Enzymatic pathways that regulate endocannabinoid signaling in the nervous system.

Chemical signals, or neurotransmitters, represent the fundamental mode for intercellular communication in the nervous system. (1) The classical model for neurotransmitter action involves the uptake and storage of these small molecules into synaptic vesicles, release of vesicular contents into the synaptic cleft in response to depolarization of the presynaptic terminal by an action potential, binding of released neurotransmitters to cognate protein receptors on the postsynaptic (and presynaptic) terminal, and, finally, termination of signaling by protein-mediated uptake and degradation of neurotransmitters from the synaptic cleft. This model applies to a large number of well-studied neurotransmitters, including glutamate, γ-amino butyric acid (GABA), acetylcholine, and the monoamines, all of which represent aqueous solution-soluble molecules. More recently, lipids have emerged as an important class of chemical messengers in the nervous system that operate by a distinct mechanism. The hydrophobic nature of lipids precludes their stable uptake and storage into synaptic vesicles. Instead, lipid messengers appear to be biosynthesized and released by neurons at the moment of their intended action, which is often referred to as “on-demand” production. Similarly, the capacity of lipids to freely cross cell membranes places the burden of signal termination largely on the action of degradative enzymes. Lipid signaling systems are thus embedded within an elaborate collection of metabolic pathways, the composition and regulation of which ultimately establish the magnitude and duration of transmitter action. Here, we will review these general concepts as they relate to a specific class of lipid transmitters, the endogenous cannabinoids (endocannabinoids), and highlight how delineation of their cognate metabolic enzymes has been translated into the development of chemical and genetic tools to test the role that the endocannabinoid system plays in nervous system signaling and behavior. Endocannabinoids are defined as endogenous small molecules that activate the cannabinoid receptors CB1 and CB2, which are G-protein-coupled receptors that also recognize Δ9-tetrahydrocannabinol (THC), the psychoactive component of marijuana. (2, 3) The CB1 receptor is the major cannabinoid receptor in the nervous system and is responsible for mediating most of the neurobehavioral effects of THC. (4, 5) The CB2 receptor is predominantly expressed in immune cells, (6) where it appears to play a role in mediating the immunosuppressive effects of cannabinoids. Two principal endocannabinoids have been identified in mammals, N-arachidonoyl ethanolamine (anandamide) (7) and 2-arachidonoylglycerol (2-AG) (8, 9) (Figure 1). Each endocannabinoid also belongs to a much larger class of lipids, termed N-acyl ethanolamines (NAEs) and monoacylglycerols (MAGs), respectively, where individual members differ in the length and degree of unsaturation of their acyl chains (Figure 1). Several NAEs and MAGs have been ascribed potential biological activities in vivo; (10) however, most of these lipids do not serve as ligands for cannabinoid receptors, a property that appears to be restricted to polyunsaturated derivatives such as anandamide and 2-AG. Figure 1 Two principle endocannabinoids, N-arachidonoyl ethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), which are members of theN-acyl ethanolamine (NAE) and monoacylglycerol (MAG) classes of lipids, respectively. In the nervous system, endocannabinoids are hypothesized to act as retrograde messengers, being released by postsynaptic neurons and traversing the synaptic cleft to stimulate CB1 receptors on presynaptic termini (11, 12) (Figure 2). This model is supported by a large number of in vitro electrophysiological studies, (12) as well as by the restricted localization of the CB1 receptor to presynaptic structures in many regions of the nervous system. (13, 14) Once activated by endocannabinoids, CB1 receptors couple principally through the G i/G o class of G proteins to regulate calcium and potassium channels and reduce the probability of neurotransmitter release. (3) This suppression of neurotransmitter release can result in the inhibition or, paradoxically, disinhibition of neuronal circuits, depending on whether the CB1 receptor is expressed on glutamatergic or GABergic neurons. Figure 2 General model for endocannabinoid-based retrograde signaling. Upon release of neurotransmitter (e.g., glutamate), postsynaptic receptors (e.g., α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl- d-aspartic acid (NMDA)) and . Despite sharing a common receptor and considerable structural similarity, anandamide and 2-AG can be distinguished by multiple factors. First, these endocannabinoids activate cannabinoid receptors to a differing degree in vitro, with anandamide displaying lower intrinsic efficacy than 2-AG, which results in the former lipid acting as a partial agonist (15) (it should be specified that the relevance of this distinction for signaling in vivo is unclear, especially when one considers that THC also acts as only a partial agonist for cannabinoid receptors in vitro (16)). Second, the endogenous quantities of anandamide and 2-AG differ dramatically, with the latter lipid being found at more than 100-fold higher concentrations in the nervous system. (16) Of course, these values are based on bulk tissue measurements of endocannabinoids, which almost certainly reflect a combination of metabolic and signaling pools of these lipids. Indeed, recent microdialysis studies have revealed that the extracellular concentrations of anandamide and 2-AG are nearly equivalent (within 2 5-fold), (17, 18) suggesting that, at least for the latter endocannabinoid, a large fraction of bulk tissue concentration may correspond to intracellular metabolic pools. Finally, and of greatest relevance for the subject of this review, anandamide and 2-AG are regulated by distinct biosynthetic and degradative pathways. Over the past decade, several excellent reviews have appeared that discuss endocannabinoid metabolism and signaling. (10, 19-23) Here, we will focus on the most recent advances in our understanding of the composition and regulation of endocannabinoid metabolic pathways, especially as pertains to the nervous system. A pervasive theme throughout this review will be the importance of developing selective genetic and pharmacological tools to specifically perturb individual enzymatic pathways to test their contribution to endocannabinoid metabolism, nervous system function, and, ultimately, mammalian physiology and behavior.