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. Author manuscript; available in PMC: 2024 Jun 21.
Published in final edited form as: Neurosci Lett. 2009 Sep 3;465(1):66–70. doi: 10.1016/j.neulet.2009.08.073

Hyperlocomotion and paw tremors are two highly quantifiable signs of SR141716-precipitated withdrawal from delta9-tetrahydrocannabinol in C57BL/6 mice

Peng Huang 1, Lee-Yuan Liu-Chen 1, Ellen M Unterwald 1, Alan Cowan 1
PMCID: PMC11192173  NIHMSID: NIHMS143778  PMID: 19733208

Abstract

Increasing evidence suggests that marijuana abstinence leads to clinically significant withdrawal symptoms in humans. In mouse models, following chronic treatment with delta9-tetrahydrocannabinol (THC), administration of the selective cannabinoid CB1 receptor antagonist SR141716 (rimonabant) elicited varying behavioral responses, depending on mouse strain and dosing regimen. In the present study, C57BL/6 mice were injected s.c. with THC (25 mg/kg) or vehicle twice daily for 4.5 days. SR141716 (15 mg/kg) was administrated i.p. 4 hrs following the last THC treatment. During a 2-h observation period immediately following the SR141716 challenge, the total locomotor, ambulatory and stereotypic activities of THC-treated mice were 4.1, 3.3, and 3.8 times those of vehicle-treated mice, respectively. The number of paw tremors elicited in THC-treated mice was 111±11 during the 45 min immediately following SR141716, whereas only 1.1±0.4 was associated with vehicle-treated animals. In contrast, the number of scratching bouts was higher in vehicle-treated (182±20) vs. THC-treated (17±4) mice. The present study is the first to demonstrate hyperlocomotion as an explicit sign of THC abstinence in mice. Together with paw tremors, the two unambiguous withdrawal signs may permit highly quantitative investigation of THC abstinence in C57BL/6 mice and may facilitate investigation of the mechanisms involved via both pharmacological and genetic manipulations, and ultimately potential treatments for cannabis dependence.

Keywords: THC, withdrawal, abstinence, locomotion, SR141716, mice

Introduction

Marijuana is the most commonly used illicit drug in the United States and worldwide [1,2]. Abstinence from marijuana smoking and oral delta9-tetrahydrocannabinol (THC, the main psychoactive component therein) in humans produces anxiety, hyperirritability, weight loss (decreased appetite), restlessness and insomnia [see [2] for review]. SR141716, a selective cannabinoid CB1 receptor antagonist, has been a particularly useful tool for precipitating cannabinoid withdrawal symptoms in laboratory animals. In contrast to the challenge of observing and quantifying spontaneous/abrupt withdrawal, SR141716 elicits immediate and quantifiable withdrawal responses in a variety of animal species, including mouse, rat, dog and non-human primates [see [2–4] for reviews].

Several groups have studied SR141716-precipitated THC withdrawal syndromes in mice [e.g., [5,6]]. Depending on the mouse strain and dosing regimen used, a range of withdrawal behaviors have been documented, such as paw tremors, head shakes, wet dog shakes, hunched posture, mastication and piloerection [see [3,4] for reviews]. The behavioral expression of THC withdrawal in mice was demonstrated to be less intense in comparison with that of opiate withdrawal, and lacked jumping, hyperlocomotion, autonomic features, and aversive/dysphoric properties [6]. Paw tremors and head shakes were the most reliable signs of THC withdrawal in some studies [5,7], and paw tremors were also observed consistently during SR 141716-precipitated withdrawal in mice that had been repeatedly exposed to marijuana smoke [8]. In the present study, in addition to paw tremors, increased locomotor activity is reported as a highly quantifiable sign of SR141716-precipitated THC withdrawal in mice.

Materials and Methods

Animals:

Male C57BL/6 mice were purchased from Ace Animals, Inc. (Boyertown, PA). Animals were allowed to acclimate to the animal facility for two weeks. They weighed 21–26 g at the start of the study. The mice were housed four animals per cage on a 12:12-h light/dark cycle (7 am-7 pm) with ad libitum access to food and water. Animal care and experimental procedures were conducted according to the Guide for the Care and Use of Laboratory Animals (National Research Council,1996). Experimental protocols were approved by the Institutional Animal Care and Use Committee of Temple University School of Medicine.

Compounds:

THC (in ethanol, 100 mg/ml) and SR141716 (freebase) were provided by the National Institute on Drug Abuse (Bethesda, MD). Both were first dissolved in ethanol, followed by addition of cremophor EL (Sigma), and then diluted with saline to form a vehicle mixture of ethanol/cremophor/saline in a ratio of 1:1:18. Injections were carried out in a final volume of 0.1 ml per 10 g of body weight.

Evaluation of behavioral signs of SR141716-precipitated THC withdrawal:

Mice were injected twice daily with either THC (25 mg/kg, s.c.) or vehicle for four consecutive days, every day between 9:00–11:00 am and again between 8:00–10:00 pm. On the 5th day, each mouse was given only the morning injection of THC. After the last injection, each animal remained in its home cage for 2 hrs, and then was transferred to and habituated to a clear activity chamber for another 2 hrs with motor activities automatically recorded as described below. Each mouse was challenged with SR141716 (15 mg/kg, i.p.) at the end of the 2-h habituation period, and motor activities were automatically recorded for another 2 hrs. In the meantime, the number of forepaw tremors (bouts of rapid repetitive up-down movements of one or both forepaw(s)), head shakes (turning and twisting of the head from side to side), writhing (stretching of the abdomen with extension of the hindlegs), jumping, and scratching (repetitive movements around the neck with the hindpaws, often preceded by hindpaw licking) was noted by either real-time or videotape counting during a 45-min period right after SR141716 injection.

Motor activities were measured using a Digiscan D Micro System (Accuscan, Columbus, OH, USA) and four individual activity monitors. A single activity monitor consists of an aluminum frame equipped with 16 infrared light beams and detectors where the activity chamber (a standard clear plastic animal cage, 42 × 20 × 20 cm) is placed. As the mouse moves within the chamber, light beams are broken and recorded by a computer connected to the Digiscan system. Activity was recorded as total activity, ambulatory activity and stereotypy. Total activity represents all beam breaks compiled by a single mouse, and is the sum of the ambulatory and stereotypy counts. Ambulatory activity represents successive beam breaks while stereotypy counts are defined as bouts of repetitive beam breaks. Stereotypic counts do not identify a specific stereotypic behavior, but rather repeated breaks of the same beam indicative of a stationary animal engaged in a repetitive behavior as opposed to ambulation. Activity was measured over 4 hrs with 5 min collection intervals under normal laboratory lighting conditions.

Data analysis:

Data are expressed as the mean±SEM. Data in Fig. 1 A–C were examined for statistical significance by repeated measures analysis of variance (ANOVA) followed by Bonferroni post-tests (GraphPad Prism v.4). Data in Fig. 1 D–F and Fig. 2 were analyzed using a two-tailed Student’s t test. Statistical significance was achieved when P<0.05.

Fig. 1. Effects of SR141716 administration on activity in mice treated with THC or vehicle.

Fig. 1.

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The time course of changes in total, ambulatory and stereotypic activities are shown in panels A, B and C, and cumulative 2-h data are shown in D, E and F, respectively. Mice were injected with either THC (25 mg/kg s.c.) or vehicle twice daily for 4.5 days as described in the Materials and Methods. On test days, 4 hrs following the last THC treatment, mice were habituated in activity-recording chambers for 2 hrs and then injected with SR141716 (15 mg/kg i.p.). Total (A), ambulatory (B) and stereotypic (C) activities were continuously monitored using a Digiscan D Micro System. Activities were recorded over a 4-h period with habituation for the first 2 hrs. Data in A-C are expressed as mean±SEM beam breaks/5-min period and analyzed by repeated measures ANOVA followed by Bonferroni post hoc analysis. Data in D-F are expressed as mean±SEM beam breaks/2-hr period and analyzed by the two-tailed Student’s t tests. n=8 per group (THC vs Vehicle, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001).

Fig. 2. Other behavioral signs after SR141716 administration to mice treated with THC or vehicle.

Fig. 2.

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Immediately after i.p. injection of the antagonist, the number of paw tremors (A), head shakes (B), writhing (C) and scratching (D) was recorded for 45 min. Results are shown as mean±SEM, and analyzed by two-tailed Student’s t tests. n=6–8 per group (**** P<0.0001).

Results

Effects of SR141716 administration on activity in mice treated with THC or vehicle

To test for precipitated withdrawal, SR141716 (15 mg/kg) was administered i.p. to THC-and vehicle-treated mice after 2 hrs of habituation in the activity chambers. Activity was continuously recorded for another 2 hrs (4 hrs in total).

Repeated measures analysis of variance (ANOVA) of total activity recorded during the 2 hrs of acclimation to the testing chambers (Fig. 1A) showed main effects of Treatment (THC vs vehicle) [F(1,308)=12.87, P<0.01], Time [F(22,308)=21.48, P<0.0001], and Treatment x Time interaction [F(22,308)=4.51, P<0.0001], respectively. Bonferroni post-tests demonstrated that THC-treated mice habituated faster than vehicle-treated mice in terms of taking less time to reach the baselines of total activity (Fig. 1A). In addition, statistical analyses of ambulatory (Fig. 1B) and stereotypic (Fig. 1C) activities in the same period revealed significant main effects of Time [F(22,308)=15.24 and 12.69, P<0.0001] and Treatment x Time interaction [F(22,308)=3.27 and 3.44, P<0.0001], while the effects of Treatment were not significant [F(1,308)=4.18 and 2.14, P>0.05].

For activity measured over 2 hrs post SR141716 administration, statistical analysis of ambulatory activity (Fig. 1B) demonstrated main effects of Treatment (THC vs vehicle) [F(1,336)=17.86, P<0.001] and Time [F(24,336)=3.88, P<0.0001], but there was no significant interaction [F(24,336)=1.31, P>0.05]. Statistical analyses of total (Fig. 1A) and stereotypic (Fig. 1C) activities after SR141716 challenge revealed main effects of Treatment [F(1,336)=47.37 and 9.99, P<0.0001], Time [F(24,336)=5.08 and 3.44, P<0.0001] as well as interaction [F(24,336)=1.90 and 2.44, P<0.0001]. Approximately 1–3 min following administration of the antagonist to THC-treated mice, dramatic enhancement was recorded in total activity, ambulation and stereotypy (Fig. 1). Activity increased sharply within the initial 10 min and reached the peak. The increased total and ambulatory activities lasted for at least 2 hrs with a moderate and fluctuated attenuation (Fig. 1A and 1B). Enhanced stereotypic activity diminished more quickly than total and ambulatory activities (Fig. 1C). In contrast, antagonist challenge did not increase motor activities in vehicle-treated mice (Fig. 1). Quantification of the sum of activity counts recorded during the 2 hrs immediately following SR141716 challenge demonstrated that total activity was 7985±857 vs 1939±245 [t(14)=6.78, P<0.0001] (Fig. 1D), ambulatory activity was 5258±897 vs 1572±369 [t(14)=3.80, P<0.01] (Fig. 1E), and stereotypic activity was 2727±558 vs 724±262 [t(14)=3.25, P<0.01] (Fig. 1F), for THC- vs vehicle-treated animals, respectively. Thus, the cumulative activities of total locomotion, ambulation and stereotypy of THC-treated mice were 4.1, 3.3 and 3.8 times those of vehicle-treated mice, respectively. It is noteworthy that the activity differences between THC- and vehicle-treated mice might be overestimated due to a possible “floor effect” which can occur with automated activity monitors to record the low counts of vehicle-treated animals.

Other behavioral signs after SR141716 administration to mice treated with THC or vehicle

During the 45 min immediately ensuing SR141716 challenge, other behavioral expressions were counted manually in real-time or from videotapes. Results demonstrate the following numbers of occurrences of these behaviors in THC- vs vehicle-treated animals following injection of SR141716: (1) for forepaw tremors, 111±11 vs 1.1±0.4 [t(14)=9.78, P<0.0001] (Fig. 2A); (2) for head shakes, 9.2±2.7 vs 4.3±1.7 [t(12)=1.43, p>0.05] (Fig. 2B); (3) for writhing, 2.6±0.9 vs 1.5±0.8 [t(12)=0.89, P>0.05] (Fig. 2C); (4) for scratching, 17±4 vs 182±20 [t(12)=6.96, P<0.0001] (Fig. 2D). Thus, following the antagonist challenge, THC-treated mice exhibited numerous bouts of forepaw tremors, and this behavior was rarely observed in vehicle-treated animals. In contrast, a high incidence of scratching was evident in vehicle-treated mice after SR141716 administration. This behavior occurred about 10 times less frequently in THC-treated mice. In addition, head shakes and writhing were observed only sporadically in both groups of mice, and 2 vertical jumps were detected in only one THC-treated mouse upon the antagonist challenge.

Discussion

This is the first report showing that hyperlocomotion is a highly quantifiable sign of THC withdrawal precipitated by SR141716 in a mouse model. The present study also confirms paw tremor as a reliable sign of THC withdrawal in mice, which is consistent with previous findings [5,7,9]. Because SR141716 failed to produce similar behaviors in vehicle-treated mice, these two signs were deemed “precipitated withdrawal” rather than an intrinsic effect of the antagonist itself.

In addition, in terms of the total activity before the SR141716 injection, the THC-treated mice habituated to the activity chamber faster than the vehicle-treated animals. This observation might suggest a change in response to novelty in THC-dependent mice. THC treatment-induced sedation and/or catalepsy could also be another explanation of this difference.

Our locomotor activity results are reminiscent of the findings by Tsou et al. [10] in rats demonstrating a similar increase in horizontal and vertical activities following SR141716-precipitated THC withdrawal. In addition, Oliva et al. [11] reported that spontaneous withdrawal from a more potent cannabinoid (CP-55,940) in male Swiss albino mice was characterized by a modest increase (~1.8 fold of control) in ambulatory activity, which was evaluated one day after the last administration of CP-55,940 in a 15-min observation period after a 5-min habituation. In contrast, Hutcheson et al. [6] and Tourino et al. [17] demonstrated that, after SR141716 challenge (10 mg/kg i.p.), male CD-1 mice pretreated i.p. repeatedly with 20 mg/kg of THC, but not with 10 mg/kg, showed a significant hypolocomotion compared to vehicle-treated animals, and it should be noted that locomotor activity was evaluated in a low luminosity room [6][17]. The discrepancy might be due to differences in experimental conditions such as the mouse strain, dosing regimen and lighting conditions during testing. It is also noteworthy that SR141716 alone was found to increase [12,13] and decrease [14] locomotor activity in naïve mice although the effects were not likely mediated by the CB1 cannabinoid receptor [13,14]. Thus, it is still not known whether hyperlocomotion can be established as a common sign to evaluate SR141716-precipitated abstinence from THC in mouse strains other than C57BL/6.

SR141716 at dose of 10 mg/kg has been commonly used to precipitate THC-withdrawal in animals including mice [see [3,4] for reviews]. We chose SR141716 of 15 mg/kg for the following reasons: (1) SR141716 dose ranges to block various THC-induced responses in mice have been determined [12]. The maximum observed antagonism of THC-mediated effect was 82% at 10 mg/kg of SR141716 i.p. in the PPQ-stretch procedure (an antinociceptive evaluation). The sigmoidal curve-fitting analysis revealed that 15~20 mg/kg dose was needed for SR141716 to antagonize the THC effect completely in the PPQ-stretch test, although the i.p. dose of 3 mg/kg SR141716 produced a nearly complete (95%) inhibition of THC-induced hypothermia and an almost complete (89%) inhibition of the antinociceptive effect of THC in the tail-flick procedure; (2) SR141716 was characterized at doses up to 30 mg/kg i.p. to precipitate THC-withdrawal in mice [5]. SR141716 at 30 mg/kg appeared to induce higher frequencies of paw tremors and head shakes than the dose of 10 mg/kg. In addition, for THC we used the dose of 25 mg/kg, because the behavioral responses to precipitated withdrawal in mice have been shown to be THC (3~30 mg/kg) dose-dependent [e.g., [5][7]] and high doses (20 or 30 mg/kg) of THC were used to yield high levels of responses [e.g., [5–7]]. Further experiments performed with different doses of THC and SR141716 are needed to define the dose ranges required to manifest hyperlocomotion in mice and to understand the nature of the interaction between drugs and their target(s).

Besides paw tremor, head shakes have also been monitored as a THC withdrawal sign in some mouse studies [5–7], but not in others, due to the observation that SR141716 elicited a comparable level of head shaking regardless of THC treatment [9]. In the present study, head shaking was not a prominent withdrawal sign in C57BL/6 mice, whereas opposite results have been shown by Lichtman et al. [7] who used the same mouse strain but different dosing regimens. In addition, vehicle-treated mice displayed very high episodes of scratching upon SR141716 challenge in contrast to THC-treated animals, which appear to be in line with two findings: (1) SR141716 increases scratching in vehicle-treated mice in a dose-dependent manner, but fails to do so in THC-treated mice [5]; and (2) activation of CB1 receptors by THC or anandamide reduces the incidence of scratching in an acute allergenic murine model [15].

As SR141716 provokes locomotion and paw tremors to high levels in THC-treated mice, and the recording of locomotor activity is automated, these two signs represent readily measurable endpoints of THC withdrawal. C57BL/6 is one of the most widely used strains in terms of genetic background for genetically modified (knockout and knockin) mouse lines [16]. The finding that this strain of mice displayed hyperlocomotion and paw tremors as unambiguous THC withdrawal signs allows future studies on the underlying mechanisms of THC withdrawal by not only pharmacological but also by genetic manipulations.

Acknowledgements:

This work was supported by National Institute of Health Grants R01 DA17302 (LYLC) and P30DA013429 (LYLC, EU and AC). We thank Drs. Mary Abood and Ellen Walker for comments on an early version of the manuscript, and thank Chinwe Nwaneshiudu, Nicole Enman and Dr. Wei Xu for the technical supports.

Footnotes

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