Neurobehavioral and in vitro actions of cannabinoids

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Despite the decades of extensive investigations and recent developments in can-nabinoid research, the identification of specific mechanisms for the actions of cannabinoids have been slow to emerge. We therefore do not attempt to provide a comprehensive account of the numerous in vivo and in vitro effects of canna-binoids but a few examples from our studies and those of others. The discovery of endocannabinoids such as anandamide and 2-arachidonyly glycerol and the widespread localization of Cnrs in the brain and peripheral tissues, suggests that the cannabinoid neurochemical system represents a previously unrecognized ubiquitous network in the nervous system, whose biology and function is unfolding. We have tested the hypothesis that some of the actions of anandamide are independent of a Cnr mechanism (Akinshola et al., 1999b). In the first series of experiments, the effects of anandamide or methanandamide on behavior and CBX Cnr gene expression in three mouse strains was determined. This was accomplished by the use of cannabinoid agonist and antagonist interaction in in vitro and in vivo test systems. The effects of acute administration of anandamide to C57BL/6, DBA/2 and ICR mice were evaluated in motor function and emotionality tests. The C57BL/6 and ICR mouse strains were more sensitive than the DBA/2 strain to the depression of locomotor and stereotyped behavior caused by anandamide. Although anandamide produced catalepsy in all three strains, anandamide induced ataxia in the minus-maze test only in the C57BL/6 animals and at the lowest dose used. In the plus-maze test, anandamide produced a mild averse response, which became intense aversion to the open arms of the plus-maze following repeated daily treatment. Northern analysis data using the CBX cDNA as a probe indicated that there was greater expression of the CBX gene in the whole brain of the ICR mouse than in the brains of the C57BL/6 and DBA/2 strains with or without pretreatment with anandamide. Since the anandamide induced neurobehavioral changes that did not correspond to the CBX Cnr gene expression in the mouse strains, it is unlikely that the CBX Cnr mediates all the cannabimimetic effects of anandamide in the brain. In vitro, we

Ampa Current

Figure 1.8 Current traces of anandamide action on AMPA receptor subunits. Current traces of anandamide inhibition of kainate-activated currents in Xenopus oocytes expressing homomeric and heteromeric AMPA receptor subunit combinations. Each set of traces shows the effect of 100 |J,M anandamide on currents activated by 200 |J,M kainic acid in oocytes expressing the GluR subunits indicated. The bars above current records represent the time duration of kainic acid and/or anandamide application.

Figure 1.8 Current traces of anandamide action on AMPA receptor subunits. Current traces of anandamide inhibition of kainate-activated currents in Xenopus oocytes expressing homomeric and heteromeric AMPA receptor subunit combinations. Each set of traces shows the effect of 100 |J,M anandamide on currents activated by 200 |J,M kainic acid in oocytes expressing the GluR subunits indicated. The bars above current records represent the time duration of kainic acid and/or anandamide application.

used Xenopus laevis oocytes and two-voltage clamp technique (as described above) in combination with differential display polymerase chain reaction to determine whether the differential display genes following treatment with anandamide may be linked to the AMPA glutamate receptor. The differential expression of genes in vivo after the sub-acute administration of anandamide could not be directly linked with AMPA glutamate receptor. For the antagonist studies in vivo, SR141716A, the CBj antagonist, induced anxiolysis that was dependent on the mouse strain used in the anxiety model and blocked the anxiogenic effects of anandamide or methanandamide whereas, SR141716A had no effect on the anandamide inhibition of kainite activated currents in vitro.

We tested another hypothesis that there might exist in the central nervous system a multiplicity of Cnrs. The basis for the hypothesis has been the myriad neuro-behavioral effects produced after smoking marijuana or the administration of cannabinoids to humans and animals. We therefore studied the neurobehavioral specificity of CB1 Cnr gene expression and whether A9-THC induced neurobehavioral changes are attributable to genetic differences (Onaivi et al., 1996). We also examined whether some of these neurobehavioral changes were mediated by specific brain regions in the mouse model. We found that the differential sensitivity following the administration A9-THC to three mouse strains, C57BL/6, DBA/2 and ICR mice indicated that some of the neurobehavioral changes might be attributable to genetic differences. The objective of the study was to determine the extent to which the CB1 Cnr is involved in the behavioral changes following A9-THC administration. This objective was addressed by experiments using the following strategies: DNA-PCR and reverse PCR; systemic administration of A9-THC and intracerebral microinjection of A9-THC. The site specificity of action of A9-THC in the brain was determined using stereotaxic surgical approaches. The intracerebral microinjection of A9-THC into the nucleus accumbens (ACB) was found to induce catalepsy, while injection of A9-THC into the central nucleus of amygdala resulted in the production of an anxiogenic - like response. Although the DNA-PCR data indicated that the CB1 Cnr gene appeared to be identical and intronless in all the three mouse strains, the reverse PCR data showed two additional distinct CB1 mRNAs in the C57BL/6 mouse which also differed in pain sensitivity and rectal temperature changes following the administration of A9-THC (Onaivi et al., 1996). We therefore suggested that the diverse neurobehavioral alterations induced by A9-THC may not be mediated by CB1 Cnrs in the brain and that the CB1 Cnr genes may not be uniform in the mouse strains used. The potential promise of antisense oligonu-cleotides as research tools and therapeutic agents has been the subject of close scrutiny and attention, particularly the application of gene therapy in the clinic. Thus, a number of problems have been identified with their use as research tools. Our use of CB2 antisense oligonucleotide indicated that CB2 may be present in the brain to influence behavior. The ICV administration of the CB2 antisense induced a significant anti-aversive response in the elevated plus-maze test of anxiety. A response similar to that following the administration of the CB1 Cnr antagonist SR141716A. Knowing that this might be a non-specific effect, it is interesting and thought provoking that CB2 Cnr or CB2-like Cnrs might be in the brain. While a number of laboratories have not been able to detect CB2 expression in the brain, a demonstration of CB2 expression in the rat microglial cells (Kearn and Hilliard, 1997) in cerebral granule cells (Skaper et al., 1996) and mast cells (Facci et al., 1995) have been reported. We utilized a CB2 antisense, 5'-TGTCTCCCGGCATC-CCTC-3', CB2 sense was 5'-GAGGGATGCCGGGAGACA-3'. The use of the antagonists that are selective for the CB1 and CB2 Cnrs in these behavioral tests will also contribute to a further understanding of the role of these Cnr subtypes in the behavioral effects of cannabinoids.

Other behavioral effects of cannabinoid agents in animal models has been reviewed by Chaperon and Thiebot (1999) and extensively reviewed in this book. Briefly, cannabinomimetics produce complex behavioral and pharmacological effects that probably involve numerous neuronal substrates. Interactions with acetylcholine, dopamine, serotonin, adrenergic, opiate, glutamatergic and GABAergic systems have been demonstrated in several brain structures. In animals, cannabinoid agonists such as, WIN 55,212-2 and CP 55,940 produce a characteristic combination of four prototypic profiles, sometimes referred to as response to the tetrad tests, including, catalepsy, analgesia, hypoactivity and hypothermia. These effects are reversed by the selective CBX Cnr antagonist, SR141716A, providing evidence for the involvement of CBX Cnr-related mechanisms. Accumulating evidence indicates that endocannabinoids have cannabinoid and non-Cnr mediated effects in these classical cannabinomimetic actions. Cnr-related processes seem also involved in cognition, memory, anxiety, control of appetite, emesis, inflammatory and immune response covered in various sections in this volume. Cannabinoid agonist may induce biphasic effects, for example, hyperactivity at low doses and severe motor deficits at larger doses have been documented.

The conditioned place preference (CPP) paradigm has been used extensively to study brain mechanisms of reward and reinforcement. Marijuana (cannabinoid) interactions with the brain substrates for reward and reinforcement were excellently reviewed by Gardner in this book and also by Tzschentke (1998). Although the paradigm has been criticized because of some inherent methodological problems, it is clear that the place preference conditioning has become a valuable and firmly established and a widely used tool in addiction research, Tzschentke (1998). The rewarding properties of cannabinoids and A9-THC are difficult to demonstrate in rodents using standard place preference procedures (Tzschentke, 1998; Valjent and Maldonado, 2000). Furthermore, only few studies have examined the effects of marijuana and hashish, and inconsistent results have been reported. Sanudo-Pena et al. (1997) found no CPP at a low dose of THC (1.5mg/kg) and a conditioned place aversion (CPA) at a high dose (15 mg/kg) while the Cnr antagonist SR141716A induced a CPP at a low and high dose (0.5 and 5mg/kg). In contrast, Mallet and Beninger (1998) found a significant CPA for the same low doses of THC (1.0 and 1.5 mg/kg), and neither CPP or CPA was found for anandamide. Lepore et al. (1995) however, reported THC-induced CPP for 2 and 4 mg/kg, but not for 1 mg/kg, when animals received one conditioning session per day. But, when conditioning took place only every other day (to allow for a 24 h washout period for THC), the dose of 1 mg/kg THC was sufficient to produce CPP, whereas the higher doses (2 and 4 mg/kg) produced CPA. The synthetic cannabinoid CP 55940 was reported to produce CPA, (McGregor et al., 1996). The synthetic Cnr agonist WIN 55212-2 produced a robust CPA while the CBX antagonist SR141716A produced neither CPP nor CPA (Chaperon et al., 1998). The emerging consensus appear to be that cannabinoid antagonism produces CPP while cannabinoid agonism induces place aversion, (Sanudo-Pena et al., 1997). Taken together the effects of cannabinoids in the CPP paradigm suggest that the effects of cannabinoids and perhaps marijuana may be complex and conclusions about their rewarding and adverse actions deserve further intensive study.

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