Paul E. Mallet

Chronic cannabinoid exposure produces lasting memory impairment and increased anxiety in adolescent but not adult rats.

Although many studies have examined the acute behavioural effects of cannabinoids in rodents, few have examined the lasting effects of cannabinoids at different developmental ages. This study compared lasting effects of cannabinoid exposure occurring in adolescence to that occurring in early adulthood. Forty, 30-day old (adolescent) and 18, 56-day old (adult) female albino Wistar rats were injected with vehicle or incremental doses of the cannabinoid receptor agonist (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol (CP 55,940) once per day for 21 consecutive days (150, 200 and 300 μg/kg i.p. for 3, 8 and 10 days, respectively). Following a 21-day drug-free period, working memory was assessed using an object recognition task. Locomotor activity was also measured in the object recognition apparatus via a ceiling-mounted passive infrared sensor. Three days later, anxiety was assessed using a social interaction test. In the object recognition task, significantly poorer working memory was observed in the adolescent but not adult CP 55,940-treated rats. Adolescent, but not adult CP 55,940-treated rats, also exhibited a significant decrease in social interaction with a novel conspecific. These results suggest that chronic exposure to a cannabinoid receptor agonist well after the immediate postnatal period, but before reaching sexual maturity, can lead to increased anxiety and a lasting impairment of working memory.

Adolescent rats find repeated Delta(9)-THC less aversive than adult rats but display greater residual cognitive deficits and changes in hippocampal protein expression following exposure.

The current study examined whether adolescent rats are more vulnerable than adult rats to the lasting adverse effects of cannabinoid exposure on brain and behavior. Male Wistar rats were repeatedly exposed to Δ-9-tetrahydrocannabinol (Δ9-THC, 5 mg/kg i.p.) in a place-conditioning paradigm during either the adolescent (post-natal day 28+) or adult (post-natal day 60+) developmental stages. Adult rats avoided a Δ9-THC-paired environment after either four or eight pairings and this avoidance persisted for at least 16 days following the final Δ9-THC injection. In contrast, adolescent rats showed no significant place aversion. Adult Δ9-THC-treated rats produced more vocalizations than adolescent rats when handled during the intoxicated state, also suggesting greater drug-induced aversion. After a 10–15 day washout, both adult and adolescent Δ9-THC pretreated rats showed decreased social interaction, while only Δ9-THC pretreated adolescent rats showed significantly impaired object recognition memory. Seventeen days following their last Δ9-THC injection, rats were euthanased and hippocampal tissue processed using two-dimensional gel electrophoresis proteomics. There was no evidence of residual Δ9-THC being present in blood at this time. Proteomic analysis uncovered 27 proteins, many involved in regulating oxidative stress/mitochondrial functioning and cytoarchitecture, which were differentially expressed in adolescent Δ9-THC pretreated rats relative to adolescent controls. In adults, only 10 hippocampal proteins were differentially expressed in Δ9-THC compared to vehicle-pretreated controls. Overall these findings suggest that adolescent rats find repeated Δ9-THC exposure less aversive than adults, but that cannabinoid exposure causes greater lasting memory deficits and hippocampal alterations in adolescent than adult rats.

Repeated cannabinoid exposure during perinatal, adolescent or early adult ages produces similar longlasting deficits in object recognition and reduced social interaction in rats.

There is mounting evidence that chronic cannabis use might result in lasting neurobehavioural changes, although it remains unclear whether vulnerability diminishes with age. The current study compared the effects of cannabinoid exposure at three developmental periods on subsequent measures of memory and anxiety. Male rats aged 4 days (perinatal), 30 days (adolescent) and 56 days (young adult) were injected with vehicle or incremental doses of the cannabinoid receptor agonist CP 55940, daily for 21 consecutive days (0.15, 0.20 or 0.30mg/kg for 7 days per dose, respectively). Following a 28-day drug-free period, working memory was assessed in an object recognition task. One week later, social anxiety was assessed in a social interaction test. Two days later, generalized anxiety was assessed in an emergence test. Results revealed that CP 55940 impaired working memory and social interaction similarly at all three ages. CP 55940 had no effects in five of six emergence test measures, but a modest but significant reduction in anxiety was noted in one measure following adolescent exposure. We conclude that chronic cannabinoid exposure leads to long-term memory impairments and increased anxiety, irrespective of the age at which drug exposure occurrs.

Increased anxiety and impaired memory in rats 3 months after administration of 3,4-methylenedioxymethamphetamine ("ecstasy").

Male Wistar rats were administered either (a) a high dose regime of 3,4-methylenedioxymethamphetamine (MDMA) (4 x 5 mg/kg, i.p. over 4 h on each of 2 consecutive days), (b) a moderate dose regime of MDMA (1 x 5 mg/kg on each of 2 consecutive days), (c) D-amphetamine (4 x 1 mg/kg over 4 h on each of 2 days), or (d) vehicle injections. The high MDMA dose regime and the amphetamine treatment both produced acute hyperactivity and hyperthermia. Twelve weeks later, all rats were tested in the drug-free state on a battery of anxiety tests (elevated plus maze, emergence and social interaction tests). A further 2 weeks later they were tested on a novel object recognition memory task. Rats previously given the neurotoxic dose of MDMA showed greater anxiety-like behaviour on all three anxiety tests relative to both controls and D-amphetamine-treated rats. Rats given the moderate MDMA dose regime also showed increased anxiety-like behaviour on all three tests, although to a lesser extent than rats in the high dose group. In the object recognition task, rats given the high MDMA dose regime showed impaired memory relative to all other groups when tested at a 15-min delay but not at a 60-min delay. Rats previously exposed to amphetamine did not differ from saline controls in the anxiety or memory tests. These data suggest that moderate to heavy MDMA exposure over 48 h may lead to increased anxiety and memory impairment 3 months later, possibly through a neurotoxic effect on brain serotonin systems.

Consumption of high carbohydrate, high fat, and normal chow is equally suppressed by a cannabinoid receptor antagonist in non-deprived rats.

Administration of the CB1 receptor antagonist SR 141716 [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide] suppresses intake of highly palatable (high carbohydrate) food. However, the effects of SR 141716 on intake of foods with varying macronutrient compositions, and in non-deprived animals have not been well studied. In the present study, non-deprived rats were injected intraperitoneally with SR 141716 (0.03-3.0 mg/kg) and presented with a high carbohydrate, high fat, or normal chow diet. Food intake and locomotor activity were recorded for 120 min. Results showed that SR 141716 significantly suppressed food intake irrespective of the composition of the test food without affecting locomotor activity. These data suggest that food deprivation or high palatability of the ingesta are not necessary to observe the suppressive effects of SR 141716 on food intake.

The distribution of cannabinoid-induced Fos expression in rat brain: differences between the Lewis and Wistar strain.

Previous studies have suggested that cannabis-like drugs produce mainly aversive and anxiogenic effects in Wistar strain rats, but rewarding effects in Lewis strain rats. In the present study we compared Fos expression, body temperature effects and behavioral effects elicited by the cannabinoid CB(1) receptor agonist CP 55,940 in Lewis and Wistar rats. Both a moderate (50 microg/kg) and a high (250 microg/kg) dose level were used. The 250 microg/kg dose caused locomotor suppression, hypothermia and catalepsy in both strains, but with a significantly greater effect in Wistar rats. The 50 microg/kg dose provoked moderate hypothermia and locomotor suppression but in Wistar rats only. CP 55,940 caused significant Fos immunoreactivity in 24 out of 33 brain regions examined. The most dense expression was seen in the paraventricular nucleus of the hypothalamus, the islands of Calleja, the lateral septum (ventral), the central nucleus of the amygdala, the bed nucleus of the stria terminalis (lateral division) and the ventrolateral periaqueductal gray. Despite having a similar distribution of CP 55,940-induced Fos expression, Lewis rats showed less overall Fos expression than Wistars in nearly every brain region counted. This held equally true for anxiety-related brain structures (e.g. central nucleus of the amygdala, periaqueductal gray and the paraventricular nucleus of the hypothalamus) and reward-related sites (nucleus accumbens and pedunculopontine tegmental nucleus). In a further experiment, Wistar rats and Lewis rats did not differ in the amount of Fos immunoreactivity produced by cocaine (15 mg/kg). These results indicate that Lewis rats are less sensitive to the behavioral, physiological and neural effects of cannabinoids. The exact mechanism underlying this subsensitivity requires further investigation.

A cannabinoid receptor antagonist attenuates conditioned place preference but not behavioural sensitization to morphine

The present study compared the effects of the cannabinoid receptor antagonist SR 141716 on morphine-induced locomotor sensitization (Experiment 1) and conditioned place preference (CPP, Experiment 2) in male albino Wistar rats. In Experiment 1, rats received seven consecutive daily treatments with morphine (10 mg/kg, SC) in combination with either SR 141716 (0, 0.1, 0.5 or 3.0 mg/kg, IP), or naloxone (10 mg/kg, IP). Three days later, all rats were challenged with a lower dose of morphine (5 mg/kg, SC). Rats pre-treated with morphine showed significantly elevated locomotor activity during the challenge session compared to vehicle-pre-treated animals indicating behavioural sensitization. Prior naloxone, but not SR 141716, co-administration with morphine, significantly attenuated the locomotor sensitization observed. In Experiment 2A, SR 141716 (0.1 mg/kg, IP), co-administered during conditioning, significantly attenuated the place preference produced by morphine (4 mg/kg, SC) in a standard unbiased two compartment place conditioning task. In Experiment 2B, the timing of drug administration and drug doses used were altered to be similar to Experiment 1, such that a comparison between the sensitization and CPP paradigms could be made. Thus, rats were conditioned with morphine (10 mg/kg, SC) combined with SR 141716 (0, 0.1, 0.5 or 3.0 mg/kg, IP) and tested for place preference under the influence of morphine (5 mg/kg, SC). SR 141716 attenuated morphine place preference at a dose (3.0 mg/kg) that did not itself affect place conditioning. Morphine also induced locomotor sensitization in the drug-paired compartment in Experiment 2B which was not blocked by any dose of SR 141716. We conclude that CB1 receptor antagonism modulates the rewarding value of opioids, but not the behavioural sensitization induced by chronic opioid administration.

Cannabinoids prevent the acute hyperthermia and partially protect against the 5-HT depleting effects of MDMA (“Ecstasy”) in rats

Cannabinoid-MDMA interactions were examined in male Wistar rats. MDMA (4 x 5 mg/kg or 2 x 10 mg/kg over 4 h on each of 2 days) was administered with or without Delta 9-tetrahydrocannabinol (THC) (4 x 2.5 mg/kg), the synthetic cannabinoid receptor agonist CP 55,940 (2 x 0.1 or 0.2 mg/kg) or the cannabinoid receptor antagonist SR 141716 (2 x 5 mg/kg). Co-administered Delta 9-THC and CP 55,940 but not SR 141716 prevented MDMA-induced hyperthermia, causing a powerful hypothermia. Co-administered Delta 9-THC, CP 55,940 and SR 141716 all tended to decrease MDMA-induced hyperactivity. Co-administered Delta 9-THC provided protection against the long-term increases in anxiety seen in the emergence test, but not the social interaction test, 6 weeks after MDMA treatment. Co-administered Delta 9-THC and CP 55,940, but not SR 141716, partly prevented the long-term 5-HT and 5-HIAA depletion caused by MDMA in various brain regions. SR 141716 administered with CP 55,940 and MDMA prevented the hypothermic response to the CP 55,940/MDMA combination but did not alter the CP 55,940 attenuation of MDMA-induced 5-HT depletion. These results suggest a partial protective effect of co-administered cannabinoid receptor agonists on MDMA-induced 5-HT depletion and long-term anxiety. This action appears to operate independently of cannabinoid CB1 receptors.

Evidence for an interaction between CB1 cannabinoid and oxytocin receptors in food and water intake

Oxytocin and CB(1) cannabinoid receptors independently modulate food intake. Although an interaction between oxytocin and cannabinoid systems has been demonstrated with respect to the cannabinoid withdrawal syndrome, the interaction between these systems in modulating food intake has not yet been examined. The present study had three primary purposes: (1) to determine whether oxytocin and a CB(1) receptor antagonist block food and fluid intake in a supra-additive manner, (2) to determine the relative position of the CB(1) receptors in the chain of control of food intake in relation to the oxytocin system, and (3) to determine whether the increase in fluid intake induced by an oxytocin antagonist is mediated via cannabinoid receptors. Rats were habituated to the test environment and injection procedure, and then received intracerebroventricular (ICV) injections of various combinations of the oxytocin receptor antagonist tocinoic acid, the cannabionid receptor agonist delta(9)-tetrahydrocannabinol (THC), oxytocin, or the cannabinoid receptor antagonist SR 141716. Food and water intake and locomotor activity were then measured for 120 min. When administrated alone, SR 141716 and oxytocin dose-dependently attenuated baseline food intake, while oxytocin but not SR 141716 reduced water intake. Sub-anorectic doses of SR 141716 and oxytocin attenuated baseline feeding beyond what would be expected by the sum of the individual drug effects without affecting baseline water intake. THC stimulated feeding but not water intake. THC-induced feeding was not blocked by oxytocin, however, the oxytocin did attenuate water intake during such feeding. SR 141716 dose-dependently reduced tocinoic-acid-stimulated food intake and partially attenuated water intake. Locomotor activity was not significantly affected by any drug treatments, suggesting that effects on feeding were not due to a non-specific reduction in motivated behaviour. These findings reveal an interaction between cannabinoid and oxytocin systems in food intake. Results further reveal that the oxytocin system effects on water intake are partially mediated via CB(1) receptors, CB(1) receptors are located downstream from oxytocin receptors, and CB(1) receptor signalling is necessary to prevent oxytocin from altering food intake.

The dopamine receptor antagonist SCH 23390 attenuates feeding induced by Δ9-tetrahydrocannabinol

A large body of evidence supports the notion that Delta9-tetrahydrocannabinol (THC) stimulates food intake by its actions on CB1 cannabinoid receptors. Indirect evidence also suggests a role for dopamine (DA) receptors in mediating THC-induced feeding. In the present study, a series of experiments involving intraperitoneal drug administration in rats were conducted to further investigate the interaction between cannabinoid and dopamine receptors in feeding behaviour. Male Wistar rats were habituated to the test environment and injection procedure, and then were injected with vehicle alone, the dopamine D1-like receptor antagonist SCH 23390 (0.005, 0.01, 0.5 or 0.1 mg/kg), THC (0.1, 0.5 or 1.0 mg/kg) or SCH 23390 and THC combined. Food intake and locomotor activity were then measured for 120 min. Results revealed that administration of SCH 23390 dose-dependently decreased food intake while THC dose-dependently increased feeding. Furthermore, SCH 23390 attenuated feeding induced by THC at a dose that did not affect feeding on its own. These findings provide direct evidence for the existence of cannabinoid-dopamine interactions in feeding behaviour and suggest that dopamine D1 signalling is necessary for cannabinoids to stimulate food intake.