More surprises lying ahead. The endocannabinoids keep us guessing

• This article outlines several unanswered questions about the endocannabinoid system.
• The enzymes involved in anandamide biosynthesis are unknown.
• This knowledge gap makes it difficult to localize neurons that use anandamide as a transmitter.
• The enzymes involved in 2-AG biosynthesis and degradation have been identified.
• 2-AG production is localized to a signaling complex, whose exact composition remain unknown.

The objective of this review is to point out some important facts that we don't know about endogenous cannabinoids — lipid-derived signaling molecules that activate CB1 cannabinoid receptors and play key roles in motivation, emotion and energy balance. The first endocannabinoid substance to be discovered, anandamide, was isolated from brain tissue in 1992. Research has shown that this molecule is a bona fide brain neurotransmitter involved in the regulation of stress responses and pain, but the molecular mechanisms that govern its formation and the neural pathways in which it is employed are still unknown. There is a general consensus that enzyme-mediated cleavage, catalyzed by fatty acid amide hydrolase (FAAH), terminates the biological actions of anandamide, but there are many reasons to believe that other as-yet-unidentified proteins are also involved in this process. We have made significant headway in understanding the second arrived in the endocannabinoid family, 2-arachidonoyl-sn-glycerol (2-AG), which was discovered three years after anandamide. Researchers have established some of the key molecular players involved in 2-AG formation and deactivation, localized them to specific synaptic components, and showed that their assembly into a multi-molecular protein complex (termed the ‘2-AG signalosome’) allows 2-AG to act as a retrograde messenger at excitatory synapses of the brain. Basic questions that remain to be answered pertain to the exact molecular composition of the 2-AG signalosome, its regulation by neural activity and its potential role in the actions of drugs of abuse such as Δ9-THC and cocaine.This article is part of a Special Issue entitled ‘NIDA 40th Anniversary Issue’.