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Published in final edited form as: Pharmacol Biochem Behav. 2017 Mar 31;156:10–15. doi: 10.1016/j.pbb.2017.03.007

Depression of home cage wheel running is an objective measure of spontaneous morphine withdrawal in rats with and without persistent pain

Ram Kandasamy 1,*, Andrea T Lee 2,*, Michael M Morgan 1,2
PMCID: PMC5484634  NIHMSID: NIHMS866557  PMID: 28366799

Abstract

Opioid withdrawal in humans is often subtle and almost always spontaneous. In contrast, most preclinical studies precipitate withdrawal by administration of an opioid receptor antagonist such as naloxone. These animal studies rely on measurement of physiological symptoms (e.g., wet dog shakes) in the period immediately following naloxone administration. To more closely model the human condition, we tested the hypothesis that depression of home cage wheel running will provide an objective method to measure the magnitude and duration of spontaneous morphine withdrawal. Rats were allowed access to a running wheel in their home cage for 8 days prior to implantation of two 75 mg morphine or placebo pellets. The pellets were removed 3 or 5 days later to induce spontaneous withdrawal. In normal pain-free rats, removal of the morphine pellets depressed wheel running for 48 hours compared to rats that had placebo pellets removed. Morphine withdrawal-induced depression of wheel running was greatly enhanced in rats with persistent inflammatory pain induced by injection of Complete Freund’s Adjuvant (CFA) into the hindpaw. Removal of the morphine pellets following 3 days of treatment depressed wheel running in these rats for over 6 days. These data demonstrate that home cage wheel running provides an objective and more clinically relevant method to assess spontaneous morphine withdrawal compared to precipitated withdrawal in laboratory rats. Moreover, the enhanced withdrawal in rats with persistent inflammatory pain suggests that pain patients may be especially susceptible to opioid withdrawal.

Keywords: opioid withdrawal, dependence, addiction, tolerance, locomotor activity

The increased use of opioids to treat pain has caused a simultaneous increase in the incidence of opioid dependence (Volkow and McLellan, 2016). Opioid withdrawal symptoms in opioid-dependent pain patients contributes to the continued use and subsequent abuse of opioids (Brodner and Taub, 1978; Fishbain et al., 1992; Hou et al., 2015). Although opioid withdrawal has been well studied in animals, these studies rarely assess spontaneous withdrawal as it occurs in humans (Papaleo and Contarino, 2006; Schulteis, 2010). Administration of an opioid receptor antagonist such as naloxone precipitates immediate and severe physiological withdrawal symptoms (e.g., wet dog shakes, teeth chattering, diarrhea) in opioid-dependent animals (Maldonado et al., 1996), but this phenomenon bears little resemblance to spontaneous opioid withdrawal which appears to induce greater psychological than physiological symptoms and occurs over a prolonged period of time (Schaefer and Michael, 1983; Schulteis et al., 1998; Cicero et al., 2002).

A number of methods have been used to assess spontaneous withdrawal in rodents. These include assessment of physical withdrawal symptoms (Cicero et al., 2002; Kalinichev and Holtzman, 2003; Cobuzzi and Riley, 2011), saccharin consumption (Cobuzzi and Riley, 2011), food intake (van der Laan et al., 1991), anxiety (Schulteis et al., 1998), conditioned place aversion (Vargas-Perez et al., 2009), intracranial self-stimulation (Schaefer and Michael, 1983; Holtz et al., 2015), working memory (Sala et al., 1994), and startle responses (Kalinichev and Holtzman, 2003). Depending on the behavior observed, these studies indicate that spontaneous opioid withdrawal ranges from 3 – 72 hours. Unfortunately, capturing the complete time course for withdrawal is not possible with these approaches either because testing is confined to specific times (e.g., physical withdrawal symptoms) or the time of the observed behavior is not evident (e.g., food intake). These tests can also be difficult to use because of prolonged training procedures, invasive surgeries, or the introduction of confounds from removing an animal from its home cage for testing. Given that spontaneous withdrawal signs may occur infrequently and at unpredictable times in the hours or days following cessation of opioid use (Schulteis, 2010), an objective method to assess withdrawal continuously is needed.

Wheel running is a natural and voluntary rodent behavior. We have previously shown that home cage wheel running provides an objective and clinically relevant measure of the duration and magnitude of pain in rats (Kandasamy et al., 2016; 2017a; 2017b). If depression of home cage wheel running is a measure of an abnormal physiological state as these previous studies indicate, then spontaneous opioid withdrawal should also cause depression of home cage wheel running. Although almost all previous preclinical studies of opioid withdrawal examine symptoms in naïve animals, the high incidence of prescription opioid abuse in pain patients (Ballantyne and LaForge, 2007) suggests that pain may exacerbate opioid withdrawal symptoms. These hypotheses will be tested by assessing spontaneous morphine withdrawal in rats with and without persistent inflammatory pain using home cage wheel running.

2. Materials and Methods

2.1 Subjects

Data were collected from 43 adult male Sprague-Dawley rats bred at Washington State University Vancouver. All rats weighed 260 – 410 grams at the start of the study and were randomly assigned to treatment groups. Prior to wheel exposure, rats were housed in pairs in a colony room (22 – 24 °C) on a reverse 12/12-hour light/dark cycle (lights off at 1700 h). All procedures were approved by the Washington State University Animal Care and Use Committee and conducted in accordance with the International Association for the Study of Pain’s Policies on the Use of Animals in Research.

2.2 Running wheel

Rats were housed individually to assess wheel running. A Kaytee Run-Around Giant Exercise Wheel (diameter = 27.9 cm; Kaytee Products, Inc., Chilton, WI, USA) was suspended from the top of the rat’s home cage. The floor of the cage was covered with cellulose bedding (BioFresh™, Ferndale, WA, USA). A thin aluminum plate (0.8 mm × 5.08 cm × 3.81 cm; K&S Precision Metals, Chicago, IL, USA) was attached to one spoke of the running wheel to interrupt a photobeam projecting across the cage with each rotation. The beam was set 18 cm above the floor of the cage so that only the rotation of the wheel, not the normal activity of the rat, would interrupt the beam. The number of wheel revolutions were summed over 5 min bins for 23 hrs each day using Photobeam Activity System software (San Diego Instruments, San Diego, CA, USA). Recordings began at the beginning of the dark phase (1700 h) of the day/night cycle when rats are most active. A full description of the running wheel with video is available in our previous publication (Kandasamy et al., 2016). Rats were allowed unrestricted access to the wheel for 23 hours/day for 8 days prior to the experimental manipulation. The number of wheel revolutions that occurred during the 23 hours during the eighth day was used as the baseline activity. Rats that ran less than 400 revolutions on the baseline day were not included in further testing (n = 13/56) (Kandasamy et al., 2016).

2.3 Pellet Implantation Surgery

Animals were briefly anesthetized with isoflurane (3–5 minutes) and implanted with two 75 mg morphine or placebo pellets (National Institute on Drug Abuse, Bethesda, MD, USA). The pellets were wrapped in nylon and implanted subcutaneously in the upper back. The morphine dose was consistent with previous studies examining dependence (Gold et al., 1994). The incision was closed with wound clips and a topical antibiotic was applied. While anesthetized, a subset of animals (Experiments 2 and 3) received an injection of Complete Freund’s Adjuvant (CFA, 0.1 mL; Sigma-Aldrich, Inc., St. Louis, MO, USA) into the right hindpaw to induce inflammatory pain. Rats woke up from anesthesia in their home cages where they had continuous access to a running wheel.

2.4 Experiment 1: Spontaneous withdrawal in naïve rats

The objective of this experiment was to determine whether wheel running could be used to measure tolerance and withdrawal from continuous morphine administration in normal pain-free rats. Wheel running was recorded for three consecutive days following pellet implantation. At the end of the third day, rats were re-anesthetized, the incision was opened, and the nylon covered pellets were removed. The incision was closed with wound clips and the rat was returned to its home cage. Wheel running was assessed 23 hrs a day for the next 6 days.

2.5 Experiment 2: Spontaneous withdrawal in inflamed animals after 3 days of morphine

The objective of this experiment was to determine whether chronic inflammatory pain alters the magnitude and/or duration of spontaneous morphine withdrawal as assessed by depression of home cage wheel running. The methodology for this experiment was identical to Experiment 1 except rats received a unilateral injection of CFA into the right hindpaw on the day the pellets were implanted. The effect of implanting and removing two morphine pellets on wheel running was compared to a control group that received two placebo pellets.

2.6 Experiment 3: Spontaneous withdrawal in inflamed animals after 5 days of morphine

The objective of this experiment was to determine whether prolonged morphine exposure would enhance the magnitude of spontaneous morphine withdrawal in rats with hindpaw inflammation. The methodology for this experiment was identical to Experiment 2 except rats were exposed to morphine or placebo pellets for five instead of three consecutive days.

2.7 Data Analysis

Baseline activity was defined as the total number of wheel revolutions during the 23 hours preceding the first injection. Given individual differences in wheel running, all wheel running data are presented as a percent change from each rat’s baseline value. Nearly all running occurred during the dark phase of the daily cycle. The average hourly nighttime running rate was used to calculate the percent change in running when hourly data are reported. All data are expressed as mean ± SEM. Data were analyzed using an independent samples t-test (Experiment 1) to compare placebo- and morphine-treated groups or repeated measures ANOVA for day-by-day and hour-by-hour running analyses (Experiments 1–3). Statistical significance was defined as a probability of <0.05.

3. Results

The average baseline running for the 43 rats over 23 hrs was 1110 ± 672 revolutions (988 meters). The median number of revolutions was 807 with a range of 415 to 3278 (369 – 2917 meters). Almost all (96.4%) of the running occurred during the 12 hrs of the dark phase.

3.1 Experiment 1: Spontaneous withdrawal in naïve rats

Median baseline running levels was 804 revolutions (Range: 636 to 1451) for the morphine-treated animals and 852 revolutions (Range: 415 to 1692) for the placebo-treated animals. Implantation of morphine, but not placebo pellets caused a pronounced decrease in wheel running in the 23 hours post-implantation (Fig. 2A). Although the surgery to implant the placebo pellet caused a drop in wheel running, the magnitude of the reduction in morphine treated rats was significantly greater (t(16) = 3.524, p = 0.003). Wheel running was relatively stable for placebo-treated animals across the three days, whereas a significant recovery of wheel running occurred in morphine-treated animals across days as indicated by a group × time interaction (F(1,16) = 7.808, p = 0.013).

Figure 2. Morphine administration and withdrawal transiently decreases wheel running in pain-free rats.

Figure 2

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A) Implantation of two morphine pellets (n = 8) significantly decreased wheel running in the first 24 hours compared to rats implanted with two placebo pellets (n = 10). Tolerance to morphine is evident in that rats gradually recovered to normal running levels by Day 3. B) Removal of the pellets depressed wheel running in both morphine- and placebo-treated rats. Wheel running recovered within 10 hours following removal of placebo pellets, but remained low in animals in which morphine pellets were removed. C) Analysis of daily wheel running revealed that withdrawal was greatest 48 hours after removal of the morphine pellets. * indicates p < 0.05 difference between groups

A decrease in wheel running occurred when the morphine and placebo pellets were removed. The first indication of morphine withdrawal was evident approximately 6 hours after pellet removal (Fig. 2B). Analysis of wheel running presented over 2 hour blocks shows a gradual recovery in wheel running in rats following surgery to remove the placebo pellets compared to the sustained depression of wheel running in rats following removal of the morphine pellets (F(1,16) = 5.586, p = 0.031). Post hoc analysis revealed that placebo- and morphine-treated rats differed at hours 10 and 12 (Bonferroni: p < 0.05). There was a significant recovery of wheel running in both groups by Day 3 (F(1,16) = 8.937, p = 0.009; Fig 2C). Although the difference between groups did not reach statistical significance when examined across the first three days (F(1,16) = 3.914, p = 0.065), there was a significant difference in wheel running between groups on Day 2 (t(16) = 3.163, p = 0.006).

3.2 Experiment 2: Spontaneous withdrawal in inflamed animals after 3 days of morphine

The median baseline running level was 598 revolutions (Range: 488 to 1644) for the morphine-treated animals and 649 revolutions (Range: 475 to 1727) for the placebo-treated animals. A near complete inhibition of wheel running occurred in both placebo- and morphine-treated animals in the 23 hours following injection of CFA into the hindpaw whether morphine or placebo pellets were implanted (Fig. 3A). Wheel running gradually increased with each passing day in both groups, but never fully recovered to pre-CFA baseline levels (Fig. 3A). Although morphine would be expected to alleviate CFA-induced pain, Experiment 1 showed that continuous morphine administration also reduces wheel running. Thus there was no significant difference between groups over the 3 days following pellet implantation (F(1,9) = 0.923, p = 0.362).

Figure 3. Termination of morphine administration after 3 days produces prolonged depression of wheel running in rats with hindpaw inflammation.

Figure 3

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A) Wheel running was significantly reduced following unilateral hindpaw injection of CFA and implantation of two morphine pellets (n = 6) or two placebo pellets (n = 5). Wheel running gradually increased to roughly 50% of baseline levels over three days. B) In the 12 hours following removal of the morphine or placebo pellets, wheel running was reduced in both groups as a result of hindpaw inflammation and pellet removal surgery. C) This decrease in wheel running persisted for 6 days following removal of morphine pellets, whereas much higher levels of running occurred in rats following removal of placebo pellets. * indicates p < 0.05 difference between groups

A decrease in wheel running occurred when the morphine and placebo pellets were removed (Fig. 3B). There was no significant difference between groups in the 12 hours following pellet removal (F(1,9) = 0.719, p = 0.419). Wheel running was depressed in both hindpaw inflammation groups in the 23 hours following removal of the morphine or placebo pellets (Fig. 3C). Wheel running remained depressed for over 6 days in rats previously treated with morphine, whereas rats previously treated with placebo pellets showed a significant recovery of wheel running by the second day (Fig. 3C). Analysis of variance revealed a significant difference between groups (F(1,9) = 15.505, p = 0.003) that persisted throughout the 6 days of testing. Post hoc analysis revealed significant differences between groups on Days 2–6 (Bonferroni: p < 0.05).

3.3 Experiment 3: Spontaneous withdrawal in inflamed animals after 5 days of morphine

Median baseline running levels was 1721 revolutions (Range: 572 to 3278) for the morphine-treated animals and 1877 revolutions (Range: 669 to 3005) for the placebo-treated animals. As in the previous experiment, the combination of hindpaw injection of CFA and surgical implantation of the pellets depressed wheel running in both placebo- and morphine-treated animals (Fig. 4A). A consistent incremental increase in wheel running occurred in both groups over the next 5 days. There was no significant main effect of group (F(1,12) = 0.838, p = 0.378) or group by day interaction (F(1,12) = 0.211, p = 0.654).

Figure 4. Termination of morphine administration after 5 days depresses wheel running for 3 days in rats with hindpaw inflammation.

Figure 4

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A) Wheel running was significantly reduced following unilateral hindpaw injection of CFA and implantation of two morphine pellets (n = 7) or two placebo pellets (n = 7). Both groups of animals returned to baseline levels of running by 5 days post-implantation. B) Wheel running was significantly reduced in the first 12 hours after pellet removal in morphine-treated animals compared to placebo-treated controls. C) Day-by-day analysis of wheel running revealed that removal of morphine pellets depressed wheel running for 3 days compared to rats in which placebo pellets were removed. * indicates p < 0.05 difference between groups

Removal of the pellets caused a decrease in running in both placebo- and morphine-treated animals (Fig. 4B). Analysis of the wheel running in the first 12 hours following pellet removal revealed that wheel running was almost completely inhibited in rats undergoing morphine withdrawal compared to the transient and less severe depression in placebo-pretreated rats [(F(1,12) = 37.396, p < 0.001); Fig. 4B]. Post hoc analysis revealed a significant difference between groups from hours 4–12 (Bonferroni: p < 0.05). The day-to-day magnitude of this decrease in running was significantly greater in rats pretreated with morphine compared to placebo pellets (F(1,12) = 7.348, p = 0.019). The depression of wheel running in the rats undergoing morphine withdrawal gradually recovered until Day 4 when the statistically significant difference in wheel running between rats pretreated with morphine and placebo was no longer evident (Bonferroni: p < .05 for Days 2 and 3; Fig. 4C).

4. Discussion

The two main findings of these studies are that spontaneous morphine withdrawal causes depression of home cage wheel running and that this depression is exacerbated in animals with persistent hindpaw inflammation. These data indicate that home cage wheel running is a sensitive method to assess the duration and magnitude of opioid withdrawal in the rat.

Rats were given morphine continuously to induce dependence. The sedative effects of morphine were evident during the first day of morphine administration in that almost no wheel running occurred. Running recovered on Days 2 and 3 as would be expected with the development of tolerance. Tolerance was only evident in pain-free animals because the induction of hindpaw inflammation also depressed home cage wheel running as we have reported previously (Kandasamy et al., 2017a). Wheel running gradually recovered across days so that when the morphine pellets were removed a spontaneous withdrawal-induced decrease in wheel running occurred.

Our withdrawal-induced decrease in wheel running is consistent with other studies that show spontaneous withdrawal from opioids reduces movement (van der Laan and de Groot, 1988; Stinus et al., 1998). The difference is that we studied voluntary running on a wheel as opposed to movement around the rat’s home cage. Spontaneous opioid withdrawal has also been shown to cause somatic withdrawal symptoms (Koga and Inukai, 1981; Cicero et al., 2002; Papaleo and Contarino, 2006; Allahverdiyev et al., 2015), although these tend to be mild and are not evident in all studies (Palma et al., 2015). Wheel running improves on home cage monitoring and assessment of somatic symptoms by continuously assessing withdrawal-induced decreases in voluntary behavior, a common clinical phenomenon observed in patients undergoing opioid withdrawal (Wesson and Ling, 2003).

A wide range of other methods have been used to assess spontaneous withdrawal (body weight, defecation, anxiety, taste aversion, ultrasonic vocalization, intracranial self-stimulation, conditioned place aversion, and startle) (Schaefer and Michael, 1983; Schulteis et al., 1998; Kalinichev and Holtzman, 2003; Papaleo and Contarino, 2006; Vargas-Perez et al., 2009; Cobuzzi and Riley, 2011; Holtz et al., 2015). These studies vary greatly in terms of when withdrawal symptoms were assessed and show that spontaneous withdrawal can occur within hours or days of termination of opioid treatment. Home cage wheel running has the distinct advantage of objectively revealing the entire time course for withdrawal. For example, depression of home cage wheel running lasted for 2 days in pain free rats and for over 6 days in rats with hindpaw inflammation. Another advantage of home cage wheel running is that it prevents confounds caused by handling or testing the animal in a novel environment.

Clinical evidence indicates that non-physical signs of opioid withdrawal may be more relevant to drug craving and relapse than somatic symptoms (Jasinski et al., 1985; Maldonado et al., 1996). A low-grade protracted withdrawal syndrome characterized by discomfort and anhedonia is present in patients in the absence of physical symptoms (Højsted and Sjøgren, 2007). Opioid withdrawal can even occur in the complete absence of overt outward physical symptoms (Higgins and Sellers, 1994). Home cage wheel running provides a method to assess the affective aspect of withdrawal. Although simultaneous assessment of somatic or anxiety symptoms of withdrawal is ideal, the nature of our home cage wheel running involves testing animals in an isolated room with no experimenter or other disruptions. As such, we did not assess physical withdrawal symptoms. However, our results are consistent with other studies demonstrating physiological symptoms of withdrawal using the same approach to generate morphine dependence (Yoburn et al., 1985; Gold et al., 1994).

It has been argued that pain limits the develop of opioid dependence (Portenoy, 1996). Our data suggest the opposite is true. The magnitude and duration of spontaneous morphine withdrawal was much greater in rats with inflammatory pain compared to pain-free rats. Morphine withdrawal caused depression of home cage wheel running that lasted for at least 3 days in rats with hindpaw inflammation, whereas withdrawal was only evident for 2 days in pain-free rats. Previous studies showing allodynia and hyperalgesia following opioid withdrawal provide a possible explanation for the enhanced withdrawal in rats with hindpaw inflammation (Li et al., 2001; Liang et al., 2008).

One problem in studying withdrawal in an animal with a pain condition is that the pain condition changes with time and may affect the expression of withdrawal symptoms. For example, spontaneous opioid withdrawal was shorter in rats treated with morphine for 5 days compared to 3 days. Given that plasma morphine levels are stable 3–12 days post-pellet implantation (Gold et al., 1994), a decrease in morphine concentration with prolonged treatment is unlikely to explain this difference. A more likely explanation is that the impact of hindpaw inflammation on wheel running is reduced with each passing day. That is, CFA-induced depression of wheel running is greater on Day 3 than Day 5 (Kandasamy et al., 2016). Further analysis of the interaction between pain state and opioid withdrawal is needed.

In conclusion, continuous recording of home cage wheel running following chronic morphine treatment is an objective and reliable method for assessing the duration and magnitude of spontaneous opioid withdrawal in the rat. This method may be particularly useful in evaluating the efficacy of treatments for withdrawal because it captures subtle aspects of withdrawal such as affective states that are not readily apparent in observational studies. Furthermore, these studies suggest that the sustained use of opioids may increase the risk of dependence in chronic pain patients.

Figure 1. Experimental timeline.

Figure 1

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Rats in Experiment 1 had pellets removed after 3 days and did not receive CFA injections. Rats in Experiment 2 had pellets removed after 3 days and received CFA injections. Rats in Experiment 3 had pellets removed after 5 days and received CFA injections.

Highlights.

  • Current methods to assess spontaneous withdrawal are limited

  • Spontaneous withdrawal depresses wheel running in pain-free rats

  • Withdrawal-induced depression of wheel running is exacerbated in injured rats

  • Home cage wheel running is a valid and sensitive measure of spontaneous withdrawal

  • Opioid withdrawal may be worse in chronic pain patients resulting in opioid abuse

Acknowledgments

The authors thank Hailey Smith, Shauna Schoo, and Rebecca Wescom for technical assistance. This investigation was supported in part by funds provided by medical and biological research by the State of Washington Initiative Measure No. 171 to ATL and NIH grant NS095097 to MMM.

Footnotes

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None of the authors declare a conflict of interest.

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