The neuroprotective actions of cannabidiol and other cannabinoids were examined in rat cortical neuron cultures exposed to toxic levels of the excitatory neurotransmitter glutamate. Glutamate toxicity was reduced by both cannabidiol, a nonpsychoactive constituent of marijuana, and the psychotropic cannabinoid (؊)⌬ 9 -tetrahydrocannabinol (THC). Cannabinoids protected equally well against neurotoxicity mediated by N-methyl-D-aspartate receptors, 2-amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid receptors, or kainate receptors. N-methyl-D-aspartate receptorinduced toxicity has been shown to be calcium dependent; this study demonstrates that 2-amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid͞kainate receptor-type neurotoxicity is also calcium-dependent, partly mediated by voltage sensitive calcium channels. The neuroprotection observed with cannabidiol and THC was unaffected by cannabinoid receptor antagonist, indicating it to be cannabinoid receptor independent. Previous studies have shown that glutamate toxicity may be prevented by antioxidants. Cannabidiol, THC and several synthetic cannabinoids all were demonstrated to be antioxidants by cyclic voltametry. Cannabidiol and THC also were shown to prevent hydroperoxide-induced oxidative damage as well as or better than other antioxidants in a chemical (Fenton reaction) system and neuronal cultures. Cannabidiol was more protective against glutamate neurotoxicity than either ascorbate or ␣-tocopherol, indicating it to be a potent antioxidant. These data also suggest that the naturally occurring, nonpsychotropic cannabinoid, cannabidiol, may be a potentially useful therapeutic agent for the treatment of oxidative neurological disorders such as cerebral ischemia.Cannabinoid components of marijuana are known to exert behavioral and psychotropic effects but also to possess therapeutic properties including analgesia (1), ocular hypotension (2), and antiemesis (3). This report examines another potential therapeutic role for cannabinoids as neuroprotectants and describes their mechanism of action in rat cortical neuronal cultures.During an ischemic episode, large quantities of the excitatory neurotransmitter glutamate are released. This event causes neuronal death by over-stimulating N-methyl-Daspartate receptors (NMDAr) and 2-amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid (AMPA) and kainatetype receptors and results in metabolic stress and accumulation of toxic levels of intracellular calcium (4). In vitro and in vivo studies (4,5,6) have demonstrated that such neurotoxicity can be reduced by antioxidants or antagonists to NMDAr and AMPA͞kainate receptors. Antioxidants such as ␣-tocopherol (5, 6) are effective neuroprotectants because of their ability to reduce the toxic reactive oxygen species (ROS) formed during ischemic metabolism. Cannabinoids like (Ϫ)⌬ 9

Binge alcohol consumption in the rat induces substantial neurodegeneration in the hippocampus and entorhinal cortex. Oxidative stress and cytotoxic edema have both been shown to be involved in such neurotoxicity, whereas N-methyl-D-aspartate (NMDA) receptor activity has been implicated in alcohol withdrawal and excitoxic injury. Because the nonpsychoactive cannabinoid cannabidiol (CBD) was previously shown in vitro to prevent glutamate toxicity through its ability to reduce oxidative stress, we evaluated CBD as a neuroprotectant in a rat binge ethanol model. When administered concurrently with binge ethanol exposure, CBD protected against hippocampal and entorhinal cortical neurodegeneration in a dose-dependent manner. Similarly, the common antioxidants butylated hydroxytoluene and ␣-tocopherol also afforded significant protection. In contrast, the NMDA receptor antagonists dizocilpine (MK-801) and memantine did not prevent cell death. Of the diuretics tested, furosemide was protective, whereas the other two anion exchanger inhibitors, L-644,711 [(R)-(ϩ)-(5,6-dichloro2,3,9,9a-tetrahydro 3-oxo-9a-propyl-1H-fluoren-7-yl)oxy acetic acid] and bumetanide, were ineffective. In vitro comparison of these diuretics indicated that furosemide is also a potent antioxidant, whereas the nonprotective diuretics are not. The lack of efficacy of L-644,711 and bumetanide suggests that the antioxidant rather than the diuretic properties of furosemide contribute most critically to its efficacy in reversing ethanol-induced neurotoxicity in vitro, in our model. This study provides the first demonstration of CBD as an in vivo neuroprotectant and shows the efficacy of lipophilic antioxidants in preventing binge ethanol-induced brain injury.

Anandamide is an endogenous ligand of cannabinoid receptors that induces pharmacological responses in animals similar to those of cannabinoids such as z9 -tetrahydrocannabinol (THC). Typical pharmacological effects of cannabinoids include disruption of pain, memory formation, and motor coordination, systems that all depend on NMDA receptor mediated neurotransmission. We investigated whether anandamide can influence NMDA receptor activity by examining NMDA-induced calcium flux (L~Ca2~NMDA) in rat brain slices. The presence of anandamide reduced L~.Ca2~NMDA and the inhibition was disrupted by cannabinoid receptor antagonist, pertussis toxin treatment, and agatoxin (a calcium channel inhibitor). Whereas these treatments prevented anandamide inhibiting z~Ca2~NMDA, they also revealed another, underlying mechanism by which anandamide influences Ca2~NMDA. In the presence of cannabinoid receptor antagonist, anandamide potentiated L~Ca2~NMDA in cortical, cerebellar, and hippocampal slices. Anandamide (but not THC) also augmented NMDA-stimulated currents in Xenopus oocytes expressing cloned NMDA receptors, suggesting a capacity to directly modulate NMDA receptor activity. In a similar manner, anandamide enhanced neurotransmission across NMDA receptor-dependent synapses in hippocampus in a manner that was not mimicked by THC and was unaffected by cannabinoid receptor antagonist. These data demonstrate that anandamide can modulate NMDA receptor activity in addition to its role as a cannabinoid receptor ligand. Key Words: Anandamide-Cannabinoid-NMDA-Glutamate-Calcium channel ischemia.

A BSTRACT : Cannabidiol and other cannabinoids were examined as neuroprotectants in rat cortical neuron cultures exposed to toxic levels of the neurotransmitter, glutamate. The psychotropic cannabinoid receptor agonist ⌬ 9tetrahydrocannabinol (THC) and cannabidiol, (a non-psychoactive constituent of marijuana), both reduced NMDA, AMPA and kainate receptor mediated neurotoxicities. Neuroprotection was not affected by cannabinoid receptor antagonist, indicating a (cannabinoid) receptor-independent mechanism of action. Glutamate toxicity can be reduced by antioxidants. Using cyclic voltametry and a fenton reaction based system, it was demonstrated that Cannabidiol, THC and other cannabinoids are potent antioxidants. As evidence that cannabinoids can act as an antioxidants in neuronal cultures, cannabidiol was demonstrated to reduce hydroperoxide toxicity in neurons. In a head to head trial of the abilities of various antioxidants to prevent glutamate toxicity, cannabidiol was superior to both ␣ -tocopherol and ascorbate in protective capacity. Recent preliminary studies in a rat model of focal cerebral ischemia suggest that cannabidiol may be at least as effective in vivo as seen in these in vitro studies.