This chapter has described methods for the assessment of cannabinoid-induced behavioral changes. In view of the widespread presence of cannabinoid receptors and their ligands (see for example refs. 84a, 84b), it is hardly surprising that the cannabinoids are involved in virtually every mode of behavior. Moreover, it has recently become evident that the endocannabinoids play a pivotal role as the retrograde messengers in glutamatergic and GABA-ergic synapses in a number of brain structures (84b-84d). With new technologies, in this era of the genome, the creation and availability of transgenics and knockout and knockin mice will allow the determination of the mechanisms associated with specific behaviors induced by cannabinoids. As the identity of previously unknown components of the EPCS become available, these behavioral tests and new methods to tease out the contribution of cannabinoid genetics in mediating the behavioral effects of cannabinoids will be needed. Although several decades of irrational prejudice may have hampered basic and clinical research on the therapeutic potential of cannabinoids, new knowledge of the evolutionary conservation of the components of the EPCS and high copy number of cannabinoid receptors in the brain underscores the numerous behaviors induced by cannabinoids. Undoubtedly future research will uncover the role of cannabinoids in diverse pathways associated with apoptosis, neurogenesis, epigenesis, neuroinflammation, and neuroprotection and how these processes affect behavior and how such behaviors can be evaluated. Thus, cannabinoid research has experienced major breakthroughs over the last 25 yr since the discovery of the CBi and CB2 receptors for the plant-derived A9-THC and their endogenous ligands. No specific behavioral methods have been devised to capture cannabinoid-induced behaviors except for the "tetrad," which describes a relatively selective "cannabinoid profile." This is not surprising, however, in view of the ubiquitous presence of CB1 receptors and their ligands in the central nervous system and the rich interactions between the EPCS system and other neurotransmitter/modulator systems such as the opiates and dopamine. We therefore envision progress in behavioral cannabi-noid research as going in tandem with the progress made in neurobehavioral techniques designed to measure the endocannabinoid as interacting with a variety of other neurochemical systems. When the smoke clears, the influence of the EPCS in feeding behaviors for example, may be exploited in the treatment of obesity and other disturbances regulated by endocannabinoid signaling.
1. Compton et al. (6) have shown a high degree of correlation between performance in the mouse tetrad and cannabinoid receptor (CB1) binding in rat brain membranes. These authors also described a high degree of correlation between CB1 receptor binding and cannabinoid potencies in the rat drug discrimination test, which is taken to be predictive of cannabimimetic effects in humans (19). Because they also found a significant correlation between CB1 receptor binding and psychoactive effect in humans, they suggested that the mouse model might be used to investigate potential abuse potential of cannabinoids (6). The endogenous ligands for the cannabinoid receptors discovered thus far (the endocannabinoids) include the anandamides (20,21), 2-arachidonoyl glycerol (2-AG) (22), noladin (arachi-donyl glyceryl ether) (23), the antagonist/partial agonist virodhamine (24), and N-arachidonoyl-dopamine (NADA) (25). Thus far, the prototypical anandamide, arachidonoyl ethanolamide (20), and 2-AG are the most thoroughly studied endo-cannabinoids. Although the overall pharmacological activity is similar to the psy-choactive plant constituent A8-THC (11,26), there are clear differences between anandamide and plant-derived and synthetic cannabinoids (26-30). Behaviorally, it was clear from the initial description of anandamide's effects in the tetrad that it has partial effects for some of its components (hypothermia and analgesia) (see Fig. 1) (11,26). Moreover, when different routes of anandamide administration were compared, a complex pattern of full and partial activities was observed (29). Furthermore, A9-THC but not anandamide produced conditioned place avoidance (31). Additional behavioral differences include the effects of very low doses of anandamides (0.0001-0.01 mg/kg).
2. These biphasic effects of anandamide suggest the possibility that the physiological functions of the endocannabinoids may be opposite those depicted by many of the experimental, pharmacological observations performed with high doses of cannabinoids. A speculative explanation was offered as a linkage of the CB1 receptor to Gs proteins at different levels of CB1 receptor activation. Thus, when agonist concentrations are high (which is usually the case in pharmacological experiments or after intake of high amounts of cannabis), only Gi protein activation is observable, resulting overall in behavioral depression. In contrast, when agonist concentrations are low, activation of Gs proteins become apparent (27), in an analogous fashion to what has been found for opiate receptors (33). Direct evidence for such Gs linkage to the CB1 receptor has been demonstrated, at least in neurons from the corpus striatum and in CB1-transfected cells (34).
3. These observations are compatible with the assessments (see above) that cannabinoids, although they are not associated with one unique type of behavior, produce a characteristic pattern of effects on the central nervous system. Moreover, both discriminative stimulus effects of various cannabinoids and marijuana-intoxication symptoms in humans were found to highly correlate with CB1 receptor binding. Consequently it was suggested that the rat model of drug discrimination may be used to predict cannabinoid intoxication in humans (19).
4. ICSS was discovered in the 1950s, and a wide variety of ICSS techniques have been developed since then. In 1988 Gardner's group showed that A9-THC enhances brain stimulation reward in rats (40,41).
5. Although early attempts to induce THC self-administration failed (40), more recent studies, using more potent and specific CB1 agonists and the CB1 antagonist SR141716, have unequivocally shown that cannabinoids are readily self-adminis tered and that SR141716 will block this behavior in mice (45,46), rats (47 ) and monkeys (48). The latter study in monkeys was performed using A9-THC, which makes these data even more pertinent to the human condition.
6. Conditioned place preference is a behavioral model currently used to measure the rewarding or aversive properties induced by the administration of various drugs. The administration of cannabinoid agonists is known to produce variable rewarding and aversive responses in the place-conditioning paradigm, while the endogenous cannabinoid anandamide does not induce any behavioral response. Conditioned place aversion induced by cannabinoid agonists is abolished by the co-administration of SR141716. SR141716 administration can produce some intrinsic motivational responses in this conditioned paradigm. Thus, some specific doses of SR141716 are able to produce conditioned place preference in rats and conditioned place aversion in mice.
7. The plus-maze test was developed in 1984 (50) as a behavioral assay for testing antianxiety effects in benzodiazepine drugs. Indeed, this test has been associated most specifically with this class of drugs. However, it can also be used to assess the level of anxiety influenced by other agents such as antidepressants (3) or to assess levels of anxiety as affected by prenatal stress, (51), isolation rearing (93), or to determine differences in basal levels of anxiety in rat strains (94). A9-THC (52), HU210 (53), and anandamide (54) all have been shown to cause anxiety-like behavior in the plus-maze, depending on the dose administered. The involvement of the EPCS in anxiety and the response to stress has been documented thoroughly. For example, intracerebroventricular injections of anandamide or A9-THC activated the hypothalamic-pituitary-adrenal stress axis in rats (55), while the potent cannabinoid receptor agonist HU210 produced a stress-like behavioral response in the defensive withdrawal test, which was accompanied by a rise in plasma corti-costerone (56).
8. In rodents USVs are interpreted as distress calls and are therefore used to assess anxiety (57). Benzodiazepines and antidepressants effectively inhibit stress-induced USVs in rodents (57). We have observed in mice that although swim stress increases USVs in normal C57BL/6, the response in CB1-/- knockout mice is blunted (92).
9. The Morris water-maze task with the hidden platform is widely accepted for examining spatial learning and memory, and a number of studies have shown that some phytocannabinoids and cannabinoids can impair memory (89) or enhance memory (89) function in rodent models. Genetic and pharmacological manipulation have been used to evaluate the CB1 mutant mice in the Morris water maze and the antagonist SR141716 in a delayed radial maze task (90,91).
10. Cannabis is well known to stimulate appetite (61-64). There is evidence that suggests that cannabinoid-induced enhancement of appetite is selective for snack foods (62,65). Thus, in human studies the use of higher doses of cannabinoids as well as different routes of administration, including the rectal (66) or the sublingual (67) route, should be further investigated.
11. Studies in laboratory animals have confirmed observations in humans and unequivocally shown that cannabinoid CB1 receptors mediate cannabinoid-induced increase in food ingestion (72), especially of highly palatable foods (73). Thus, both exogenous cannabinoids (A9-THC) and the endocannabinoid anandamide-induced enhancement of appetite were reversed by the specific CB1 antagonist SR141716A (68,72). SR141716A injected by itself reduced appetite and body weight. Whether palatability is required for the antagonist's anorectic effect is controversial (71,74,75). In a chronic study in mice, very low doses of anandamide (0.001 mg/kg) were effective in enhancing food intake (76), in accordance with a stimulatory effect of very low doses of anandamide in a series of cannabimimetic assays (32).
12. These findings were replicated in three strains of mice (Table 1). Interestingly, the strain greatly influences the size of the antagonist-induced effect. A large majority of Sabra and C57BL/6 mice perish as a result of SR141716 application on d 1 of life, while more than 75% of ICR pups survive. It is not clear whether the strain differences are related to CB1 receptor distributions and/or to strain differences in general development. Interestingly, uninjected CB1-/- pups only start milk suckling on d 2-3 (78,87) (Table 1) and display an overall greater mortality and depressed growth curve compared to wild-type C57BL/6 pups (Table 1). The possibility of a compensatory role of a putative "CB3" receptor has been discussed (78). Taken together, the poor development of CB1 receptor knockout mice supports a role for differential CB1 receptor development in explaining the strain differences in the effects of SR141716 on pup suckling and development.
13. Ultrasonic vocalizations in rodent pups are increased after maternal separation, suggesting that the neonates emit USVs to attract retrieval and maternal care. USVs emitted by pups have also been anthropomorphically described as "separation cries," homologous to those of human infants (79,80). However, more severe stress suppresses USVs (80). Cannabinoid receptors have been linked to stress (55,81) and to USV emission (82). Thus, SR141716 injected into 12-d-old rat pups increased ultrasonic vocalizations without effect on body temperature (82). We have observed that ultrasonic vocalizations in pups treated with SR141716 on the first day of life are inhibited (a preliminary report of these data was presented in ref. 83). Untreated CB1-/- knockout pups had lower USVs compared to wild-type mouse pups (92). Thus, in agreement with Morgan et al. (80), there seems to be a bimodal USV response to stress: moderate stress increases USV rates, severe stress suppresses the emission of USVs. Perhaps when the organism is faced with a life and death danger, it is more beneficial for survival not to be heard at all.
14. Behavioral effects of cannabinoids are usually assessed in adult rodents. However, in vivo assessment of cannabinoid effects in developing animals is important in order to assess the correlation between CB1 receptor development with age and their functional (in vivo) expression (84). In addition, in vivo developmental studies help evaluate therapeutic and/or side effects of putative cannabinoid-based (medicinal) drugs in children (85,86). The tetrad developed for assessment of cannabinoid-like activity in adult mice is not suitable for assaying cannabinoid effects in immature rodents. However, several aspects of such function do develop sufficiently that valid assessments of cannabinoid-induced behaviors may be meas ured before puberty (86). Thus, although newborn rodents do not rear and cannot balance themselves on an elevated ring, we found that significant locomotion is expressed by d 6 and gradually increases over the next 2 wk, while analgesia on a hot plate is present from d 18. Moreover, using an external thermocouple probe (TES 1307 k/J thermometer, Bioseb, France), axilliary temperature can be reliably measured in newborns (87). Thus, we have found that suppression of locomotion and the analgesic response to anandamide and A9-THC is not present at least until 3 wk of age. Because these parameters are indices of central cannabinoid activity, this finding has important implications for the therapeutic use of cannabinoids in children (85,88).
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