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International Journal of Neuropsychopharmacology (2013), 16, 2273–2284. CINP 2013 Biphasic effects of Δ9-tetrahydrocannabinol onbrain stimulation reward and motor activity Vicky Katsidoni, Andreas Kastellakis and George Panagis Laboratory of Behavioral Neuroscience, Department of Psychology, School of Social Sciences, University of Crete, Crete, Greece Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive ingredient of marijuana, has led to equivocalresults when tested with the intracranial self-stimulation (ICSS) procedure or the open-ﬁeld test formotor activity, two behavioural models for evaluating the reward-facilitating and locomotor stimulatingeffects of drugs of abuse, respectively. Therefore, in the present study, the effects of high and low dosesof Δ9-THC were compared in the ICSS procedure and the open-ﬁeld test. Moreover, the involvement ofCB1 receptors in tentative Δ9-THC-induced effects was investigated by pre-treating the animals with theCB1 receptor antagonist SR141716A (rimonabant). The results obtained show that low doses of Δ9-THCinduce opposite effects from high doses of Δ9-THC. Speciﬁcally, 0.1 mg/kg Δ9-THC decreased ICSSthresholds and produced hyperactivity, whereas 1 mg/kg increased ICSS thresholds and produced hypo-activity. Both effects were reversed by pre-treatment with SR141716A, indicating the involvement of CB1receptors on these actions. Altogether, our results indicate that Δ9-THC can produce acute activating effectsin locomotion that coincide with its reward-facilitating effects in the ICSS paradigm. The present ﬁndingsprovide further support that Δ9-THC induces behaviours typical of abuse and substantiate the notion thatmarijuana resembles other drugs of abuse.
Received 19 April 2013; Reviewed 8 May 2013; Revised 21 May 2013; Accepted 21 May 2013;First published online 5 July 2013 Key words: Δ9-tetrahydrocannabinol, cannabinoids, intracranial self-stimulation, locomotion, SR141716A.
important issue addressed by researchers and mayhave implications for public policy and health.
Cannabis products are the most widely abused drugs Despite the clear evidence for rewarding effects of among illicit compounds currently available for rec- cannabis preparations and Δ9-THC in humans (Hart reational use (Ramo et al., ). The main psycho- et al., ), rewarding effects of Δ9-THC or other active ingredient of these preparations is the alkaloidΔ9 cannabinoids in animal models of drug abuse and -tetrahydrocannabinol (Δ9-THC), which is well dependence have been controversial and appear to be known to produce feelings of euphoria and relaxation very much dependent on the experimental conditions in human users (Haney et al., ; Justinova et al., (Parolaro et al., ; Solinas et al., Panagis These feelings may play a central role in the et al., ). Additionally, the lack of a pronounced reinforcement of repeated use and abuse of cannabis withdrawal syndrome following abrupt cessation of preparations and, in some cases, the development of cannabis has reinforced the notion that cannabinoids dependence. However, the popularity of marijuana are only mildly addictive (Smith, However, it and other cannabis products may also stem from the is very likely that such ﬁndings reﬂect the chemistry fact that they are more socially tolerated and often and pharmacokinetics of these compounds (i.e. high perceived as harmless and non-habit-forming drugs.
lipophilicity and long duration of action) rather Thus, assessment of cannabis abuse potential is an than their low abuse potential (Maldonado, ).
Moreover, studies examining the rewarding effects ofΔ9-THC may be further confounded by the drug's Address for correspondence: Dr G. Panagis, School of Social Sciences, tendency to produce aversive feelings at higher Department of Psychology, Laboratory of Behavioral Neuroscience, doses (Lepore et al., Sanudo-Pena et al., University of Crete, 74100 Rethymno, Crete, Greece.
Hutcheson et al., ; Mallet and Beninger, Tel.: +30 28310 77544 Fax: +30 28310 77578 Email: [email protected] Robinson et al., ). Indeed, several studies using 2274 V. Katsidoni et al.
lower doses have found that Δ9-THC is self- In the present study, effects of high and low doses administered by experimental animals (Justinova of Δ9-THC were compared in the ICSS procedure and et al., ; Braida et al., Le Foll et al., the open-ﬁeld test for motor activity. Finally, the and produces conditioned place preference (Lepore involvement of CB1 receptors in tentative Δ9-THC- et al., ; Valjent and Maldonado, ; Braida induced effects was investigated by pre-treating the et al., Interestingly, microinjections of Δ9-THC animals with the CB1 receptor antagonist SR141716A into the posterior ventral tegmental area and the posterior shell of the nucleus accumbens also produceconditioned place preference (Zangen et al., An animal behavioural model commonly used to determine the effects of psychotropic drugs in reward Animals and surgery processes is the intracranial self-stimulation (ICSS) Male Sprague–Dawley rats (n = 46) weighing 300–350 g paradigm (Wise, Carlezon and Chartoff, were used. Animals were housed two or three per cage Vlachou and Markou, Most drugs of abuse are under a 12 h light–12 h dark cycle (lights on 08:00 able to lower ICSS thresholds, an effect that supports hours) with free access to food and water. Surgery the notion that they activate the same substrate with for self-stimulation followed previously described electrical stimulation in a synergistic manner (Wise, procedures (Katsidoni et al., Experiments were ). Only a few studies have been conducted on conducted in accordance with the National Institutes the effects of Δ9-THC in the ICSS paradigm. According of Health Guide for the Care and Use of Laboratory to Gardner and colleagues, 1 and 1.5 mg/kg Δ9-THC Animals. All efforts were made to minimize animal decrease the ICSS threshold in Lewis rats but not in suffering and to reduce the number of animals used.
Fisher 344 rats, whereas in Sprague–Dawley rats theeffect was only marginal (Gardner et al., ; Lepore Apparatus and procedures for ICSS et al., ). In contrast, studies from our group failedto show an enhancement of brain stimulation reward After 1 wk recovery, the rats were tested for self- with Δ9-THC in the dose range from 0.5 to 2 mg/kg stimulation in an operant chamber made of trans- (Vlachou et al., ; Fokos and Panagis, parent Plexiglas (25-cm wide, 25-cm deep and 30-cm Thus, a major problem regarding the robustness of high). A stainless steel rodent lever protruded 2 cm Δ9-THC in ICSS is the lack of agreement between from the left wall at a height of 4 cm from the ﬂoor.
different studies. This is quite contrary to the con- Each bar-press triggered a constant current stimulator sistency of the ﬁndings with other abused substances, (Med Associates, USA) that delivered a 0.4-s train such as psychostimulants, nicotine and opioids (Wise, of rectangular cathodal pulses of constant duration (0.1 ms) and intensity (250 mA) and variable frequency The measurement of motor activity is another be- (15–100 Hz, i.e. 6–40 number of pulses/0.4 s). The pulse havioural test commonly used in the study of drugs frequency, i.e. the number of pulses within a train, was of abuse (Geyer and Paulus, Most drugs of progressively increased up to 40 per stimulation train abuse tend to stimulate motor activity, an effect that until the animal showed vigorous self-stimulation.
may become sensitized and contribute to drug addic- During the acquisition phase, the animals were trained tion (Wise, ). Several studies have shown that to self-stimulate for at least 3 consecutive days (1 h cannabinoids, including Δ9-THC, suppress ambulation daily), using stimulation parameters that maintained and rearing in higher doses (Jarbe et al., Wiley near maximal bar-pressing rates. After self-stimulation and Martin, Smirnov and Kiyatkin, was acquired and stabilized for a given pulse fre- Polissidis et al., whereas in lower doses, quency, rats were trained to self-stimulate using four increases in such measurements have been reported alternating series of ascending and descending pulse (Sanudo-Pena et al., ; Polissidis et al., frequencies. The pulse frequency was varied by steps The degree by which locomotor stimulating effects of approximately 0.1 log units. Each frequency was of Δ9-THC can be detected relates to experimental tested within trials of 60 s in duration, followed by design (i.e. strain of animal, rat phenotype, habituation an extinction period of 30 s. For each trial, there was and reaction to novelty, inﬂuence of the light/dark an initial ‘priming' phase, during which the animals cycle) and dose regimen. Thus, although a biphasic received three trains of stimulation at the frequency stimulatory/inhibitory effect of Δ9-THC on motor that was available for the speciﬁc trial. A rate– activity has been reported, the literature is lacking a frequency determination session lasted approximately detailed time resolution of this effect.
45 min. One rate–frequency curve was established Actions of Δ9-THC on brain reward and locomotion 2275 daily, for 10–14 d, depending on the period when the was registered. The behavioural testing was performed self-stimulation indices (i.e. curve shift and threshold between 08:00 and 16:00 hours. One day before the measure) were stable. The stimulation parameters, drug testing, each rat was gently handled for 15 min ICSS sessions and data collection were controlled by and habituated to the experimental room and the open-ﬁeld for 1 h.
Drug testing began for each animal when the rate–frequency function was stable for at least 3 con- Data analysis and statistics for locomotor activity secutive days.
In the motor activity experiments, total ambulatory Data analysis and statistics for ICSS studies distance and rearing counts over the 3 h observation The analysis was performed on two aspects of data period were evaluated. In the ﬁrst experiment, the sig- obtained from the rate–frequency curve: the ICSS niﬁcance of the drug effect and time was statistically threshold and the maximum rate of responding or evaluated initially using two-way (ANOVA) with asymptote, as it has previously been described repeated measures. In the second experiment, three- (Katsidoni et al., The post-treatment threshold way ANOVA with repeated measures was performed and asymptote values were expressed as percentage to evaluate statistically signiﬁcant interactions and of pre-treatment values. In the ﬁrst experiment, the main effects. In both cases, when the interaction the signiﬁcance of the drug effect and time was in the two-way ANOVA was signiﬁcant we considered statistically evaluated initially using two-way analysis Bonferroni's inequality approach or paired sample of variance (ANOVA) with repeated measures. In the t test, dependent on the case.
second experiment, three-way ANOVA with repeatedmeasures was performed to evaluate statistically Drugs and drug administration signiﬁcant interactions and the main effects of the Δ9-THC (Sigma-Aldrich, USA) and SR141716A two drugs and the time. In both cases, when the inter- (Cayman, USA) were dissolved into a vehicle solution action in the two-way ANOVA was signiﬁcant, we that consisted of 5% dimethylsulfoxide, 5% cremophor considered Bonferroni's inequality approach or paired EL and 90% of 0.9% NaCl and injected i.p. at a volume sample t test – dependent on the case – and the of 3 ml/kg body weight. Control animals received, analysis of simple effects was tested: i.p., the corresponding vehicle solutions in thesame injection volume. Based on the reports of behav- p = The sum of p′s for the main plus interaction effects Number of simple effects ioural studies that effects of Δ9-THC and other canna-binoid agonists follow a biphasic mode, a low dose The signiﬁcance of simple effects was evaluated of 0.1 mg/kg and a high dose of 1 mg/kg were selected using repeated measures ANOVA followed, whenever based on their possible stimulatory and inhibitory appropriate, by correlated t test using Bonferroni's adjustment for multiple comparisons. The level of sig- All animals took part in only one experiment and niﬁcance was set at 0.05. Statistical analyses were con- received all drug treatments of the experiment. The ducted using the Statistical Package for the Social order of testing for various doses of each drug treat- Sciences v.19.0 (SPSS, USA).
ment was counterbalanced according to a Latin-squaredesign and a 3-d period was allowed between Assessment of locomotor activity Spontaneous motor activity was measured using anactivity recording system (Model 7445; Ugo Basile, Behavioural studies: ICSS studies Italy). Each system consists of an animal cage and an Expt 1: effects of systematically administrated Δ9-THC on electronic unit incorporating a counter and a printer.
brain stimulation reward The rectangular animal cage (56 × 56 × 30 cm) has trans-parent sides and lid to allow observation. The cage In the ﬁrst experiment, a group of animals (n = 8) was ﬂoor has horizontal and vertical infrared sensors.
used to evaluate the effects of the acute administration The counter sums up the photocell disruptions and of Δ9-THC (0, 0.1 and 1 mg/kg i.p.) on brain stimu- a printer displays the results at preset intervals. In lation reward. Each drug or vehicle self-stimulation our studies, a summation of photocell disruptions test consisted of a pre-drug and two post-drug rate– of ambulatory distance and rearing, for each 10-min frequency function determinations (for 45 min each).
interval period, during the 3 h observation period The injection of the compound was given immediately 2276 V. Katsidoni et al.
following the pre-drug rate–frequency function deter- of the ﬁrst post-injection showed a statistical signiﬁcant mination. The ﬁrst session began 20 min post-injection, effect of Δ9-THC (F2,14 = 282.828, p < 0.001). Paired- while the second session started 80 min after Δ9-THC sample t test using Bonferroni's adjustment for mul- tiple comparisons revealed that Δ9-THC at thedose of 0.1 mg/kg signiﬁcantly decreased (p < 0.001), Expt 2: effects of the CB1 receptor antagonist SR141716A while at the dose of 1 mg/kg signiﬁcantly increased on Δ9-THC-induced changes on brain stimulation reward (p < 0.001) the ICSS threshold. Repeated measureson the simple effect of the second post-injection The purpose of the second experiment was to examine showed a statistically signiﬁcant effect of Δ9-THC whether the CB1 receptor antagonist SR141716A could reverse the reward-facilitating effect of Δ9-THC. Thus, 2,14 = 471.248, p < 0.001). Paired-sample t test using Bonferroni's adjustment for multiple comparisons a group of animals (n = 8) received SR141716A (0.02 revealed that Δ9-THC at the dose of 0.1 mg/kg mg/kg i.p.) or vehicle followed 5 min later by Δ9-THC signiﬁcantly decreased (p < 0.001) while at the dose of (0.1 mg/kg i.p.) or vehicle. Each drug or vehicle 1 mg/kg signiﬁcantly increased (p < 0.001) the ICSS self-stimulation test consisted of a pre-drug and two threshold. Repeated measures on the simple effect of post-drug rate–frequency function determinations the dose of 0.1 mg/kg Δ9-THC showed a statistically (for 45 min each). The ﬁrst session began 20 min post- signiﬁcant effect of time (F injection, while the second session started 80 min 1,7 = 11.436, p < 0.05). Paired sample t test using Bonferroni's adjustment for mul- after Δ9-THC injection.
tiple comparisons revealed that the dose 0.1 mg/kgsigniﬁcantly decreased the ICSS threshold during Behavioural studies: locomotor activity studies the ﬁrst post-injection (p < 0.001), while the decrease Expt 1: effects of systematically administrated Δ9-THC on was more pronounced during the second post-injection locomotor activity (p < 0.05). Repeated measures on the simple effect ofthe doses of 0 and 1 mg/kg Δ9-THC did not reveal A group of animals (n = 10) was used to examine the any statistically signiﬁcant effect of time.
effects of Δ9-THC (0, 0.1 and 1 mg/kg i.p.) on locomotor Two-way ANOVA with repeated measures showed activity. Animals were injected with Δ9-THC or its no statistically signiﬁcant effect of Δ9-THC (F vehicle and placed immediately in the centre of the 0.429, p > 0.05), time (F activity box. Locomotor activity was recorded for 3 h.
2,14 = 1.772, p > 0.05) or their inter- action (F2,14 = 1.214, p > 0.05) on the asymptotic rate ofresponding.
Expt 2: reversal of Δ9-THC-induced changes in locomotionwith the CB1 receptor antagonist SR141716A Expt 2: effects of the CB Two groups of animals (n = 20) were injected with 1 receptor antagonist SR141716A on Δ9-THC-induced changes on brain stimulation reward SR141716A (0.02 mg/kg) or its vehicle, and 5 minlater the ﬁrst group (n = 10) received 0.1 mg/kg of The changes in ICSS threshold and asymptotic rate of Δ9-THC or its vehicle and the second group (n=10) responding after acute administration of SR141716A received 1 mg/kg of Δ9-THC or its vehicle, and (0, 0.02 mg/kg i.p.) and Δ9-THC (0 and 0.1 mg/kg i.p.) placed immediately in the centre of the activity box.
are presented in d respectively. Three-way Locomotor activity was recorded for 3 h.
ANOVA with repeated measures showed a statistic-ally signiﬁcant SR141716A × Δ9-THC × time interaction(F 1,7 = 49.075, p < 0.01) on the ICSS threshold. Two-way ANOVA with repeated measures for the ﬁrst post- Behavioural studies: ICSS studies injection showed a statistically signiﬁcant SR141716A ×Δ9-THC interaction (F Expt 1: effects of systematically administrated Δ9-THC on p < 0.001). Paired sample t test using Bonferroni's adjustment for mul- brain stimulation reward tiple comparisons revealed that SR141716A blocked The changes in ICSS threshold and asymptotic rate the reward-facilitating effect of Δ9-THC at the dose of responding after acute administration of Δ9-THC of 0.1 mg/kg (p < 0.001). Similarly, two-way ANOVA (0, 0.1 and 1 mg/kg i.p.) are presented in b, with repeated measures for the second post-injection respectively. Two-way ANOVA with repeated mea- showed a statistically signiﬁcant SR141716A × Δ9-THC sures showed a statistically signiﬁcant Δ9-THC × time interaction (F2,14 = 187.699, p < 0.001). Paired sample (F2,14 = 13.279, p < 0.001) on the ICSS t test using Bonferroni's adjustment for multiple threshold. Repeated measures on the simple effect comparisons revealed that SR141716A blocked the Actions of Δ9-THC on brain reward and locomotion 2277 (a) Threshold (b) Asymptote (c) Threshold (d ) Asymptote 0.02 0.02 (mg/kg) Fig. 1. Changes in intracranial self-stimulation (ICSS) threshold (a, c) and asymptotic rate of responding (b, d) expressed aspercentage of pre-drug values, following acute Δ9-tetrahydrocannabinol (Δ9-THC; 0, 0.1 and 1 mg/kg i.p.) administration.
Vertical bars represent the means ± S.E.M. * Signiﬁes an ICSS threshold signiﬁcantly different from the respective control group(vehicle): *** p < 0.001. # Signiﬁes a statistically signiﬁcant effect compared to the ﬁrst post-injection effect of the same dose:# p < 0.05. + Signiﬁes a statistically signiﬁcant effect compared to the SR141716A 0 mg/kg – Δ9-THC 0.1 mg/kg group:+++ p < 0.001. The dose of 0.1 mg/kg decreased, whereas the dose of 1 mg/kg increased ICSS thresholds. The effects ofΔ9-THC on ICSS thresholds remained for 2 h post-injection. SR141716A antagonized the reward-facilitating effect of Δ9-THC.
reward-facilitating effect of Δ9-THC at the dose of 0.1 (F1,7 = 99.311, p < 0.001). Paired sample t test using mg/kg (p < 0.001).
Bonferroni's adjustment for multiple comparisons Three-way ANOVA with repeated measures did not revealed that the dose of 0.1 mg/kg signiﬁcantly reveal any statistically signiﬁcant effect of SR141716A increased the ambulatory distance at 60 min (p < 0.01), (F1,7 = 1.745, p > 0.05), Δ9-THC (F1,7 = 5.623, p > 0.05), 90 min (p < 0.001) and 120 min (p < 0.001). Repeated time (F1,7 = 1.419, p > 0.05) or their interaction (F2,14 = measures on the simple effect of the dose of 1 mg/kg 1.214, p > 0.05) on the asymptotic rate of responding.
of Δ9-THC showed a statistically signiﬁcant effectof time (F1,7 = 85.513, p < 0.001). Paired sample t test Behavioural studies: locomotor activity studies using Bonferroni's adjustment for multiple compari-sons revealed that the dose of 1 mg/kg signiﬁcantly Expt 1: effects of systematically administrated Δ9-THC on decreased the ambulatory distance at 60 min (p < 0.05) locomotor activity and 90 min (p < 0.01).
The changes on ambulatory distance and rearing after Two-way ANOVA with repeated measures showed acute administration of Δ9-THC (0, 0.1 and 1 mg/kg a statistically signiﬁcant Δ9-THC × time interaction i.p.) are presented in b, respectively. Two-way on rearing (F1,6 = 25.397, p < 0.01). Repeated measures ANOVA with repeated measures showed a statistically on the simple effect of the dose of 0.1 mg/kg signiﬁcant Δ9-THC × time interaction (F -THC showed a statistically signiﬁcant effect of p < 0.001) on ambulatory distance. Repeated measures time (F1,7 = 73.134, p < 0.01). Paired sample t test using on the simple effect of the dose 0.1 mg/kg of Δ9-THC Bonferroni's adjustment for multiple comparisons showed a statistically signiﬁcant effect of time revealed that the dose of 0.1 mg/kg signiﬁcantly 2278 V. Katsidoni et al.
(a) Ambulatory distance (b) Rearing 30 min 60 min 90 min 120 min 150 min 180 min 30 min 60 min 90 min 120 min150 min180 min (c) Ambulatory distance (d) Rearing 30 min 60 min 90 min 120 min150 min180 min 30 min 60 min 90 min 120 min 150 min 180 min (e) Ambulatory distance (f ) Rearing 30 min 60 min 90 min 120 min 150 min 180 min 30 min 60 min 90 min 120 min 150 min 180 min Fig. 2. Effects of Δ9-tetrahydrocannabinol (Δ9-THC; 0, 0.1 and 1 mg/kg i.p.) on locomotor activity and effect of SR141716A(0.02 mg/kg) on Δ9-THC 0.1 mg/kg-induced hyperactivity and Δ9-THC 1 mg/kg-induced hypoactivity. Histograms representthe photocell disruptions caused by the animals' ambulatory distance travelled (a, c, e) and rearing (b, d, f) (mean±S.E.M.).
* Signiﬁes a statistically signiﬁcant effect compared to the vehicle (Veh) group: * p < 0.05, ** p < 0.01, *** p < 0.001. + Signiﬁes astatistically signiﬁcant effect compared to the Veh – Δ9-THC 0.1 mg/kg group (c, d) and the Veh – Δ9-THC 1 mg/kg group(e, f): + p < 0.05, ++ p < 0.01, +++ p < 0.001. The dose of 0.1 mg/kg increased, whereas the dose of 1 mg/kg decreased spontaneousmotor activity. These effects were reversed by pre-treatment with SR141716A.
increased rearing at 90 min (p < 0.001). Repeated Expt 2: reversal of Δ9-THC-induced changes in locomotion measures on the simple effect of the dose of 1 mg/kg with the CB1 receptor antagonist SR141716A Δ9-THC showed no statistically signiﬁcant effect oftime. Repeated measures on the simple effect of all The changes on ambulatory distance and rearing after time-points did not reveal any statistically signiﬁcant acute administration of SR141716A (0, 0.02 mg/kg i.p.) effect for the dose of 1 mg/kg Δ9-THC.
and Δ9-THC (0 and 0.1 mg/kg i.p.) are presented in Actions of Δ9-THC on brain reward and locomotion 2279 (p < 0.001) and 120 min (p < 0.001). Two-way ANOVA with repeated measures for the dose of 0 mg/kg 1st post-injection2nd post-injection Δ9-THC did not reveal any statistically signiﬁcant SR141716A × time interaction (F1,7 = 0.297, p > 0.05).
showed a statistically signiﬁcant SR141716A × Δ9-THC Bar pressing/min 20 0.1 mg/kg) × time p < 0.01) on rearing. Two-way ANOVA with repeated 0.8 1.0 1.2 1.4 1.6 1.8 Log N Pulse/train measures for the dose of 0.1 mg/kg Δ9-THC showed astatistically signiﬁcant SR141716A × time interaction SR141716A 0.02-∆9-THC 0.1 p < 0.01). Repeated measures on the 1st post-injection 1st post-injection simple effect of the dose of 0.02 mg/kg SR141716A 2nd post-injection 2nd post-injection showed a statistically signiﬁcant effect of time (F1,7 = 71.003, p < 0.01). Paired sample t test using Bonferroni's adjustment for multiple comparisons revealed that the dose 0.02 mg/kg SR141716A blocked the increased rearing induced by 0.1 mg/kg Δ9-THC 0.8 1.0 1.2 1.4 1.6 1.8 0.8 1.0 1.2 1.4 1.6 1.8 at the time-point of 90 min (p < 0.05).
Log N Pulse/train Log N Pulse/train The changes on ambulatory distance and rearing SR141716A 0.02- Veh after acute administration of SR141716A (0, 0.02 mg/kg i.p.) and Δ9-THC (0 and 1 mg/kg i.p.) are presented 1st post-injection 1st post-injection 2nd post-injection 2nd post-injection in e, f, respectively. Three-way ANOVA with repeated measures showed a statistically signiﬁcant SR141716A × Δ9-THC × time interaction (F1,6 = 122.336, p < 0.001) on ambulatory distance. Two-way ANOVA with repeated measures for the dose of 1 mg/kg 0.8 1.0 1.2 1.4 1.6 1.8 0.8 1.0 1.2 1.4 1.6 1.8 Δ9-THC showed a statistically signiﬁcant SR141716A× Log N Pulse/train Log N Pulse/train time interaction (F1,7 = 89.378, p < 0.01). Repeated mea-sures on the simple effect of the dose of 0.02 mg/kg Fig. 3. Rate–frequency functions (rate of lever pressing as a SR141716A showed a statistically signi function of stimulation frequency) taken from representative ﬁcant effect of animals for each drug treatment. Each plot represents data time (F1,7 = 92.587, p < 0.001). Paired sample t test using from a single animal under pre-drug and drug conditions.
Bonferroni's adjustment for multiple comparisons Rate frequency functions were obtained by logarithmically revealed that SR141716 blocked the hypolocomotion decreasing the frequency of the stimulation pulses from a induced by 1 mg/kg Δ9-THC at the time-points of 60 min value that sustained maximal lever pressing to one that (p < 0.05) and 90 min (p < 0.05). Two-way ANOVA with failed to sustain lever pressing. The dose of 0.1 mg/kg repeated measures for the dose of 0 mg/kg Δ9-THC caused parallel leftward shifts in the rate–frequency did not reveal any statistically signiﬁcant SR141716A × function, whereas the dose of 1 mg/kg caused rightward time interaction (F1,7 = 1.897, p > 0.05).
shifts. Veh, Vehicle; Δ9-tetrahydrocannabinol (Δ9-THC).
Three-way ANOVA with repeated measures did d, respectively. Three-way ANOVA with not reveal any statistically signiﬁcant effect of repeated measures showed a statistically signiﬁcant SR141716A (F1,7 = 0.998, p > 0.05), Δ9-THC (F1,7 = 2.545, SR141716A × Δ9-THC × time interaction (F1,6 = 61.287, p > 0.05), time (F1,7 = 2.785, p > 0.05) or their interaction p < 0.001) on ambulatory distance. Two-way ANOVA (F1,6 = 2.001, p > 0.05) on rearing.
with repeated measures for the dose of 0.1 mg/kgΔ9-THC showed a statistically signiﬁcant SR141716A× time interaction (F1,7 = 102.586, p < 0.001). Repeated mea-sures on the simple effect of the dose of 0.02 mg/kg The ﬁrst ﬁnding of the present study is that Δ9-THC SR141716A showed a statistically signiﬁcant effect of is able to induce both rewarding and anhedonic time (F1,7 = 97.138, p < 0.001). Paired sample t test using effects in the ICSS paradigm in Sprague–Dawley rats, Bonferroni's adjustment for multiple comparisons depending on the dose used. Indeed, a low dose of revealed that the dose of 0.02 mg/kg SR141716A 0.1 mg/kg decreased ICSS thresholds and caused par- blocked the hyperlocomotion induced by 0.1 mg/kg allel leftward shifts in the rate–frequency function, Δ9-THC at the time-points of 60 min (p<0.05), 90 min whereas a higher dose of 1 mg/kg increased ICSS 2280 V. Katsidoni et al.
thresholds, producing rightward shifts (see ). In produced a clear anhedonic effect in Sprague–Dawley other words, the low dose of Δ9-THC reduced the rats. Although this ﬁnding is in contradiction with pre- amount of stimulation necessary to sustain responding vious ICSS results reported in the literature, it could be at a given criterion level (Miliaressis et al., due to differences in methodology and the experi- increasing the rewarding efﬁcacy of the stimulation.
mental design, as has been detailed by Vlachou et al.
The observed effects of Δ9-THC on ICSS thresholds (Apart from this slight discordance, our were relatively long-lasting, since they remained for results conﬁrm data obtained from conditioned place 2 h post-injection.
preference studies in rats, in which 0.1 mg/kg Δ9-THC produces preference (Le Foll et al., whereas 0.1 mg/kg Δ9-THC was more pronounced in the second 1 mg/kg produces aversion (Lepore et al., post-injection trial and was nearly equivalent to that Parker and Gillies, Mallet and Beninger, produced by low doses of cocaine (5 mg/kg) (Vlachou It is fundamental to note that other behavioural et al., Katsidoni et al., Moreover, models of drug reward, such as the self-administration this study replicated our previous ﬁndings that and the conditioned place preference paradigm, 1 mg/kg Δ9-THC increases ICSS thresholds. Δ9-THC have provided inconsistent results with Δ9-THC did not signiﬁcantly affect the maximal rates of (Panagis et al., ). Indeed, many of the studies responding at any of the doses tested. There is strong have shown Δ9-THC self-administration in rodents evidence that the presently used ICSS paradigm pro- only under a limited set of conditions, such as vides ICSS threshold estimates that are unaffected by previous drug exposure, food and water deprivation performance effects (Miliaressis and Rompre, (Deneau and Kaymakcalan, Takahashi and This is also evident in the present study, in which the Singer, Tanda et al., However, Justinova increases in ICSS thresholds produced by Δ9-THC and colleagues showed beyond any doubt that low were not accompanied by signiﬁcant changes in doses of Δ9-THC can initiate and sustain high rates of asymptotic rates of responding.
i.v. self-administration in drug-naive squirrel monkeys SR141716A administered in a dose that by itself was (Justinova et al., The self-administration of ineffective in altering ICSS thresholds (0.02 mg/kg) Δ9-THC in the latter study has been attributed to signiﬁcantly antagonized the reward-facilitating effect the rapid rate at which Δ9-THC was infused and the of Δ9-THC, indicating that the rewarding effects range of the doses tested. In a more recent study, the observed herein are speciﬁcally mediated by cannabi- self-administration of Δ9-THC was antagonized by a noid CB1 receptors. Remarkably, the anhedonic effects systemic injection of SR141716A, indicating that it of Δ9-THC are also mediated via CB1 receptor stimu- was mediated by the CB1 receptor (Justinova et al., lation, since they have been blocked by pre-treatment ). Importantly, the pattern of self-administration with SR141716A (0.02 mg/kg) in a previous study with other cannabinoids also reveals a biphasic effect, from our group (Vlachou et al., showing both positive reinforcing and aversive effects, It has been suggested that cannabinoids exhibit depending on the dose used (Martellotta et al., rewarding and hedonic-like properties in experimental Braida et al., ).
animals mostly under particular experimental con- As previously mentioned, several studies have ditions. However, in the present study, a low dose of shown that Δ9-THC and other cannabinoids produce Δ9-THC induced clear and dose-dependent reward- dose-dependent conditioned effects in the conditioned facilitating effects in ICSS, as already reported for place preference paradigm. Thus, at high doses, other recreational and abused drugs (Wise, both Δ9-THC and synthetic cannabinoid agonists pro- This substantiates previous ﬁndings in the literature.
duce conditioned place aversion (Lepore et al., Indeed, according to Gardner and colleagues, Δ9-THC McGregor et al., ; Sanudo-Pena et al., in a dose range of 1 and 1.5 mg/kg lowered ICSS Chaperon et al., ; Hutcheson et al., Mallet thresholds in Lewis rats but not in Fisher 344 rats, and Beninger, ; Cheer et al., ; Valjent and whereas in Sprague–Dawley the effect was very lim- Maldonado, Robinson et al., ), whereas ited and signiﬁcant only when in the analysis of the lower doses have been shown to produce conditioned data the Θ0 criterion and not the M50 criterion for place preference (Lepore et al., Valjent and threshold measure was used (Gardner et al., Maldonado, Braida et al., Both Lepore et al., ). It can thus be suggested that the conditioned place preference observed at low Lewis rats may have a differential sensitivity to doses and the conditioned place aversion observed Δ9-THC compared to Sprague–Dawley and Fisher at high doses have been blocked by the CB1 receptor 344 rats. Nevertheless, in our study 1 mg/kg Δ9-THC Actions of Δ9-THC on brain reward and locomotion 2281 Braida et al., This bidirectional effect in Biphasic dose-dependent effects of Δ9-THC and reward is also reported in the present study. Overall, other cannabinoid agonists on spontaneous motor these ﬁndings indicate that the motivational responses activity have been reported by several studies of Δ9-THC are dose-dependent and directly mediated (Sanudo-Pena et al., Jarbe et al., Wiley by the CB1 receptor.
and Martin, ; Le Foll et al., Smirnov and Interestingly, biphasic effects have been also Kiyatkin, Polissidis et al., Thus, typically described with Δ9-THC and other cannabinoid ago- low doses of cannabinoids increase and higher nists in other emotional-related behaviours. For in- doses decrease motor activity and produce catalepsy, stance, several studies that have been carried out although several pharmacological (i.e. dose and route using various animal models of anxiety in rodents of administration) and non-pharmacological (i.e. rat report that Δ9-THC and other cannabinoid agonists phenotype, habituation and reaction to novelty, inﬂu- display a dose-dependent biphasic proﬁle, with low ence of the light/dark cycle) factors signiﬁcantly inﬂu- doses producing anxiolytic-like responses, whereas ence these effects. In most studies demonstrating higher doses produce anxiogenic-like and aversive effects of Δ9-THC on motor activity, the behavioural responses (Onaivi et al., ; Berrendero and responses were examined for 1 h after drug adminis- Maldonado, Valjent et al., Patel and tration. McMahon and Koek report that the hypo- Hillard, Braida et al., Similarly, in human activity induced by Δ9-THC was maximal from 1 to users, cannabis derivatives can produce opposite 2 h after drug administration and was not detected effects, varying from euphoria (high) to dysphoria after 4 h (McMahon and Koek, In line with this and from relaxation to anxiety or even panic (Hart ﬁnding, we observed that the effects of Δ9-THC on et al., Wachtel et al., spontaneous motor activity were maximal from 1 to Importantly, biphasic effects of cannabinoids have 2 h after drug administration. Moreover, we observed been also reported on other aspects of brain function, that hyperlocomotion induced by the low dose of such as neurotransmitter release (Tzavara et al., Δ9-THC lasts longer than the hypolocomotion induced Moreover, a biphasic effect of Δ9-THC has by the higher dose.
been reported on cerebral metabolism using the The hyperlocomotion and the hypolocomotion 2-deoxyglucose autoradiographic imaging technique, observed after low and high doses of Δ9-THC, with a low dose of 0.2 mg/kg causing an increase of respectively, were reversed by pre-treatment with metabolism in cortical and limbic structures, whereas SR141716A. Importantly, there were no statistically sig- higher doses of 2 mg/kg cause a reduction of meta- niﬁcant changes in motor activity when SR141716A bolism in these regions (Margulies and Hammer, (0.02 mg/kg) was administered alone. Similar results Thus, we hypothesize that the reward- have been obtained in a previous study by our group facilitating effect of the low dose of Δ9-THC could be with the CB1 receptor agonist WIN55,212-2 (Vlachou related to the reported increase in metabolism in limbic et al., . These ﬁndings indicate that Δ9-THC structures (Margulies and Hammer, and the exhibited its actions through CB1 receptor stimulation.
increased dopamine release in the shell of the nucleus The neuroanatomical substrate that mediates the accumbens (Tanda et al., rewarding and psychomotor stimulant effects of can- According to the results of the second study, nabinoids has been identiﬁed by intracranial micro- Δ9-THC inﬂuenced locomotion in a dose-dependent injections in rats. Microinjections of Δ9-THC into the biphasic manner. The low dose of 0.1 mg/kg increased, posterior ventral tegmental area and the posterior whereas the higher dose of 1 mg/kg decreased, spon- shell of the nucleus accumbens increase locomotion taneous motor activity. The hyperactivity produced and produce conditioned place preference, an effect by the low dose of Δ9-THC was accompanied by a pro- that is blocked by SR141716A (Zangen et al., ).
found increase in investigatory responses, as indicated Moreover, Δ9-THC is directly self-administered into by the increased rearing counts. In contrast, the high the posterior ventral tegmental area and the shell of dose of Δ9-THC, although it produced hypoactivity, the nucleus accumbens of rats (Zangen et al., ).
did not inﬂuence investigatory behaviour, since Noteworthy, in the present study, the stimulating the rearing counts were not further suppressed.
electrodes for ICSS have been implanted in the medial Interestingly, dose inducing hyperactivity coincides forebrain bundle, which connects these structures.
with the dose that decreased ICSS thresholds (present In conclusion, we have demonstrated in two study) and produces place preference (Le Foll et al., separate experimental paradigms that low doses of Thus, the relationship between these effects Δ9-THC induce opposite effects from high doses of induced by the low dose of Δ9-THC is explicit.
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Investors‟ decisions – both conscious and subconscious – have an important bearing on their long-term wealth. Through simple illustrations, this month‟s Strategy Talk looks at the Power of Compounding and the benefits of reinvestment. Our regular health update focuses on how to maintain healthy brain functioning with some helpful information and tips. 1.

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