a lack of sensi- phine (0.5 mg/kg) or GR89,696 (1.25 × 10 mg/kg), there tivity of current reflex-based analgesic assays for detecting was a partial analgesic effect on the acetic acid/rearing analgesic effects at the lower doses of morphine. Our assessment produced by morphine, as the reaching and group has shown that 0.5 mg/kg of morphine was suffi- duration values were lower than baseline, but higher than cient to suppress nociceptive activity [39,41]. In contrast, no treatment or GR89696. This dose of morphine was not when we completed analgesic assessment using hindpaw effective in blocking normal heat sensitivity (Fig. 3B), withdrawal time as the outcome, a 10-fold higher dose which is consistent with the lack of antinociceptive effect was required to block thermal pain. demonstrated using the rearing assay. Recently, the kappa-2 opioid receptor has become a target As a second pain model, we applied a low dose of capsai- for pain control, with drugs such as GR89,696 being con- cin cream to the facial region to produce neurogenic sidered. In contrast to mu-receptor agonism, kappa-2 inflammatory pain. Previously, we demonstrated that receptor binding produces an antihyperalgesic effect, but morphine at doses ≤ 0.5 mg/kg produced a significant Page 8 of 10 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:49 http://www.behavioralandbrainfunctions.com/content/3/1/49 3. 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