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Effects of physostigmine on the conditioned hyperactivity and locomotor sensitization to morphine in rats
Behavioural Brain Research 206 (2010) 223–228
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Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Research report
Effects of physostigmine on the conditioned hyperactivity and locomotor sensitization to morphine in rats Xinwang Li a,∗ , Jun-Xu Li b,∗ , Xiaolin Zhu a , Ruisi Cui a , Jingjing Jiao a a b
Department of Psychology, Capital Normal University, Beijing, China Department of Pharmacology, University of Texas Health Science Center at San Antonio, San Antonio TX, USA
a r t i c l e
i n f o
Article history: Received 4 June 2009 Received in revised form 7 September 2009 Accepted 10 September 2009 Available online 15 September 2009 Keywords: Morphine Physostigmine Conditioned hyperactivity Locomotor sensitization Rats
a b s t r a c t Repetitive exposure to opioids elicits sensitization to its psychomotor stimulating effect and environmental stimuli contribute to this effect. This study first developed a procedure that simultaneously measures conditioned hyperactivity and locomotor sensitization, and then investigated the effects of physostigmine on the development and expression of conditioned hyperactivity and locomotor sensitization in rats. Five groups of rats (10–12 rats each) were conditioned with a conditioned stimulus (CS) for 20 min and then drug or saline paired with CS for 2 h daily for 10 days. Rats were tested 20 min on day 18. On day 25, rats were tested 20 min and subsequently 2 h (immediately after morphine injection). Although the 20 min locomotion was not different among the rats on day 1, rats that received 5 mg/kg morphine during conditioning showed higher locomotion than those received saline or 5 mg/kg morphine in the home cage on day 18 and day 25. Rats received 0.1 mg/kg physostigmine and 5 mg/kg morphine during conditioning showed higher locomotion than those received 5 mg/kg morphine on day 18. On day 25, 0.1 mg/kg physostigmine attenuated the conditioned hyperactivity and expression of morphine locomotor sensitization. In contrast, rats received 0.1 mg/kg physostigmine and 5 mg/kg morphine during conditioning showed higher locomotion during 2 h test period than those received 5 mg/kg morphine. In conclusion, this study established a procedure that simultaneously study conditioned hyperactivity and locomotor sensitization. Physostigmine attenuates the expressions but enhances the development of conditioned hyperactivity and sensitization and the possible mechanisms are discussed. Published by Elsevier B.V.
1. Introduction Environmental stimuli can become strongly associated with and predictive of the drug effects after they are repeatedly paired with many drugs of abuse and thereby acquire the ability to evoke conditioned responses by classical conditioning process. Conditioning effects are one of the factors believed to contribute to the relapse of drug use even after long periods of drug abstinence [4,8,15]. For instance, cocaine abusers exhibit strong craving when presented with stimuli previously paired during cocaine use [16]. Abundant evidence suggests that sensitized psychomotor stimulating effects induced by repeated intermittent administration of some drugs (e.g. opioids and psychostimulants) may be an indication of sensitization to the underlying reward/incentive systems [1,25,26,32,33]. Drug induced locomotor sensitization can also be strongly modulated by the environmental context. The magnitude of amphetamine and cocaine-induced locomotor sensitization is greatly attenuated if drugs are injected (intravenous) via a remotely
∗ Corresponding authors. E-mail addresses: [email protected] (X. Li), [email protected] (J.-X. Li). 0166-4328/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.bbr.2009.09.016
controlled infusion apparatus, a manipulation that presumably markedly decreases the salience of environmental cues usually associated with drug administration such as handling of the animals and giving the injections by the experimenter [6,7,10,17]. In most of the earlier conditioned hyperactivity studies, a simultaneous conditioning technique is used so that immediately after the subjects receive the drug or vehicle injection, they are exposed to the environmental context (with or without the presence of conditioned stimulus, respectively) to initiate the conditioning process. Damianopoulos and Carey developed a new procedure which measures the conditioned and unconditioned drug effects in two distinct environments sequentially within the same session, raising the possibility of studying conditioned and unconditioned drug effects separately [12]. In the current study, a modified method from Damianopoulos and Carey was used in which a delay conditioning process was used to develop conditioned hyperactivity so that animals were exposed to conditioned stimulus (CS, environmental context) before the unconditioned stimulus (US; drug, morphine in this case) was given. This temporal arrangement allows simultaneous measurement of conditioned hyperactivity (before drug is administered) and the expression of locomotor sensitization (after
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drug is administered). Furthermore, since conditioned hyperactivity was successfully established with this procedure, the effect of acetylcholinesterase (AChE) inhibitor physostigmine on conditioned hyperactivity and locomotor sensitization to morphine was studied. Previous studies have reported that cholinergic systems are involved in the conditioned cue-induced behavioral effects. For example, the muscarinic receptor antagonist scopolamine blocks conditioned locomotor sensitization to cocaine [21]. Physostigmine inhibits the conditioned cue-induced reinstatement of morphine self-administration behavior [37].
injection and immediately put back into the chamber and the locomotor activity was measured for the following 2 h. Rats in the unpaired group (Group 4) were given morphine injection and rats in other groups saline injection 4 h after the daily session in the home cages. Then, they remained in their home cage without any drug treatment during days 11–17 and days 19–24. The dose of morphine used in this study (5 mg/kg) was chosen based upon previous report [3] and our pilot studies (data not shown) showing that this dose of morphine produced the most robust hyperactivity and locomotor sensitization.
2. Materials and methods
3.2. Conditioned hyperactivity and locomotor sensitization test
2.1. Subjects Adult male Wister rats (220–250 g, Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were housed in standard lab Plexiglas cages (45 × 30 × 25 cm, 3 rats/cage) in a weather-controlled ventilated colony room on a 12/12 h light/dark cycle (experiments were conducted during the light period) with free access to water and food in the home cage. All animals were maintained and experiments were conducted in accordance with the Institutional Animal Care and Use Committee, Capital Normal University and with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources on Life Sciences, National Research Council, National Academy of Sciences, Washington DC, USA). 2.2. Drugs Morphine hydrochloride (Shenyang First Pharmaceutical Factory, Shenyang, China) and physostigmine (Sigma–Aldrich, St. Louis, MO) were dissolved with saline and injected intraperitoneally (i.p.) at a volume of 1 ml/kg. 2.3. Apparatus Locomotor activity was measured by an automated video tracking system with four customer made activity chambers. The chambers were made of black Perspex plastics (40 × 40 × 50 cm, length × width × height). A video camera was mounted at the top of the chambers, which was connected to a PC to record the locomotion of rats. The video documents (stored in the computer) were analyzed by the LA analysis software (Institute of Psychology, Chinese Academy of Sciences, Beijing, China). The locomotor activity was expressed as the total distance travelled for a predetermined period of minutes. 1.5 ml of 50% acetic acid dropped on absorbent cotton served as conditioned stimulus and replaced with fresh made daily immediately before the session started. The cotton was held in a porous metal container and put in one top corner of the chamber out of the reach of the animals.
3. Behavioral procedure 3.1. Acquisition of conditioned hyperactivity Rats were randomly assigned into one of the five groups as indicated in Table 1. Conditioning training was conducted in 10 consecutive days (days 1–10). During each training session, all rats were given the first injection (i.p.) and 10 min later put into the locomotion chamber for 20 min (paired with conditioned stimulus as previously described); 20 min later rats were given the second
On day 18, all rats were put into the locomotion chamber without any treatment and the locomotor activity was measured for 20 min. Based upon the test results, rats in Group 2 (those received morphine during training sessions) were re-assigned into two groups so that the locomotor activity between the two groups were not significantly different. On day 25, rats received either saline (Groups 1, 2a, 3, 4 and 5) or 0.1 mg/kg physostigmine (Group 2b) and 10 min later were put into the locomotion chamber and the locomotor activity was measured for 20 min. Then all rats received 5 mg/kg morphine and the locomotor activity was measured 2 more hours. 3.3. Data analyses The data were expressed as means ± SEM with the exception of Fig. 2, which only showed the mean for clarity purpose). Unless otherwise noted, statistical analyses were performed by one-way ANOVA and a post hoc LSD test. For data in Fig. 2, a two-factor repeated measures ANOVA was used with groups as a betweengroup factor and days as a within-group factor. p-values less than 0.05 were considered statistically significant. 4. Results On day 1, when rats were first exposed to the test environment, the baseline locomotor activity was not significantly different among the groups. As shown in Fig. 1, one-way ANOVA revealed no significant differences among groups during the first 20 min on the first day of conditioning training [top panel, Fig. 1, F(4, 64) = 0.771, p > 0.05] and 0.1 mg/kg physostigmine did not significantly change the locomotor activity in the first 20 min (top panel, Fig. 1, compare Group 3 with Group 1). These results indicated that 0.1 mg/kg physostigmine did not significantly change the locomotor activity and this dose of physostigmine was chosen for the following experiments. Other doses of physostigmine were tested in pilot studies and it was found that 0.2 mg/kg or higher doses of physostigmine alone significantly decreased the locomotor activity whereas doses
Table 1 Group assignment, timeline and treatment. Group
Treatment Day 1–Day10
Day 18
Group 1
Sal (20 min) + Sal (2 h) (n = 10)
No treatment, only test (20 min)
Day 25 Sal (20 min) + 5 Mor (2 h)
Group 2
Sal (20 min) + 5 Mor (2 h) (n = 24)
No treatment, only test (20 min), then assigned into 2 groups based on test results
Group 2a: Sal (20 min) + 5 Mor (2 h) (n = 12)
Group 3
0.1 Physo (20 min) + 5 Mor (2 h)
No treatment, only test (20 min)
Sal (20 min) + 5 Mor (2 h)
Group 4
Sal (20 min) + Sal (2 h) (Mor injection in home cage) (n = 12)
No treatment, only test (20 min)
Sal (20 min) + 5 Mor (2 h)
Group 5
Sal (20 min) + 0.1Physo (2 h) (n = 11)
No treatment, only test (20 min)
Sal (20 min) + 5 Mor (2 h)
Group 2b: 0.1 Physo (20 min) + 5 Mor (2 h) (n = 12)
Abbreviations Sal, saline; Mor, morphine hydrochloride; Physo, physostigmine.
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smaller than 0.1 mg/kg did not markedly change the locomotion in rats (data not shown). However, one-way ANOVA revealed that there were significant differences among groups during the 2 h after receiving the second injection on the first day of conditioning training [bottom panel, Fig. 1, F(4, 64) = 10.527, p < 0.001]. Post hoc comparisons demonstrated that no significant differences between Group 2 and Group 3 (p > 0.05) but these two groups displayed higher locomotor activity compared with all other groups (p < 0.001) suggesting that acute treatment with 5 mg/kg morphine significantly increased locomotor activity and 0.1 mg/kg physostigmine did not significantly change this effect. As shown in Fig. 2, during the 10 conditioning sessions, the locomotion level of all groups of rats progressively decreased, demonstrating that habituation occurred, although rats who received morphine during conditioning sessions tended to show higher locomotion than other groups during the latter conditioning sessions (top panel, Fig. 2). However, there was no statistically significant difference among groups. During the daily 2 h sensitization tests, rats who received morphine during the session (Groups 2 and 3) showed significantly higher locomotion level than other groups (p < 0.001 for every session). Moreover, although there were fluctuations across days, the locomotion level was significantly higher 6 days after daily morphine treatment compared with day 1, demonstrating the progressive development of morphine locomotor sensitization (p < 0.05; bottom panel, Fig. 2). However, there
Fig. 2. Locomotor activity of different groups of rats on the 10 conditioning days. Top panel: locomotor activity collected during the 20 min period; bottom panel: locomotor activity collected during the 2 h period. * p < 0.05 compared with day 1. Note that the statistical significance was not shown on the bottom panel for the between group comparisons because Groups 2 and 3 were both significantly higher than Groups 1, 4 and 5 throughout the 10 days. The data were only presented as mean and the SEMs were not shown for clarity purpose. See Fig. 1 for other details.
was no systematic change of the locomotion level across days in Groups 1, 4 and 5. On day 18, rats that received morphine injection in the locomotion chamber during conditioning period showed significantly higher locomotor activity than those that received saline and those that received morphine injection in the home cage (Fig. 3, com-
Fig. 1. Locomotor activity of different groups of rats on the first conditioning day. Top panel: locomotor activity collected during the 20 min period; bottom panel: locomotor activity collected during the 2 h period. Data are presented as mean ± SEM. Ordinate: distance (cm) of rats travelled during the 20 min (top panel) or 2 h (bottom panel) period. *** p < 0.001 compared with Group 1.
Fig. 3. Locomotor activity of different groups of rats on day 18. ** p < 0.01, *** p < 0.001 compared with Group 1 and Group 4; # p < 0.05 compared with Group 2a or 2b. See Fig. 1 for other details.
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pare Groups 2a and 2b with Group 1 or Group 4, p < 0.01 for both group comparison). More importantly, the locomotor activity level of Group 2 was much higher on day 18 than on day 1, suggesting that morphine treatment during conditioning did not simply block habituation, as seen with Group 1 (compare Group 1 in Fig. 3 with top panel of Fig. 1), but did induce a conditioned hyperactivity. Rats that received physostigmine before the session showed significantly higher locomotor activity than those that received saline (Fig. 3, compare Group 3 with Group 2a or 2b, p < 0.05 for both group comparison). Rats that received saline before the session but received saline or physostigmine during the session (immediately before the 2 h test) showed similar level of locomotor activity on day 18 (Fig. 3, compare Group 5 with Group 1, p > 0.05), suggesting that chronic treatment with physostigmine alone did not elicit conditioned hyperactivity. On day 25, when the first 20 min of locomotor activity was analyzed, the increased locomotor activity remained higher for rats that received morphine during conditioning training than those that received saline and those that received morphine in the home cage (top panel, Fig. 4, compare Group 2a with Group 1 or Group 4, p < 0.05). However, 0.1 mg/kg physostigmine given 10 min before session markedly attenuated the increment (top panel, Fig. 4, compare Group 2a with Group 2b, p < 0.05). Furthermore, rats that received physostigmine before session during the conditioning training did not show significantly higher locomotor activity than those that received saline (top panel, Fig. 4, compare Group 3 with Group 2a, p > 0.05) on day 25.
Fig. 4. Locomotor activity of different groups of rats on day 25. Top panel: locomotor activity collected during the 20 min period; bottom panel: locomotor activity collected during the 2 h period after morphine injection. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with Group 1, Group 2b and Group 4; # p < 0.05 compared with Group 2a. See Fig. 1 for other details.
On day 25, when the 2 h locomotor activity data were analyzed wherein all rats received 5 mg/kg morphine, morphine elicited significantly higher locomotor activity in rats that received morphine during training sessions than those that received saline (bottom panel, Fig. 4, compare Group 2a with Group 1). Rats that received 0.1 mg/kg physostigmine 10 min before session on day 25 appeared to show higher locomotor activity than those that received saline but did not reach statistical significance (bottom panel, Fig. 4, compare Group 2b with Group 1). Rats that received 0.1 mg/kg physostigmine 10 min before the daily conditioning training sessions showed significantly higher locomotor activity than those that received saline (bottom panel, Fig. 4, compare Group 3 with Group 1). Rats that received morphine administration in the home cage during the training sessions showed similar locomotor activity with those that received saline (bottom panel, Fig. 4, compare Group 4 with Group 1). Chronic administration of 0.1 mg/kg physostigmine did not change acute morphine-induced hyperactivity (bottom panel, Fig. 4, compare Group 5 with Group 1).
5. Discussion The primary findings of this study were that delay conditioning effectively established conditioned hyperactivity to morphine in rats and that the AchE inhibitor physostigmine inhibited the expression but enhanced the development of conditioned hyperactivity and locomotor sensitization. A number of studies have used locomotion as a behavioral endpoint to investigate conditioned effects of a variety of drugs and the mechanisms [9,14,27,28,30]. However, most studies use withinsubject design so that the same group of animals is exposed to the environment with or without CS when paired with drug or saline, respectively, and the training of the pair with drug and saline is typically conducted in different sessions. Moreover, these studies almost exclusively use simultaneous conditioning technique (i.e., the CS and US are presented and terminated at the same time) [34]. However, given the simultaneous nature of the temporal arrangement of CS and US in simultaneous conditioning, it is impossible to study conditioned hyperactivity and locomotor sensitization at the same time. Thus, when the conditioned effects and locomotor sensitization are studied in the same animals, tests of the two phases are typically temporally separated and additional training is required [9,14]. Carey and co-workers developed a procedure that exposes animals in two distinct environments, one associated with and the other without US, proposing to use it for studying the conditioned and drug induced effect separately [12]. In this study, a modified procedure from Carey and co-workers and a delay conditioning technique was used so that for the first phase of the session (20 min) animals were exposed to the CS (odor stimulus) without US (morphine) pairing. The second phase (2 h) of the session was similar to other studies wherein animals were exposed to the pair of CS and the US. Ten sessions of conditioning training under this arrangement was effective to elicit conditioned hyperactivity as demonstrated on day 18 and day 25. When rats were only exposed with the CS for the first 20 min of the session on day 18 and day 25, rats that previously received morphine showed much higher locomotor activity than those that previously received saline. More importantly, although during the 10 conditioning training sessions rats who received morphine in the session appeared to decrease less than other groups, suggesting the possibility that morphine might simply have blocked the habituation to the environment, the data on day 18 did not support this hypothesis, because those rats showed substantially higher locomotion level on day 18 than on day 1. Furthermore, this increased activity did not appear to be due to morphine injection per se (i.e. 10
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days of morphine treatment might simply increased the baseline locomotor activity) but due to the pairing of morphine and the environment (conditioning) since the locomotor activity of other rats that received the same dose of morphine in the home cage without the pairing history was not significantly different from those that received saline during conditioning training. When administered with morphine on day 25, rats that received morphine during conditioning sessions showed more robust locomotor activity than those that received saline, implying that this behavior was “sensitized” by previous drug treatment history. One may posit that the observed higher locomotion in rats that received morphine than those received saline during conditioning may be due, at least partially, to the conditioning process so the “locomotor sensitization” cannot be teased apart with “conditioned hyperactivity”. This may be true since it has long been recognized that the ability of drugs to induce or express sensitization is powerfully modulated by learning and the circumstances where drug is given [7]; and conditioned effects may play a role in most behavioral sensitization studies since when animals are tested (i.e. receiving a challenge dose or drug) in an environment different from the one in which they received during prior drug treatments, sensitization is often not expressed [2,36] and this phenomenon is also observed in the current study (i.e. rats that received morphine injection in their home cage did not demonstrate locomotor sensitization). It is believed that dopamine plays a crucial part in the locomotor sensitization elicited by morphine [18,23]. Abundant evidence demonstrates robust interaction between dopaminergic and cholinergic systems. Dopamine neurons express muscarinic and nicotinic cholinergic receptors and stimulation of these receptors elicits dopamine cell firing, mesolimbic dopamine release and increases operant behavior [19,20]. Acute morphine treatment significantly increases Ach level in the brain [35]. Moreover, physostigmine, which increases brain Ach level by inhibiting the degradation, enhances the rewarding effects of morphine in a rat conditioned place preference paradigm [31], increases the antinociceptive effects of morphine [29] but inhibits conditioned cue-induced reinstatement in a heroin self administration procedure in rats [37]. Thus, the impact of physostigmine on the behavioral effects of morphine appears to be inconsistent and may be procedure dependent. In the current study, physostigmine enhanced the development of conditioned hyperactivity and locomotor sensitization, but attenuated the expression of conditioned hyperactivity and locomotor sensitization. The attenuation of conditioned hyperactivity on day 25 did not appear to be due to the locomotor suppressing effects of acute physostigmine administration, because as shown in Fig. 1, this dose of physostigmine did not suppress locomotor activity when given acutely. Another interesting finding is that physostigmine did not appear to directly facilitate conditioning process but rather enhance the conditioning effects indirectly. During the 10 conditioning training sessions, rats who received physostigmine daily also showed habituation to the conditioned environment and decreased locomotion, and the locomotion level was substantially lower than that on day 18 when rats were actually received saline injection. Thus far, the neurobiological mechanism for this indirect facilitative effect of physostigmine is not known. Although it is unclear why physostigmine shows differential effects on the two phases, three possibilities worth mention. First, the increment of development could be that physostigmine increases morphine-induced dopamine release and neuroplasticity, thus indirectly increasing the psychomotor stimulating effects of morphine. This interaction could be apparent only after repeated rather than acute treatment since converging evidence suggests that the development and expression of behavioral sensitization have different neuroplastic mechanisms [24]. Second, physostigmine could increase the development phase via facilitating the
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conditioning learning process. Physostigmine not only improves impaired learning and memory [5,11,22], but also increase contextual learning [13]. Lastly, that physostigmine injection as a novel interoceptive stimulus interferes with the expression of the established conditioned stimuli might contribute to the attenuation of the expression of conditioned hyperactivity and sensitization. In conclusion, the current study used delay conditioning technique to develop a procedure that can be used to study conditioned and drug-elicited hyperactivity and sensitization within the same session in the same animals. Moreover, the finding that physostigmine enhanced the development but attenuated the expression of conditioned hyperactivity and locomotor sensitization to morphine suggests that cholinergic systems play different role in the two phases. Further studies are warranted to use this procedure and selective cholinergic receptor ligands to better understand the involvement of cholinergic system in behavioral sensitization to repeated morphine treatment.
Acknowledgements This study was supported by a grant (KM 200710028021) from the Science and Technology Promotion program of Beijing Municipal Commission of Education, China. The authors acknowledged the invaluable comments from three anonymous reviewers, whose input greatly improved this manuscript.
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Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Research report
Effects of physostigmine on the conditioned hyperactivity and locomotor sensitization to morphine in rats Xinwang Li a,∗ , Jun-Xu Li b,∗ , Xiaolin Zhu a , Ruisi Cui a , Jingjing Jiao a a b
Department of Psychology, Capital Normal University, Beijing, China Department of Pharmacology, University of Texas Health Science Center at San Antonio, San Antonio TX, USA
a r t i c l e
i n f o
Article history: Received 4 June 2009 Received in revised form 7 September 2009 Accepted 10 September 2009 Available online 15 September 2009 Keywords: Morphine Physostigmine Conditioned hyperactivity Locomotor sensitization Rats
a b s t r a c t Repetitive exposure to opioids elicits sensitization to its psychomotor stimulating effect and environmental stimuli contribute to this effect. This study first developed a procedure that simultaneously measures conditioned hyperactivity and locomotor sensitization, and then investigated the effects of physostigmine on the development and expression of conditioned hyperactivity and locomotor sensitization in rats. Five groups of rats (10–12 rats each) were conditioned with a conditioned stimulus (CS) for 20 min and then drug or saline paired with CS for 2 h daily for 10 days. Rats were tested 20 min on day 18. On day 25, rats were tested 20 min and subsequently 2 h (immediately after morphine injection). Although the 20 min locomotion was not different among the rats on day 1, rats that received 5 mg/kg morphine during conditioning showed higher locomotion than those received saline or 5 mg/kg morphine in the home cage on day 18 and day 25. Rats received 0.1 mg/kg physostigmine and 5 mg/kg morphine during conditioning showed higher locomotion than those received 5 mg/kg morphine on day 18. On day 25, 0.1 mg/kg physostigmine attenuated the conditioned hyperactivity and expression of morphine locomotor sensitization. In contrast, rats received 0.1 mg/kg physostigmine and 5 mg/kg morphine during conditioning showed higher locomotion during 2 h test period than those received 5 mg/kg morphine. In conclusion, this study established a procedure that simultaneously study conditioned hyperactivity and locomotor sensitization. Physostigmine attenuates the expressions but enhances the development of conditioned hyperactivity and sensitization and the possible mechanisms are discussed. Published by Elsevier B.V.
1. Introduction Environmental stimuli can become strongly associated with and predictive of the drug effects after they are repeatedly paired with many drugs of abuse and thereby acquire the ability to evoke conditioned responses by classical conditioning process. Conditioning effects are one of the factors believed to contribute to the relapse of drug use even after long periods of drug abstinence [4,8,15]. For instance, cocaine abusers exhibit strong craving when presented with stimuli previously paired during cocaine use [16]. Abundant evidence suggests that sensitized psychomotor stimulating effects induced by repeated intermittent administration of some drugs (e.g. opioids and psychostimulants) may be an indication of sensitization to the underlying reward/incentive systems [1,25,26,32,33]. Drug induced locomotor sensitization can also be strongly modulated by the environmental context. The magnitude of amphetamine and cocaine-induced locomotor sensitization is greatly attenuated if drugs are injected (intravenous) via a remotely
∗ Corresponding authors. E-mail addresses: [email protected] (X. Li), [email protected] (J.-X. Li). 0166-4328/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.bbr.2009.09.016
controlled infusion apparatus, a manipulation that presumably markedly decreases the salience of environmental cues usually associated with drug administration such as handling of the animals and giving the injections by the experimenter [6,7,10,17]. In most of the earlier conditioned hyperactivity studies, a simultaneous conditioning technique is used so that immediately after the subjects receive the drug or vehicle injection, they are exposed to the environmental context (with or without the presence of conditioned stimulus, respectively) to initiate the conditioning process. Damianopoulos and Carey developed a new procedure which measures the conditioned and unconditioned drug effects in two distinct environments sequentially within the same session, raising the possibility of studying conditioned and unconditioned drug effects separately [12]. In the current study, a modified method from Damianopoulos and Carey was used in which a delay conditioning process was used to develop conditioned hyperactivity so that animals were exposed to conditioned stimulus (CS, environmental context) before the unconditioned stimulus (US; drug, morphine in this case) was given. This temporal arrangement allows simultaneous measurement of conditioned hyperactivity (before drug is administered) and the expression of locomotor sensitization (after
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drug is administered). Furthermore, since conditioned hyperactivity was successfully established with this procedure, the effect of acetylcholinesterase (AChE) inhibitor physostigmine on conditioned hyperactivity and locomotor sensitization to morphine was studied. Previous studies have reported that cholinergic systems are involved in the conditioned cue-induced behavioral effects. For example, the muscarinic receptor antagonist scopolamine blocks conditioned locomotor sensitization to cocaine [21]. Physostigmine inhibits the conditioned cue-induced reinstatement of morphine self-administration behavior [37].
injection and immediately put back into the chamber and the locomotor activity was measured for the following 2 h. Rats in the unpaired group (Group 4) were given morphine injection and rats in other groups saline injection 4 h after the daily session in the home cages. Then, they remained in their home cage without any drug treatment during days 11–17 and days 19–24. The dose of morphine used in this study (5 mg/kg) was chosen based upon previous report [3] and our pilot studies (data not shown) showing that this dose of morphine produced the most robust hyperactivity and locomotor sensitization.
2. Materials and methods
3.2. Conditioned hyperactivity and locomotor sensitization test
2.1. Subjects Adult male Wister rats (220–250 g, Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were housed in standard lab Plexiglas cages (45 × 30 × 25 cm, 3 rats/cage) in a weather-controlled ventilated colony room on a 12/12 h light/dark cycle (experiments were conducted during the light period) with free access to water and food in the home cage. All animals were maintained and experiments were conducted in accordance with the Institutional Animal Care and Use Committee, Capital Normal University and with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources on Life Sciences, National Research Council, National Academy of Sciences, Washington DC, USA). 2.2. Drugs Morphine hydrochloride (Shenyang First Pharmaceutical Factory, Shenyang, China) and physostigmine (Sigma–Aldrich, St. Louis, MO) were dissolved with saline and injected intraperitoneally (i.p.) at a volume of 1 ml/kg. 2.3. Apparatus Locomotor activity was measured by an automated video tracking system with four customer made activity chambers. The chambers were made of black Perspex plastics (40 × 40 × 50 cm, length × width × height). A video camera was mounted at the top of the chambers, which was connected to a PC to record the locomotion of rats. The video documents (stored in the computer) were analyzed by the LA analysis software (Institute of Psychology, Chinese Academy of Sciences, Beijing, China). The locomotor activity was expressed as the total distance travelled for a predetermined period of minutes. 1.5 ml of 50% acetic acid dropped on absorbent cotton served as conditioned stimulus and replaced with fresh made daily immediately before the session started. The cotton was held in a porous metal container and put in one top corner of the chamber out of the reach of the animals.
3. Behavioral procedure 3.1. Acquisition of conditioned hyperactivity Rats were randomly assigned into one of the five groups as indicated in Table 1. Conditioning training was conducted in 10 consecutive days (days 1–10). During each training session, all rats were given the first injection (i.p.) and 10 min later put into the locomotion chamber for 20 min (paired with conditioned stimulus as previously described); 20 min later rats were given the second
On day 18, all rats were put into the locomotion chamber without any treatment and the locomotor activity was measured for 20 min. Based upon the test results, rats in Group 2 (those received morphine during training sessions) were re-assigned into two groups so that the locomotor activity between the two groups were not significantly different. On day 25, rats received either saline (Groups 1, 2a, 3, 4 and 5) or 0.1 mg/kg physostigmine (Group 2b) and 10 min later were put into the locomotion chamber and the locomotor activity was measured for 20 min. Then all rats received 5 mg/kg morphine and the locomotor activity was measured 2 more hours. 3.3. Data analyses The data were expressed as means ± SEM with the exception of Fig. 2, which only showed the mean for clarity purpose). Unless otherwise noted, statistical analyses were performed by one-way ANOVA and a post hoc LSD test. For data in Fig. 2, a two-factor repeated measures ANOVA was used with groups as a betweengroup factor and days as a within-group factor. p-values less than 0.05 were considered statistically significant. 4. Results On day 1, when rats were first exposed to the test environment, the baseline locomotor activity was not significantly different among the groups. As shown in Fig. 1, one-way ANOVA revealed no significant differences among groups during the first 20 min on the first day of conditioning training [top panel, Fig. 1, F(4, 64) = 0.771, p > 0.05] and 0.1 mg/kg physostigmine did not significantly change the locomotor activity in the first 20 min (top panel, Fig. 1, compare Group 3 with Group 1). These results indicated that 0.1 mg/kg physostigmine did not significantly change the locomotor activity and this dose of physostigmine was chosen for the following experiments. Other doses of physostigmine were tested in pilot studies and it was found that 0.2 mg/kg or higher doses of physostigmine alone significantly decreased the locomotor activity whereas doses
Table 1 Group assignment, timeline and treatment. Group
Treatment Day 1–Day10
Day 18
Group 1
Sal (20 min) + Sal (2 h) (n = 10)
No treatment, only test (20 min)
Day 25 Sal (20 min) + 5 Mor (2 h)
Group 2
Sal (20 min) + 5 Mor (2 h) (n = 24)
No treatment, only test (20 min), then assigned into 2 groups based on test results
Group 2a: Sal (20 min) + 5 Mor (2 h) (n = 12)
Group 3
0.1 Physo (20 min) + 5 Mor (2 h)
No treatment, only test (20 min)
Sal (20 min) + 5 Mor (2 h)
Group 4
Sal (20 min) + Sal (2 h) (Mor injection in home cage) (n = 12)
No treatment, only test (20 min)
Sal (20 min) + 5 Mor (2 h)
Group 5
Sal (20 min) + 0.1Physo (2 h) (n = 11)
No treatment, only test (20 min)
Sal (20 min) + 5 Mor (2 h)
Group 2b: 0.1 Physo (20 min) + 5 Mor (2 h) (n = 12)
Abbreviations Sal, saline; Mor, morphine hydrochloride; Physo, physostigmine.
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smaller than 0.1 mg/kg did not markedly change the locomotion in rats (data not shown). However, one-way ANOVA revealed that there were significant differences among groups during the 2 h after receiving the second injection on the first day of conditioning training [bottom panel, Fig. 1, F(4, 64) = 10.527, p < 0.001]. Post hoc comparisons demonstrated that no significant differences between Group 2 and Group 3 (p > 0.05) but these two groups displayed higher locomotor activity compared with all other groups (p < 0.001) suggesting that acute treatment with 5 mg/kg morphine significantly increased locomotor activity and 0.1 mg/kg physostigmine did not significantly change this effect. As shown in Fig. 2, during the 10 conditioning sessions, the locomotion level of all groups of rats progressively decreased, demonstrating that habituation occurred, although rats who received morphine during conditioning sessions tended to show higher locomotion than other groups during the latter conditioning sessions (top panel, Fig. 2). However, there was no statistically significant difference among groups. During the daily 2 h sensitization tests, rats who received morphine during the session (Groups 2 and 3) showed significantly higher locomotion level than other groups (p < 0.001 for every session). Moreover, although there were fluctuations across days, the locomotion level was significantly higher 6 days after daily morphine treatment compared with day 1, demonstrating the progressive development of morphine locomotor sensitization (p < 0.05; bottom panel, Fig. 2). However, there
Fig. 2. Locomotor activity of different groups of rats on the 10 conditioning days. Top panel: locomotor activity collected during the 20 min period; bottom panel: locomotor activity collected during the 2 h period. * p < 0.05 compared with day 1. Note that the statistical significance was not shown on the bottom panel for the between group comparisons because Groups 2 and 3 were both significantly higher than Groups 1, 4 and 5 throughout the 10 days. The data were only presented as mean and the SEMs were not shown for clarity purpose. See Fig. 1 for other details.
was no systematic change of the locomotion level across days in Groups 1, 4 and 5. On day 18, rats that received morphine injection in the locomotion chamber during conditioning period showed significantly higher locomotor activity than those that received saline and those that received morphine injection in the home cage (Fig. 3, com-
Fig. 1. Locomotor activity of different groups of rats on the first conditioning day. Top panel: locomotor activity collected during the 20 min period; bottom panel: locomotor activity collected during the 2 h period. Data are presented as mean ± SEM. Ordinate: distance (cm) of rats travelled during the 20 min (top panel) or 2 h (bottom panel) period. *** p < 0.001 compared with Group 1.
Fig. 3. Locomotor activity of different groups of rats on day 18. ** p < 0.01, *** p < 0.001 compared with Group 1 and Group 4; # p < 0.05 compared with Group 2a or 2b. See Fig. 1 for other details.
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pare Groups 2a and 2b with Group 1 or Group 4, p < 0.01 for both group comparison). More importantly, the locomotor activity level of Group 2 was much higher on day 18 than on day 1, suggesting that morphine treatment during conditioning did not simply block habituation, as seen with Group 1 (compare Group 1 in Fig. 3 with top panel of Fig. 1), but did induce a conditioned hyperactivity. Rats that received physostigmine before the session showed significantly higher locomotor activity than those that received saline (Fig. 3, compare Group 3 with Group 2a or 2b, p < 0.05 for both group comparison). Rats that received saline before the session but received saline or physostigmine during the session (immediately before the 2 h test) showed similar level of locomotor activity on day 18 (Fig. 3, compare Group 5 with Group 1, p > 0.05), suggesting that chronic treatment with physostigmine alone did not elicit conditioned hyperactivity. On day 25, when the first 20 min of locomotor activity was analyzed, the increased locomotor activity remained higher for rats that received morphine during conditioning training than those that received saline and those that received morphine in the home cage (top panel, Fig. 4, compare Group 2a with Group 1 or Group 4, p < 0.05). However, 0.1 mg/kg physostigmine given 10 min before session markedly attenuated the increment (top panel, Fig. 4, compare Group 2a with Group 2b, p < 0.05). Furthermore, rats that received physostigmine before session during the conditioning training did not show significantly higher locomotor activity than those that received saline (top panel, Fig. 4, compare Group 3 with Group 2a, p > 0.05) on day 25.
Fig. 4. Locomotor activity of different groups of rats on day 25. Top panel: locomotor activity collected during the 20 min period; bottom panel: locomotor activity collected during the 2 h period after morphine injection. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with Group 1, Group 2b and Group 4; # p < 0.05 compared with Group 2a. See Fig. 1 for other details.
On day 25, when the 2 h locomotor activity data were analyzed wherein all rats received 5 mg/kg morphine, morphine elicited significantly higher locomotor activity in rats that received morphine during training sessions than those that received saline (bottom panel, Fig. 4, compare Group 2a with Group 1). Rats that received 0.1 mg/kg physostigmine 10 min before session on day 25 appeared to show higher locomotor activity than those that received saline but did not reach statistical significance (bottom panel, Fig. 4, compare Group 2b with Group 1). Rats that received 0.1 mg/kg physostigmine 10 min before the daily conditioning training sessions showed significantly higher locomotor activity than those that received saline (bottom panel, Fig. 4, compare Group 3 with Group 1). Rats that received morphine administration in the home cage during the training sessions showed similar locomotor activity with those that received saline (bottom panel, Fig. 4, compare Group 4 with Group 1). Chronic administration of 0.1 mg/kg physostigmine did not change acute morphine-induced hyperactivity (bottom panel, Fig. 4, compare Group 5 with Group 1).
5. Discussion The primary findings of this study were that delay conditioning effectively established conditioned hyperactivity to morphine in rats and that the AchE inhibitor physostigmine inhibited the expression but enhanced the development of conditioned hyperactivity and locomotor sensitization. A number of studies have used locomotion as a behavioral endpoint to investigate conditioned effects of a variety of drugs and the mechanisms [9,14,27,28,30]. However, most studies use withinsubject design so that the same group of animals is exposed to the environment with or without CS when paired with drug or saline, respectively, and the training of the pair with drug and saline is typically conducted in different sessions. Moreover, these studies almost exclusively use simultaneous conditioning technique (i.e., the CS and US are presented and terminated at the same time) [34]. However, given the simultaneous nature of the temporal arrangement of CS and US in simultaneous conditioning, it is impossible to study conditioned hyperactivity and locomotor sensitization at the same time. Thus, when the conditioned effects and locomotor sensitization are studied in the same animals, tests of the two phases are typically temporally separated and additional training is required [9,14]. Carey and co-workers developed a procedure that exposes animals in two distinct environments, one associated with and the other without US, proposing to use it for studying the conditioned and drug induced effect separately [12]. In this study, a modified procedure from Carey and co-workers and a delay conditioning technique was used so that for the first phase of the session (20 min) animals were exposed to the CS (odor stimulus) without US (morphine) pairing. The second phase (2 h) of the session was similar to other studies wherein animals were exposed to the pair of CS and the US. Ten sessions of conditioning training under this arrangement was effective to elicit conditioned hyperactivity as demonstrated on day 18 and day 25. When rats were only exposed with the CS for the first 20 min of the session on day 18 and day 25, rats that previously received morphine showed much higher locomotor activity than those that previously received saline. More importantly, although during the 10 conditioning training sessions rats who received morphine in the session appeared to decrease less than other groups, suggesting the possibility that morphine might simply have blocked the habituation to the environment, the data on day 18 did not support this hypothesis, because those rats showed substantially higher locomotion level on day 18 than on day 1. Furthermore, this increased activity did not appear to be due to morphine injection per se (i.e. 10
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days of morphine treatment might simply increased the baseline locomotor activity) but due to the pairing of morphine and the environment (conditioning) since the locomotor activity of other rats that received the same dose of morphine in the home cage without the pairing history was not significantly different from those that received saline during conditioning training. When administered with morphine on day 25, rats that received morphine during conditioning sessions showed more robust locomotor activity than those that received saline, implying that this behavior was “sensitized” by previous drug treatment history. One may posit that the observed higher locomotion in rats that received morphine than those received saline during conditioning may be due, at least partially, to the conditioning process so the “locomotor sensitization” cannot be teased apart with “conditioned hyperactivity”. This may be true since it has long been recognized that the ability of drugs to induce or express sensitization is powerfully modulated by learning and the circumstances where drug is given [7]; and conditioned effects may play a role in most behavioral sensitization studies since when animals are tested (i.e. receiving a challenge dose or drug) in an environment different from the one in which they received during prior drug treatments, sensitization is often not expressed [2,36] and this phenomenon is also observed in the current study (i.e. rats that received morphine injection in their home cage did not demonstrate locomotor sensitization). It is believed that dopamine plays a crucial part in the locomotor sensitization elicited by morphine [18,23]. Abundant evidence demonstrates robust interaction between dopaminergic and cholinergic systems. Dopamine neurons express muscarinic and nicotinic cholinergic receptors and stimulation of these receptors elicits dopamine cell firing, mesolimbic dopamine release and increases operant behavior [19,20]. Acute morphine treatment significantly increases Ach level in the brain [35]. Moreover, physostigmine, which increases brain Ach level by inhibiting the degradation, enhances the rewarding effects of morphine in a rat conditioned place preference paradigm [31], increases the antinociceptive effects of morphine [29] but inhibits conditioned cue-induced reinstatement in a heroin self administration procedure in rats [37]. Thus, the impact of physostigmine on the behavioral effects of morphine appears to be inconsistent and may be procedure dependent. In the current study, physostigmine enhanced the development of conditioned hyperactivity and locomotor sensitization, but attenuated the expression of conditioned hyperactivity and locomotor sensitization. The attenuation of conditioned hyperactivity on day 25 did not appear to be due to the locomotor suppressing effects of acute physostigmine administration, because as shown in Fig. 1, this dose of physostigmine did not suppress locomotor activity when given acutely. Another interesting finding is that physostigmine did not appear to directly facilitate conditioning process but rather enhance the conditioning effects indirectly. During the 10 conditioning training sessions, rats who received physostigmine daily also showed habituation to the conditioned environment and decreased locomotion, and the locomotion level was substantially lower than that on day 18 when rats were actually received saline injection. Thus far, the neurobiological mechanism for this indirect facilitative effect of physostigmine is not known. Although it is unclear why physostigmine shows differential effects on the two phases, three possibilities worth mention. First, the increment of development could be that physostigmine increases morphine-induced dopamine release and neuroplasticity, thus indirectly increasing the psychomotor stimulating effects of morphine. This interaction could be apparent only after repeated rather than acute treatment since converging evidence suggests that the development and expression of behavioral sensitization have different neuroplastic mechanisms [24]. Second, physostigmine could increase the development phase via facilitating the
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conditioning learning process. Physostigmine not only improves impaired learning and memory [5,11,22], but also increase contextual learning [13]. Lastly, that physostigmine injection as a novel interoceptive stimulus interferes with the expression of the established conditioned stimuli might contribute to the attenuation of the expression of conditioned hyperactivity and sensitization. In conclusion, the current study used delay conditioning technique to develop a procedure that can be used to study conditioned and drug-elicited hyperactivity and sensitization within the same session in the same animals. Moreover, the finding that physostigmine enhanced the development but attenuated the expression of conditioned hyperactivity and locomotor sensitization to morphine suggests that cholinergic systems play different role in the two phases. Further studies are warranted to use this procedure and selective cholinergic receptor ligands to better understand the involvement of cholinergic system in behavioral sensitization to repeated morphine treatment.
Acknowledgements This study was supported by a grant (KM 200710028021) from the Science and Technology Promotion program of Beijing Municipal Commission of Education, China. The authors acknowledged the invaluable comments from three anonymous reviewers, whose input greatly improved this manuscript.
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