There are two possible routes of 2-AG biosynthesis in neurons, which are illustrated in Figure 3. Phospholipase C -mediated hydrolysis of membrane phospholipids may produce DAG, which may be subsequently converted to 2-AG by diacylglycerol lipase activity. Alternatively, phospholipase A1 may generate a lysophospholipid, which may be hydrolyzed to 2-AG by a lyso-PLC activity. In the intestine, where 2-AG was originally identified , this compound accumulates during the digestion of dietary triglycerides and phospholipids, catalyzed by pancreatic lipases . The fact that various, structurally distinct inhibitors of PLC and DGL activities prevent 2-AG formation in cultures of cortical neurons indicates that the PLC/DGL pathway may play a primary role in this process . The molecular identity of the enzymes involved remains undefined, although the purification of rat brain DGL has been reported . As first suggested by experiments with acutely dissected hippo campal slices, neural activity may evoke 2-AG biosynthesis in neurons by elevating intracellular Ca2 levels . In the hippo campal slice preparation, electrical stimulation of the Schaffer collaterals produces a fourfold increase in 2-AG formation, which is prevented by the Na channel blocker tetrodotoxin or by removing Ca2 from the medium. Noteworthy, the local concentrations reached by 2-AG after stimulation are in the low micromolar range , which should be sufficient to activate the dense population of CB1 receptors present on axon terminals of hippocampal GABAergic interneurons. In addition to neural activity,cannabis grower certain neurotransmitter receptors also may be linked to 2-AG formation. For example, in primary cultures of cortical neurons, glutamate stimulates 2-AG synthesis by allowing the entry of Ca2 through activated N-methyl-D-aspartate receptor channels .

Interestingly, this response is strongly enhanced by the cholinergic agonist carbachol, which has no effect on 2-AG formation when applied alone . The molecular basis of the synergistic interaction between NMDA and carbachol is unclear at present but deserves further investigation in light of the potential roles of 2-AG in hippo campal retrograde signaling .The anandamide precursor N-arachidonoyl PE belongs to a family of N-acylated PE derivatives, which contain different saturated or unsaturated fatty acids linked to their ethanolamine moieties and give rise to the corresponding fatty acid ethanolamides . These compounds generally lack CB1 receptor-binding activity but display a number of remarkable effects and possible biological functions. In this regard, two FAE have been studied in some detail, palmitoylethanolamide and oleoylethanolamide .PEA exerts profound analgesic and anti-inflammatory effects in vivo, which have been attributed to its ability to interact with a putative receptor site sensitive to the CB2-preferring antagonist SR144528 . The molecular identity of this site is unknown, although it is probably distinct from the CB2 receptor whose gene has been cloned . PEA is present at high levels in skin and other tissues where, together with locally produced anandamide, may participate in the peripheral control of pain initiation . Despite its chemical similarity with PEA, OEA shows weak analgesic properties but exerts potent appetite suppressing effects in the rat . Because these effects are prevented by sensory deafferentation, and intestinal OEA biosynthesis is linked to the feeding state , it has been suggested that OEA may be involved in the peripheral regulation of feeding .A series of close structural analogs of anandamide with activity at cannabinoid receptors have been isolated from brain tissue. These compounds, which include eicosatrienoylethanolamide and docosatetraenoylethanolamide , may be generated through the same enzymatic route as anandamide, albeit in smaller quantities.

Distinct from these polyunsaturated ethanolamides as well as from 2-AG are two recently discovered brain lipids: 2-arachidonoyl glyceryl ether and O-arachidonoyl ethanolamine. Noladin ether was isolated from porcine brain and identified by using a combination of mass spectrometry, nuclear magnetic resonance, and chemical synthesis. The compound binds to CB1 receptors with high affinity in vitro [dissociation constant 21 nM] and produces cannabinoid-like effects in the mouse in vivo, including sedation, immobility, hypothermia, and antinociception . Virodhamine was identified in rat brain by mass spectrometry and chemical synthesis and shown to weakly activate CB1 receptors in a 35S-labeled guanosine 5-O– binding assay in which the compound also displayed partial agonist activity . Moreover, virodhamine decreases body temperature in the mouse, although less effectively than anandamide, and inhibits anandamide transport in RBL-2H3 cells . A possible confounding factor in these studies is due, however, to the chemical instability of virodhamine, which in an aqueous environment is rapidly converted to anandamide. The formation and inactivation of these molecules, as well as their physiological significance, is the subject of ongoing investigations .Both anandamide and 2-AG may be generated by and released from neurons through a mechanism that does not require vesicular secretion. However, unlike classical or peptide neurotransmitters, which readily diffuse across the synaptic cleft, anandamide and 2-AG are hydrophobic molecules and, as such, are constrained in their movements through the aqueous environment surrounding cells. How may these compounds reach their receptors on neighboring neurons? Experiments with bacterial PLD suggest that, in cortical neurons, 40% of the anandamide precursor N-arachidonoyl PE is localized to the cell surface , which also contains 2-AG precursors such as phosphoinositol phosphate and bisphosphate . This suggests that both endocannabinoids may be generated in the plasmalemma, where they are ideally poised to access the external medium. As with other lipid compounds, the actual release step may be mediated by passive diffusion and/or facilitated by the presence of lipid-binding proteins such as the lipocalins .

The existence of different routes for the synthesis of anandamide and 2-AG suggests that these two endocannabinoids could in principle operate independently of each other. This idea is supported by three main findings. First, electrical stimulation of hippocampal slices increases the levels of 2-AG, but not those of anandamide . Second, activation of dopamine D2 receptors in the striatum enhances the release of anandamide, but not that of 2-AG . Third, activation of NMDA receptors in cortical neurons in culture increases 2-AG levels but has no effect on anandamide formation, which requires instead the simultaneous activation of NMDA and -7 nicotinic receptors . It is unclear at present whether these differences reflect regional segregation of the PLC/ DGL and PLD/NAT pathways, the existence of receptor activated mechanisms linked to the generation of specific endocannabinoids, or both.Carrier-mediated uptake into nerve endings and glia, probably the most frequent mechanism of neurotransmitter inactivation, is also involved in the clearance of lipid messengers. This idea may appear at first counter intuitive: why should a lipid molecule need a carrier protein to cross plasma membranes when it could do so by passive diffusion? A large body of evidence indicates, however, that even very simple lipids such as fatty acids are transported into cells by protein carriers,mobile grow system several families of which have now been molecularly characterized . Indeed, carrier-mediated transport may provide a rapid and selective means of delivering lipid molecules to specific cellular compartments . Thus it is not surprising that neural cells might adopt the same strategy to interrupt lipid-mediated signaling .Anandamide transport differs from that of amine and amino acid transmitters in that it does not require cellular energy or external Na, implying that it may be mediated through facilitated diffusion . Because anandamide is rapidly hydrolyzed within cells , it is reasonable to hypothesize that intracellular breakdown contributes to the rate of anandamide transport. Accordingly, HeLa cells that overexpress the anandamide-hydrolyzing enzyme FAAH also display higher than normal rates of [3 H]anandamide accumulation . However, in primary cultures of rat neurons and astrocytes or in adult rat brain slices, FAAH inhibitors have no effect on [3 H]anandamide transport at concentrations that completely abrogate [3 H]anandamide hydrolysis . From these results it is reasonable to conclude that anandamide transport in the CNS is largely independent of intracellular hydrolysis.

Whether persistent disruption of FAAH activity may eventually change the distribution of anandamide between intracellular and extracellular pools is an interesting question that warrants examination. The substrate selectivity of anandamide transport has been investigated in rat cortical neurons and astrocytes and, more systematically, in human astrocytoma cells . In the latter model, [3 H]anandamide uptake is not affected by a variety of lipids that bear close structural resemblance to anandamide, including arachidonic acid, PEA, ceramide, prostaglandins, leukotrienes, hydroxyeicosatetraenoic acids, and epoxyeicosatetraenoic acids. Furthermore, [3 H]anandamide accumulation in these cells is insensitive to substrates or inhibitors of fatty acid transport , organic anion transport , and P-glycoproteins . However, [3 H]anandamide uptake is competitively blocked by nonradioactive anandamide and by the anandamide analog N–arachidonamide  . A similar sensitivity to AM404 has been reported for rat cortical and cerebellar neurons , rat cortical astrocytes , and rat brain slices . Inhibitory effects of AM404 on anandamide accumulation also have been observed in a number of nonneural cells, although the concentrations of AM404 needed to produce such effects are generally higher than in neurons . Together, these data are consistent with the view that anandamide is internalized by neurons and astrocytes through a selective process of facilitated diffusion. The molecular identity of the protein responsible for this process is, however, unknown.Anandamide and 2-AG share three common structural features: 1) a highly hydrophobic fatty acid chain, 2) an amide or an ester moiety, and 3)a polar head group . Systematic modifications in the hydrophobic carbon chain indicate that the structural requisites for substrate recognition by the putative anandamide transporter may be different from those of substrate translocation. Substrate recognition may require the presence of at least one cis double bond in the middle of the fatty acid chain, indicating a preference for substrates with a fatty acid chain that can adopt an extended U-shaped conformation. In contrast, a minimum of four cis nonconjugated double bonds may be required for translocation, suggesting that a closed “hairpin” conformation is required in order for substrates to be moved across the membrane . Molecular modeling studies show that transport substrates have both extended and hairpin low-energy conformers . In contrast, extended but not hairpin conformations may be thermodynamically favored in pseudo-substrates such as oleoylethanolamide, which displace [3 H]anandamide from transport without being internalized . The effects of head group modifications on anandamide transport have also been investigated . The results suggest that ligand recognition may be maintained when the head group is removed , or replaced with substantially bulkier moieties , and when an ester bond substitutes the amide bond . Notably, ligand recognition appears to be favored by replacing the ethanolamine group with a substituted hydroxyphenyl group [as in AM404 and its derivative N-arachidonamide or a furane group ] .Biochemical experiments have demonstrated the existence of anandamide transport in primary cultures of rat cortical neurons and astrocytes , as well as rat cerebellar granule cells . But what brain regions express the transporter is still unclear, primarily due to a lack of molecular understanding of the transporter involved in this process. In one study, the CNS distribution of anandamide transport was investigated by exposing metabolically active rat brain slices to [14C]anandamide and measuring the distribution of radioactivity by autoradiography. The CB1 antagonist SR141716A was included in the incubation medium to prevent binding of [ 14C]anandamide to CB1 receptors, and AM404 was used to differentiate transport-mediated [14C]anandamide accumulation from nonspecific association with cell membranes and cell debris . These experiments suggest that the somatosensory, motor, and limbic areas of the cortex, as well as the striatum, contain substantial levels of AM404-sensitive [14C]anandamide uptake. Other brain regions showing detectable transport include the hippocampus, the amygdala, the septum, the thalamus, the substantia nigra, and the hypothalamus .Although a variety of compounds have been shown to inhibit anandamide transport, the anandamide analog AM404 remains a standard of reference, mainly because of its relatively high potency and its ability to block anandamide transport both in vitro and in vivo . AM404 inhibits [3 H]anandamide uptake in rat brain neurons and astrocytes , human astrocytoma cells , rat brain slices , and a variety of nonneural cell types . The inhibitor also enhances several CB1 receptor-mediated effects of anandamide, without directly activating cannabinoid receptors . For example, AM404 increases anandamide evoked inhibition of adenylyl cyclase activity in cortical neurons , augments the presynaptic inhibition of GABA release produced by anandamide in the midbrain periaqueductal gray , and mimics the effects of cannabinoid agonists on hippo campal depolarization induced suppression of inhibition .