Cannabinoid effects are mediated by cannabinoid receptors
For many years, researchers interested in how THC and other cannabinoids work ,in the brain were hampered by the lack of an identified cellular receptor for these compounds. In 1988, however, pharmacological characterization of a central nervous system (CNS) cannabinoid receptor was announced by a group of researchers that included William Devane and Allyn Howlett at St. Louis University and Lawrence Melvin and M. Ross Johnson at the Pfizer pharmaceutical company (Devane et al., 1988). This initial characterization was quickly followed by other studies showing significant expression of cannabinoid receptors in many brain areas such as the basal ganglia (including the striatum, globus pallidus, entopeduncular nucleus, and substantia nigra pars reticularis), cerebellum, hippocampus, and cerebral cortex. As discussed later, localization of cannabinoid receptors in these areas is consistent with the recognized behavioral effects of these compounds on locomotor activity, coordination, and memory.
Tom Bonner cloned a novel gene from rat cerebral cortex that coded for a membrane protein with the characteristics of a G protein-coupled receptor. Further studies revealed that these investigators, who were working on an unrelated problem, had actually cloned the gene for the rat brain cannabinoid receptor (Matsuda et al., 1990). This is a good example of an approach that is sometimes called reverse pharmacology, namely the cloning of a novel receptor gene, the identity of which must then be determined by more classical pharmacological methods. The CNS cannabinoid receptor is currently designated CB1 An additional cannabinoid receptor, cb2, was discovered later; however, this receptor will not be discussed further because it is found primarily in the immune system and does not seem to be expressed in the brain.
CB1 receptors belong to the broad family of metabotropic receptors. The CB1 receptors exert a variety of cellular effects, the most important of which involve inhibition of cyclic adenosine monophosphate (cAMP) formation, inhibition of voltage-sensitive Ca2+ channels, and activation of K+ channel opening. Electron microscopy in conjunction with antibodies against the CB1 receptor have been used to determine the location of these receptors within the synapse. In most instances, CB1 receptors have been shown to exist on the axon terminal instead of the postsynaptic cell. By activating these presynaptic receptors, cannabinoids can inhibit the release of many different neurotransmitters including acetylcholine, dopamine, norepinephrine, serotonin, glutamate, and GABA (y-aminobutyric acid) (Iversen, 2003).
A complete understanding of any receptor requires the development and testing of selective antagonists in addition to agonists at that receptor. Although THC, the classical cannabinoid receptor agonist, was isolated 40 years ago, it wasn’t until 1994 that the first useful antagonist was introduced. This compound, called SR 141716 (also known as rimonabant), was developed by a team of scientists at the French pharmaceutical firm Sanofi Recherche. Not only is SR 141716 a potent and selective antagonist at CB1 receptors, it is also orally active, which means that it can readily be administered to humans. Later we shall see how this compound has helped researchers clarify the behavioral and physiological functions mediated by CBj1 receptors.
Endocannabinoids are cannabinoid agonists synthesized by the brain
The discovery and characterization of cannabinoid receptors finally enabled pharmacologists to study the cellular mechanisms by which marijuana produces its behavioral effects. Yet why should our brain possess receptors for substances made by a plant? This situation is reminiscent of the quandary faced by opiate researchers when opioid receptors were first identified as mediating the actions of morphine, which comes from a poppy plant. Accordingly, the same assumption was made that there must be an endogenous neurotransmitter-like substance that acts on the newly discovered receptors. Within a few years, a group headed by Raphael Mechoulam, the same Israeli scientist involved in the discovery of THC almost 30 years earlier, announced that they had isolated a substance with cannabinoid-like activity from pig brain (Devane et al., 1992).
Chemical analysis revealed the substance to be a lipid with a structure similar to that of arachidonic acid. The formal chemical name of this substance is arachidonoyl ethanolamide, but the researchers gave it the additional name anandamide, from the Indian Sanskrit word ananda, meaning “bringer of inner bliss and tranquility” (Felder and Glass, 1998, p. 186). Later studies demonstrated the existence of other arachidonic derivatives such as 2-arachidonoylglycerol (2-AG) that also bind to and activate CBj receptors. Together, these substances have come to be known as endocannabinoids, meaning endogenous cannabinoids.
The endocannabinoids are generated from arachidonic acid, a fatty acid commonly found in membrane phospholipids. Unlike the classical neurotransmitters, however, they are too lipid soluble to be stored in vesicles since they would just pass right through the vesicle membrane. Thus researchers believe that endocannabinoids are made and released when needed. One mechanism for triggering endo-cannabinoid release is a rise in intracellular Ca2+ levels, which follows from the fact that some of the enzymes involved in the generation of these compounds are Ca2+ sensitive.
After being released, endocannabinoids are taken up from the extracellular fluid by a specific transport protein. This process appears to be important in terminating the biological action of these substances, as inhibition of the transporter by a drug called N-(4-hydroxyphenyl)arachidonylamide (AM 404) enhanced the effects of anandamide both in animals and in a cell culture system (Beltramo et al., 1997). Once inside the cell, endocannabinoids can be metabolized by several enzymes, the best known of which is fatty acid amide hydrolase (FAAH). Cravatt and colleagues (2001) demonstrated an important role for FAAH in anandamide break-down by showing that genetic knockout mice lacking this enzyme had greatly elevated anandamide levels in the brain.
Based on the discovery that many cannabinoid receptors are localized presynaptically, we might suspect that endocannabinoids are often released from postsynaptic cells to act on nearby nerve terminals. When a signaling molecule carries information in the opposite direction from normal (that is, postsynaptic to presynaptic), it is called a retrograde messenger. One such retrograde messenger discussed earlier is the gas nitric oxide. Researchers now hypothesize that endocannabinoids are also retrograde messengers at specific synapses in the hippocampus and cerebellum (Piomelli, 2003; Wilson and Nicoll, 2002). These substances are synthesized and released in response to depolarization of the postsynaptic cell due to the influx of Ca2+ through voltage-gated Ca2+ channels.
Following their release, the endocannabinoids cross the synaptic cleft, activate CBj receptors on the nerve terminal, and inhibit Ca2+-mediated neurotransmitter release from the terminal. In the hippocampus, for example, the endocannabinoids are generated by the pyramidal neurons, which are the principal output neurons of the hippocampus. The endocannabinoids diffuse to the nearby terminals of GABAergic interneurons that normally suppress the firing of the pyramidal cells. The resulting inhibition of GABA release temporarily permits the pyramidal cells to fire more rapidly. Given the widespread distribution of cannabinoid receptors in the brain, it is possible that many more examples of retrograde signaling by endocannabinoids will be discovered in future studies.
Significant progress has been made in our understanding of the mechanisms of cannabinoid action. Two cannabinoid receptors, CBj and CB2, have been identified and their genes cloned. Only the CB1 receptor is found in the brain, where it is expressed at a high density in the basal ganglia, cerebellum, hippocampus, and cerebral cortex. Cannabinoid receptors belong to the G protein-coupled receptor superfamily.
Receptor activation can inhibit cAMP formation, inhibit voltage-sensitive Ca2+ channels, and activate K+ channels. Many CB: receptors are located on axon terminals, where they act to inhibit the release of many different neurotransmitters. Studies on the function of CBj receptors have been aided by the development of a selective antagonist, SR 141716.
The brain synthesizes several substances, called endo-cannabinoids, that are neurotransmitter-like agonists at cannabinoid receptors. Anandamide was the first endo- cannabinoid to be discovered, and it is the best characterized member of the group. Endocannabinoids are generated on demand from arachidonic acid and released from the cell by a process that does not involve synaptic vesicles. They are removed from the extracellular space by an uptake process and they are degraded by several enzymes, including fatty acid amide hydrolase. In certain populations of synapses in the hippocampus and cerebellum, endocannabinoids function as important retrograde messengers. They are released from postsynaptic cells in a Ca2+-dependent manner, travel to nearby nerve terminals that carry CBj receptors, and inhibit neurotransmitter release from the terminals.