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Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary; Department of Clinical Neurobiology, University Hospital of Neurology, Heidelberg, Germany; and Department of Pharmacology, University of California Irvine, Irvine, California
ABSTRACT I. INTRODUCTION II. THE LIFE CYCLE OF THE ENDOCANNABINOIDS A. Introduction B. Biosynthetic Pathways 1. Anandamide biosynthesis 2. 2-AG biosynthesis 3. Fatty acid ethanolamides that do not interact with known cannabinoid receptors 4. Other endogenous agonists at cannabinoid receptors 5. Endocannabinoid release C. Termination of Endocannabinoid Effects: Transport and Degradation 1. Anandamide transport 2. 2-AG transport 3. Structure-activity relationship 4. Distribution of anandamide transport in the CNS 5. Inhibitors of anandamide transport 6. Anandamide hydrolysis: role of FAAH 7. Structure-activity relationship 8. FAAH distribution in the CNS 9. Inhibitors of FAAH activity 10. Physiological roles of FAAH 11. 2-AG hydrolysis: the role of monoglyceride lipase III. REGIONAL AND CELLULAR DISTRIBUTION OF NEURONAL CB1 CANNABINOID RECEPTORS A. Characteristic Differences in CB1 Receptor Distribution in the Brain B. Selective Expression of CB1 Cannabinoid Receptors by Identified Cell Types of Complex Networks 1. Methodological considerations 2. Cortical areas 3. Basal forebrain 4. Basal ganglia 5. Thalamus 6. Hypothalamus 7. Midbrain 8. Medulla and pons 9. Cerebellum 10. Spinal cord IV. ANATOMICAL, PHYSIOLOGICAL, AND PHARMACOLOGICAL EVIDENCE FOR THE PRESYNAPTIC LOCALIZATION OF CB1 CANNABINOID RECEPTORS IN THE BRAIN A. Anatomical Evidence for Presynaptic Cannabinoid Receptors B. Physiological and Pharmacological Evidence for Presynaptic Cannabinoid Receptors 1. Cortical areas 2. Basal ganglia 3. Cerebellum 4. Areas and pathways involved in pain perception C. Are There Postsynaptic CB1 Receptors? V. PHYSIOLOGICAL ROLES OF ENDOCANNABINOIDS A. The Cannabinoid Root 1. Effects on evoked potentials and long-term synaptic plasticity 2. Effects on population discharge patterns B. The DSI (DSE) Root: Control of GABAergic and Glutamatergic Synaptic Transmission via Retrograde Synaptic Signaling 1. DSI 2. DSE C. Marriage of the Two Lines of Research Explains the Mechanism of DSI (and DSE) While Endowing Endocannabinoids With Function D. Electrical Activity Patterns Required for the Release of Endocannabinoids VI. CONCLUSIONS
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I. INTRODUCTION |
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Two series of events contributed to a radical change of this view. First, motivated by the potential therapeutic applications of cannabis-like ("cannabimimetic") molecules, laboratories in academia and the pharmaceutical industry began to develop families of synthetic analogs of delta-9-THC. These agents exerted pharmacological effects that were qualitatively similar to those of delta-9-THC but displayed both greater potency and stereoselectivity. The latter feature cannot be reconciled with nonspecific membrane interactions, providing the first evidence that delta-9-THC exerts its effects by combining with a selective receptor. Second, as a result of these synthetic efforts, it became possible to explore directly the existence of cannabinoid receptors by using standard radioligand binding techniques. In 1988, Howlett and her co-workers (84, 167) described the presence of high-affinity binding sites for cannabinoid agents in brain membranes and showed that these sites are coupled to inhibition of adenylyl cyclase activity. Conclusively supporting these findings, in 1990 Matsuda et al. (236) serendipitously came across a complementary DNA encoding for the first G protein-coupled cannabinoid receptor, now known as CB1.
In heterologous expression systems, CB1 receptors were found to be functionally coupled to multiple intracellular signaling pathways, including inhibition of adenylyl cyclase activity, inhibition of voltage-activated calcium channels, and activation of potassium channels (56, 148, 221, 222, 236, 239). In situ hybridization and immunohistochemical studies have demonstrated that CB1 receptors are abundantly expressed in discrete regions and cell types of the central nervous system (CNS) (see also sect.III) but are also present at significant densities in a variety of peripheral organs and tissues (41, 225, 226, 235, 345). The selective distribution of CB1 receptors in the CNS provides a clear anatomical correlate for the cognitive, affective, and motor effects of cannabimimetic drugs.
The cloning and characterization of CB1 receptors left several important problems unsolved. Since antiquity, it has been known that the actions of Cannabis and delta-9-THC are not restricted to the CNS, but include effects on nonneural tissues such as reduction of inflammation, lowering of intraocular pressure associated with glaucoma, and relief of muscle spasms. Are these peripheral effects all produced by activation of CB1 receptors? An initial answer to this question was provided by the discovery of a second cannabinoid receptor exquisitely expressed in cells of immune origin (260). This receptor, called CB2, only shares 
44% sequence identity with its brain counterpart, implying that the two subtypes diverged long ago in evolution. The intracellular coupling of the CB2 receptor resembles, however, that of the CB1 receptor; for example, in transfected cells, CB2 receptor activation is linked to the inhibition of adenylyl cyclase activity (113).
The experience with opioid receptors and the enkephalins has accustomed scientists to the idea that whenever a receptor is present in the body, endogenous factor(s) that activate this receptor also exist. Not surprisingly, therefore, as soon as cannabinoid receptors were described, a search began to identify their naturally occurring ligand(s). One way to tackle this problem was based on the premise that, like other neurotransmitters and neuromodulators, an endogenous cannabinoid substance should be released from brain tissue in a calcium-dependent manner. Taking this route, Howlett and coworkers incubated rat brain slices in the presence of a calcium ionophore and determined whether the media from these incubations contained a factor that displaced the binding of labeled CP-55940, a cannabinoid agonist, to brain membranes. These studies demonstrated that a cannabinoid-like activity was indeed released from stimulated slices, but the minute amounts of this factor did not allow the elucidation of its chemical structure (97, 98).
Devane, Mechoulam, and co-workers (85, 243), at the Hebrew University in Jerusalem, adopted a different strategy. Reasoning that endogenous cannabinoids may be as hydrophobic as delta-9-THC, they subjected porcine brains to organic solvent extraction and fractionated the lipid extract by chromatographic techniques while measuring cannabinoid binding activity. This approach turned out to be highly successful, and the researchers were able to isolate a lipid cannabinoid-like component, which they characterized by mass spectrometry and nuclear magnetic resonance spectroscopy as the ethanolamide of arachidonic acid. They named this novel compound "anandamide" after the sanskrit "ananda," inner bliss.
The chemical synthesis of anandamide confirmed this structural identification and allowed the characterization of its pharmacological properties (112). In vitro and in vivo tests showed a great similarity of actions between anandamide and cannabinoid drugs. Anandamide reduced the electrogenic contraction of mouse vas deferens and closely mimicked the behavioral responses produced by delta-9-THC in vivo; in the rat, the compound was found to produce analgesia, hypothermia, and hypomotility. However, these effects may not be exclusively due to cannabinoid receptor activation, as anandamide is readily metabolized to arachidonic acid, which can be converted in turn to a variety of biologically active eicosanoid compounds. Subsequent studies demonstrated that anandamide is released from brain neurons in an activity-dependent manner (89, 126) and elucidated the unique biochemical routes of anandamide formation and inactivation in the CNS (25, 44, 45, 69, 89). Thus anandamide fulfills all key criteria that define an endogenous cannabinoid (endocannabinoid) substance.
In their 1992 study, Devane, Mechoulam, and coworkers (242) reported that several lipid fractions from the rat brain contained cannabinoid-binding activity, in addition to anandamide's. In characterizing these fractions, they discovered that some of them were composed of polyunsaturated fatty acid ethanolamides similar to anandamide (e.g., eicosatrienoylethanolamide), but others were instead constituted of a distinct lipid component, sn-2-arachidonoyl-glycerol (2-AG) (242). Sugiura et al. (330) arrived independently to the same conclusion. That polyunsaturated fatty acid ethanolamides should mimic anandamide, to which they are structurally very similar, does not come as a great surprise. Moreover, the pharmacological properties of these fatty acid ethanolamides, essentially indistinguishable from those of anandamide, and their scarcity in brain relegate them, at least for the moment, to a position secondary to anandamide's. We cannot say the same for 2-AG. This lipid, considered until now a mere intermediate in glycerophospholipid turnover (see sect. II), is present in the brain at concentrations that are 170-fold greater than those of anandamide and possesses two pharmacological properties that make it crucially different from the latter: it binds to both CB1 and CB2 cannabinoid receptors with similar affinities, and it activates CB1 receptors as a full agonist, whereas anandamide acts as a partial agonist.
Research of endocannabinoids begs for a conjunction of in situ biochemistry and physiology. We have learned much over the past 10 years on the behavioral effects of these molecules, on how these lipid mediators are produced physiologically, and on the functional roles that they may serve. A major step was the discovery that depolarization-induced suppression of inhibition (DSI; or excitation, DSE), a type of short-term synaptic plasticity originally discovered in the cerebellum and the hippocampus (214, 288), is mediated by endocannabinoids (199, 200, 271, 375). This discovery allowed the results of over a decade of research on retrograde synaptic signaling in these networks to be considered as functional characteristics of endocannabinoid signaling. The substrate of retrograde signaling and DSI is the predominantly presynaptic distribution of CB1 receptors on axon terminals in the hippocampus (188), as well as throughout the brain, where activation of CB1 by endocannabinoids can efficiently veto neurotransmitter release in many distinct types of synapses (see sect. IV). The conditions of synthesis, release, distance of diffusion, duration of effect, and site of action were all extensively characterized for the mediator of DSI (for review, see Ref. 10) that turned out to be an endocannabinoid (271, 375). The fact that neurons are able to control the efficacy of their own synaptic input in an activity-dependent manner (a phenomenon called retrograde synaptic signaling) is functionally very important, since this mechanism may subserve several functions in information processing by neuronal networks from temporal coding and oscillations to group selection and the fine tuning of signal-to-noise ratio. The crucial involvement of endocannabinoids in these functions just began to emerge from recent studies, which are reviewed in section V. Due to the exceptionally rapid expansion of this field in recent years (and to our special interest in neuronal signaling in complex integrative centres of the brain), we decided to focus the present review on questions related to the composition of the endocannabinoid system and its physiological roles in controlling brain activity at the regional and cellular levels as synaptic signal molecules. We did not aim to provide detailed accounts of studies dealing with other, similarly important, aspects of cannabinoid research, which have been dealt with in excellent recent reviews, e.g., about the relation of the endocannabinoid system to pain modulation (281, 366), the immune system (194), neuroprotection (136), and addiction (228).
The final message of the present review is that to understand the possible physiological functions of the endogenous cannabinoids, their roles in normal and pathological brain activity, pharmacological agents targeting the cascade of anandamide and 2-AG formation, release, uptake, and degradation will have to be developed. Such drugs, which undoubtedly will become invaluable research tools to study the potential functions listed above, may also provide novel therapeutic approaches to diseases whose clinical, biochemical, and pharmacological features suggest a link with the endogenous cannabinoid system.
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II. THE LIFE CYCLE OF THE ENDOCANNABINOIDS |
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A basic principle that has emerged from the last two decades of research on cellular signaling is that simple phospholipids such as phosphatidylcholine or phosphatidylinositol should be regarded not only as structural components of the cell membrane, but also as precursors for transmembrane signaling molecules. Intracellular second messengers like 1,2-diacylglycerol (DAG) and ceramide are familiar examples of this concept. Along with their intracellular roles, however, lipid compounds may also serve important functions in the exchange of information between cells. Indeed, biochemical mechanisms analogous to those involved in the generation of DAG or ceramide give rise to biologically active lipids that leave their cell of origin to activate G protein-coupled receptors located on the surface of neighboring cells. Traditionally overshadowed by amino acid, amine, and peptide transmitters, biologically active lipids are now emerging as essential mediators of cell-to-cell communication within the CNS, where G protein-coupled receptors for multiple families of such compounds, including lysophosphatidic acid and eicosanoids, have been identified (67, 285).
In this section, we discuss the biochemical properties of endogenous lipids that activate brain cannabinoid receptors. These compounds share two common structural motifs: a polyunsaturated fatty acid moiety (e.g., arachidonic acid) and a polar head group consisting of ethanolamine or glycerol (Fig. 1). Because of these features, endocannabinoid substances seemingly resemble the eicosanoids, ubiquitous bioactive lipids generated through the enzymatic oxygenation of arachidonic acid. However, the endocannabinoids are clearly distinguished from the eicosanoids by their different biosynthetic routes, which do not involve oxidative metabolism. The two best characterized endocannabinoids, anandamide (arachidonoylethanolamide) (85) and 2-AG (242, 330), may be produced instead through cleavage of phospholipid precursors present in the membranes of neurons, glia, and other cells. In the following sections, we will first focus on the biochemical pathways that lead to the formation of endocannabinoids in neurons and then turn to the mechanism by which these compounds are deactivated.
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Anandamide formation via energy-independent condensation of arachidonic acid and ethanolamine was described in brain tissue homogenates soon after the discovery of anandamide and was attributed to an enzymatic activity that was termed "anandamide synthase" (81, 83, 201). Subsequent work has demonstrated, however, that this reaction is in fact catalyzed by fatty acid amide hydrolase (FAAH), the primary enzyme of anandamide hydrolysis, acting in reverse (203). Since FAAH requires high concentrations of arachidonate and ethanolamine to synthesize anandamide, higher than those normally found in cells, this enzyme is unlikely to play a role in the physiological formation of anandamide (for further discussion, see sect. IIC6).
Another model for anandamide biosynthesis is illustrated schematically in Figure 2. According to this model, anandamide may be produced via hydrolysis of the phospholipid precursor N-arachidonoyl phosphatidylethanolamine (PE), catalyzed by a phospholipase D (PLD)-type activity (89, 331, 332). The precursor consumed in this reaction may be resynthesized by a separate enzyme activity, N-acyltransferase (NAT), which may transfer an arachidonate group from the sn-1 glycerol ester position of phospholipids to the primary amino group of PE (89). The validity of this model was initially questioned, because previous studies had failed to detect N-arachidonoyl PE in mammalian tissues (266, 267, 318). More recent chromatographic and mass spectrometric analyses have unambiguously shown, however, that N-arachidonyl PE is present in brain and other tissues, where it may serve as a physiological precursor for anandamide (44, 46, 89, 332).
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Although biochemically distinct, anandamide formation and N-arachidonoyl PE synthesis are thought to proceed in parallel. Both reactions may be initiated by intracellular Ca2+ rises (44, 45, 89, 315, 331, 332) and/or by activation of neurotransmitter receptors (125, 327). For example, administration of dopamine D2-receptor agonists to rats in vivo causes a profound stimulation of anandamide release in the striatum (125), which is likely mediated by de novo anandamide synthesis (A. Giuffrida and D. Piomelli, unpublished observations). Unfortunately, the two key enzyme activities responsible for these reactions, PLD and NAT, have only been partially characterized, and their molecular properties are still unknown (44, 45, 282, 283).
There are two possible routes of 2-AG biosynthesis in neurons, which are illustrated in Figure 3. Phospholipase C (PLC)-mediated hydrolysis of membrane phospholipids may produce DAG, which may be subsequently converted to 2-AG by diacylglycerol lipase (DGL) activity. Alternatively, phospholipase A1 (PLA1) 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 (242), this compound accumulates during the digestion of dietary triglycerides and phospholipids, catalyzed by pancreatic lipases (39). 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 (328). The molecular identity of the enzymes involved remains undefined, although the purification of rat brain DGL has been reported (100, 101).
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As first suggested by experiments with acutely dissected hippocampal slices, neural activity may evoke 2-AG biosynthesis in neurons by elevating intracellular Ca2+ levels (327, 328). In the hippocampal slice preparation, electrical stimulation of the Schaffer collaterals (a glutamatergic fiber tract that projects from CA3 to CA1 neurons) 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 (328), which should be sufficient to activate the dense population of CB1 receptors present on axon terminals of hippocampal GABAergic interneurons (187, 188). The possible significance of this process for hippocampal network activity is discussed in sections IV and VC.
In addition to neural activity, 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 (NMDA) receptor channels (327). Interestingly, this response is strongly enhanced by the cholinergic agonist carbachol, which has no effect on 2-AG formation when applied alone (327). 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 hippocampal retrograde signaling (see sect. VC).
3. Fatty acid ethanolamides that do not interact with known cannabinoid receptors
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 (FAE). 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 (PEA) and oleoylethanolamide (OEA).
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 (48, 99, 174, 238). The molecular identity of this site is unknown, although it is probably distinct from the CB2 receptor whose gene has been cloned (260). 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 (48).
Despite its chemical similarity with PEA, OEA shows weak analgesic properties (49) but exerts potent appetite-suppressing effects in the rat (303). Because these effects are prevented by sensory deafferentation, and intestinal OEA biosynthesis is linked to the feeding state (increasing in fed and decreasing in starved animals), it has been suggested that OEA may be involved in the peripheral regulation of feeding (303).
4. Other endogenous agonists at cannabinoid receptors
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 (144), 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 (noladin ether) (143) and O-arachidonoyl ethanolamine (virodhamine) (291) (Fig. 1). 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 (KD) 21 nM] and produces cannabinoid-like effects in the mouse in vivo, including sedation, immobility, hypothermia, and antinociception (143). 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-(3-thiotriphosphate) (GTP
S) binding assay (half-maximal effective concentration, 1.9 µM) in which the compound also displayed partial agonist activity (291). Moreover, virodhamine decreases body temperature in the mouse, although less effectively than anandamide, and inhibits anandamide transport in RBL-2H3 cells (291). 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 (105).
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 (45), which also contains 2-AG precursors such as phosphoinositol phosphate and bisphosphate (341). 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 (9).
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 (328). Second, activation of dopamine D2 receptors in the striatum enhances the release of anandamide, but not that of 2-AG (125). 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 (327). 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.
C. Termination of Endocannabinoid Effects: Transport and Degradation
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 counterintuitive: 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, several families of which have now been molecularly characterized (2, 160, 316). Indeed, carrier-mediated transport may provide a rapid and selective means of delivering lipid molecules to specific cellular compartments (for example, enzyme complexes implicated in lipid metabolism). Thus it is not surprising that neural cells might adopt the same strategy to interrupt lipid-mediated signaling (Fig. 4).
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Anandamide transport meets four key criteria of a carrier-mediated process: saturability, fast rate, temperature dependence, and substrate selectivity (25, 89, 156). In rat cortical neurons in primary culture, the uptake of exogenous [3H]anandamide is saturable at 37°C, reaches 50% of its maximum within 4 min, and displays a Michaelis constant (Km) of 1.2 µM and a maximum accumulation rate (Vmax) of 90.9 pmol · min-1 · mg protein-1 (25). Comparable kinetic values are observed in rat cortical astrocytes (Km = 0.32 µM; Vmax = 171 pmol · min-1 · mg protein-1) and human astrocytoma cells (Km = 0.6 µM; Vmax = 14.7 pmol · min-1 · mg protein-1) (25, 286), as well as in a variety of nonneural cells (for a review, see Ref. 108). For example, RBL-2H3 basophilic leukemia cells accumulate [3H]anandamide with a Km of 11.4 µM and a Vmax of 17.5 x 10-17 mol/cell (293).
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 (25, 156, 286, 293). Because anandamide is rapidly hydrolyzed within cells (see sect. IIC6), 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 [3H]anandamide accumulation (73). However, in primary cultures of rat neurons and astrocytes or in adult rat brain slices, FAAH inhibitors have no effect on [3H]anandamide transport at concentrations that completely abrogate [3H]anandamide hydrolysis (23, 25, 124). 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 (25, 89) and, more systematically, in human astrocytoma cells (286). In the latter model, [3H]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, [3H]anandamide accumulation in these cells is insensitive to substrates or inhibitors of fatty acid transport (phloretin), organic anion transport (p-amino-hippurate and digoxin), and P-glycoproteins (verapamil, quinidine) (286). However, [3H]anandamide uptake is competitively blocked by nonradioactive anandamide (IC50 = 15.1 µM) and by the anandamide analog N-(4-hydroxyphenyl)-arachidonamide (AM404) (IC50 = 2.2 µM) (24, 286). A similar sensitivity to AM404 has been reported for rat cortical and cerebellar neurons (25, 176), rat cortical astrocytes (25), and rat brain slices (24). 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 (for a review, see Ref. 108). 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(s) responsible for this process is, however, unknown.
Nonradioactive 2-AG prevents [3H]anandamide uptake in various cell types, suggesting that the two endocannabinoids may compete for the same transport system. Three observations support this hypothesis. First, in astrocytoma and other cells, [3H]anandamide and [3H]2-AG are accumulated with similar kinetic properties (26, 286). For example, in C6 glioma cells, [3H]2-AG uptake displays a Km of 1.7 µM and a Vmax of 240 pmol · min-1 · mg protein-1, values that are very close to those obtained with [3H]anandamide (26). Second, anandamide and 2-AG can prevent each other's transport (24, 26). Third, the accumulation of either endocannabinoid is blocked with similar potencies by the transport inhibitor AM404 (24, 26). Thus AM404 inhibits [14C]anandamide and [3H]2-AG accumulation in C6 glioma cells with IC50 values of 7.5 and 10.2 µM, respectively (26).
Despite these similarities, differences in the properties of anandamide and 2-AG uptake also have been documented. For example, incubation with arachidonic acid causes a marked reduction in [3H]2-AG uptake by astrocytoma cells, but it has no effect on [3H]anandamide accumulation (24). Two alternative explanations may be offered for this discrepancy. Arachidonic acid may directly interfere with a 2-AG carrier distinct from anandamide's, or the fatty acid may indirectly prevent the facilitated diffusion of [3H]2-AG by inhibiting its conversion to arachidonic acid (possibly through product inhibition) in the intracellular compartment. If the latter explanation is correct, agents that interfere with the incorporation of arachidonic acid into phospholipids, such as triacsin C (an inhibitor of acyl-CoA synthesis), also should decrease [3H]2-AG uptake. Accordingly, triacsin C selectively prevents the uptake of [3H]2-AG by astrocytoma cells, but not that of [3H]anandamide (24). Thus, although anandamide and 2-AG may utilize similar transport mechanisms, or even share a common one, they may differ in how their intracellular degradation affects the rate of transport.
3. Structure-activity relationship
Anandamide and 2-AG share three common structural features: 1) a highly hydrophobic fatty acid chain, 2) an amide (anandamide) or an ester (2-AG) moiety, and 3) a polar head group (Fig. 1). 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 (or competitive inhibitors) 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 (286). Molecular modeling studies show that transport substrates (such as anandamide and 2-AG) have both extended and hairpin low-energy conformers (286). In contrast, extended but not hairpin conformations may be thermodynamically favored in pseudo-substrates such as oleoylethanolamide, which displace [3H]anandamide from transport without being internalized (286, 295).
The effects of head group modifications on anandamide transport have also been investigated (176, 286). The results suggest that ligand recognition may be maintained when the head group is removed (as in arachidonamide), or replaced with substantially bulkier moieties (as in AM404), and when an ester bond substitutes the amide bond (as in 2-AG). Notably, ligand recognition appears to be favored by replacing the ethanolamine group with a substituted hydroxyphenyl group [as in AM404 and its derivative N-(2-methyl-4-hydroxy-phenyl)arachidonamide (25, 79) or a furane group (215)] (Fig. 1).
4. Distribution of anandamide transport in the CNS
Biochemical experiments have demonstrated the existence of anandamide transport in primary cultures of rat cortical neurons and astrocytes (25), as well as rat cerebellar granule cells (156). But what brain regions express the transporter is still unclear, primarily due to a lack of molecular understanding of the transporter(s) 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 (rimonabant) 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 (124). 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 (124).
5. Inhibitors of anandamide transport
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 (24, 127, 156, 176, 286, 293).
AM404 inhibits [3H]anandamide uptake in rat brain neurons and astrocytes (25), human astrocytoma cells (286), rat brain slices (24), and a variety of nonneural cell types (see, for review, Ref. 108). The inhibitor also enhances several CB1 receptor-mediated effects of anandamide, without directly activating cannabinoid receptors (24, 25). For example, AM404 increases anandamide-evoked inhibition of adenylyl cyclase activity in cortical neurons (25), augments the presynaptic inhibition of GABA release produced by anandamide in the midbrain periaqueductal gray (PAG) (358), and mimics the effects of cannabinoid agonists on hippocampal depolarization-induced suppression of inhibition (375) (see sect. VC). The fact that the cannabinoid antagonist SR141716A prevents these effects suggests that AM404 may act by preventing anandamide inactivation and enhancing its interactions with cannabinoid receptors. Importantly, however, AM404 also can be transported inside cells (286), where it may reach levels that are sufficient to inhibit anandamide degradation by FAAH (176).
The target selectivity of AM404 has been investigated in some detail. Initial studies showed that AM404 has no affinity for a panel of 36 potential targets, including G protein-coupled receptors, ligand-gated channels, and voltage-dependent channels (22). Subsequent work suggested, however, that AM404 may activate the capsaicin-sensitive VR1 vanilloid receptor in vitro (27, 325, but see Ref. 215 for opposing results). It is unlikely that this effect occurs in vivo, since AM404 does not display any of the pharmacological properties of a vanilloid agonist (see below). Yet, these findings underscore the need to design novel inhibitors of anandamide transport endowed with greater target selectivity. Ongoing research efforts in this direction have led to the development of several arachidonic acid derivatives that are equivalent or slightly superior to AM404 in inhibiting anandamide transport in vitro (79, 215) and in vivo, with effects similar to those of AM404 (77).
Consistent with its low affinity for CB1 receptors, AM404 does not act as a direct cannabinoid agonist when administered to live animals. The compound has no antinociceptive effects in the mouse hot-plate test (25) and does not reduce arterial blood pressure in the urethane-anesthetized guinea pig (47). In the same models, however, AM404 magnifies the responses elicited by exogenous anandamide, actions that are prevented by the CB1 antagonist SR141716A (25, 47). Furthermore, when administered alone, AM404 reduces motor activity (22), attenuates apomorphine-induced yawning (22), decreases the levels of circulating prolactin (132), and alleviates the motor hyperactivity induced in the rat by striatal 3-nitro-propionic acid lesions (206). These actions resemble those of anandamide and are blocked by SR141716A (22, 127), suggesting that endogenous anandamide may be involved. In keeping with this notion, systemic administration of AM404 in the rat causes a time-dependent increase in circulating anandamide levels (127).
The participation of anandamide in the effects of AM404 in vivo has been questioned (108) based on the ability of this compound to interact with vanilloid receptors in vitro (27, 325; but see Ref. 215). Yet, the fact that SR141716A blocks the motor inhibitory actions of AM404 at doses that are selective for CB1 receptors strongly argues for a predominant, if not unique, role of the endocannabinoid system in the behavioral response to AM404 administration. Furthermore, the pharmacological properties of AM404 are very different, often opposite to those of capsaicin and other vanilloid agonists. For example, capsaicin produces pain and bronchial smooth muscle constriction (336), whereas AM404 has no such effect when administered alone, and in fact enhances anandamide's analgesic and bronchodilatory actions (22, 49). The ability of intraperitoneal capsaicin to inhibit movement, described by Di Marzo et al. (91), superficially mimics one property of AM404, but should be viewed with caution, as it most likely results from the strong visceral pain and subsequent "freezing response" elicited by capsaicin. In conclusion, current evidence suggests that AM404 may magnify the actions of anandamide primarily by inhibiting the clearance of this compound from its sites of action.
6. Anandamide hydrolysis: role of FAAH
Almost a decade before anandamide was discovered, Schmid and collaborators (265) identified a hydrolase activity in rat liver that catalyzes the hydrolysis of fatty acid ethanolamides to free fatty acid and ethanolamine (265). That anandamide may be a substrate for such an activity was first suggested by biochemical experiments (80, 81, 89, 159, 351) and then demonstrated by molecular cloning, heterologous expression, and genetic disruption of the enzyme involved (68, 69).
FAAH (previously called anandamide amidohydrolase and oleamide hydrolase) is an intracellular membrane-bound protein whose primary structure displays significant homology with the "amidase signature family" of enzymes (69, 119). It acts as a hydrolytic enzyme for fatty acid ethanolamides such as anandamide, but also for esters such as 2-AG (134, 204) and primary amides such as oleamide (70). Site-directed mutagenesis experiments indicate that this unusually wide substrate preference may be due to a novel catalytic mechanism involving the amino acid residue lysine-142. This residue may act as a general acid catalyst, favoring the protonation and consequent detachment of reaction products from the enzyme's active site (279). This mechanism was recently confirmed by the solution of the crystal structure of FAAH complexed with the active site-directed inhibitor methoxy arachidonyl fluorophosphonate (34).
In addition to FAAH, other enzymes may participate in the breakdown of anandamide and its fatty acid ethanolamide analogs. A PEA-hydrolyzing activity distinct from FAAH was described in rat brain membranes (80) and human megakaryoblastic cells (352). This activity was purified to homogeneity from rat lung and shown to possess a marked substrate preference for PEA over anandamide (353). PEA does not bind to any of the known cannabinoid receptors but produces profound analgesic and anti-inflammatory effects (48, 238), which are prevented by the CB2-preferring antagonist SR144528 (48, 49). Future studies will undoubtedly address the relative roles of FAAH and this newly discovered enzyme in the biological disposition of PEA and anandamide.
The ability of FAAH to act in reverse (i.e., to synthesize anandamide from arachidonic acid and ethanolamine) has generated some confusion as to the mechanism of anandamide formation. Early reports of anandamide synthesis from free arachidonate and ethanolamine (81, 83) have now been unambiguously attributed to the reverse of the FAAH reaction (16, 181, 203). Because high concentrations of arachidonic acid and ethanolamine are needed to drive FAAH to work in reverse, it is unlikely that this reaction plays a physiological role in anandamide generation (see sect. IIB1). One possible exception is represented by the rat uterus, where substrate concentrations in the micromolar range are required for the synthetic reaction to occur, implying that FAAH or a similar enzyme might contribute to anandamide biosynthesis in this tissue (319).
7. Structure-activity relationship
Systematic structure-activity relationship investigations have identified several general requisites for substrate recognition by FAAH. First, FAAH accommodates a wide range of fatty acid amide substrates, but reducing the number of double bonds in the fatty acid chain generally results in a decrease in hydrolysis rate (29, 30, 80, 351). Second, replacing the ethanolamine moiety with a primary amide yields excellent substrates. For example, the rate of hydrolysis of arachidonamide is two to three times greater than anandamide's (29, 204). Third, anandamide congeners containing a tertiary nitrogen in the ethanolamine moiety are poor substrates (204). Fourth, introduction of a methyl group at the C2, C1', or C2' positions of anandamide yields analogs that are resistant to hydrolysis, probably due to increased steric hindrance around the carbonyl group (1, 204). Fifth, substrate recognition at the FAAH active site is stereoselective, at least with fatty acid ethanolamide congeners containing a methyl group in the C1' or C2' positions (1, 204). Finally, fatty acid esters such as 2-AG also are excellent substrates for FAAH activity in vitro (134, 279).
8. FAAH distribution in the CNS
Early biochemical experiments showed that FAAH activity is abundant throughout the CNS, with particularly high levels in the neocortex, the hippocampus, and the basal ganglia (80, 159). Subsequent investigations have confirmed this wide distribution. Thus, in situ hybridization studies in the rat have found that FAAH mRNA expression is higher in the neocortex and hippocampus; intermediate in the cerebellum, thalamus, olfactory bulb, and striatum; and lower in the hypothalamus, brain stem, and pituitary gland (340). Immunohistochemical experiments suggest that large principal neurons in the cerebral cortex, hippocampus, cerebellum, and olfactory bulb have the highest levels of FAAH immunoreactivity (95, 347). For example, large pyramidal neurons in the neocortex are prominently stained together with their apical and basal dendrites in layer V (347). Moderate immunostaining is observed also in the amygdala, the basal ganglia, the ventral and posterior thalamus, the deep cerebellar nuclei, the superior colliculus, the red nucleus, and motor neurons of the spinal cord (347). A more recent study reported staining of principal cells and astrocytes in various regions of the human brain (307). However, the protein recognized by the antibody utilized in these experiments has an apparent molecular mass of 50 kDa (by SDS-polyacrylamide gel electrophoresis), which does not correspond to that of native FAAH (60 kDa) (307).
Many FAAH-positive neurons throughout the brain are found in close proximity to axon terminals that contain CB1 cannabinoid receptors (see sect. III), providing important evidence for a role of FAAH in anandamide deactivation. Yet, there are multiple other regions of the brain where there is no such correlation, a discrepancy that likely reflects the participation of FAAH in the catabolism of other bioactive fatty acid ethanolamides, such as OEA (302) and PEA (48, 49).
9. Inhibitors of FAAH activity
A number of inhibitors of anandamide hydrolysis have been described, including fatty acid trifluoromethylketones, fluorophosphonates, -keto esters and -keto amides (30, 82, 198), bromoenol lactones (23), and nonsteroidal anti-inflammatory drugs (109, 110). These compounds lack, in general, target selectivity and biological availability; thus attempts to use them in vivo (64) should be interpreted with caution.
An emerging second generation of FAAH inhibitors comprises three groups of molecules. The first are fatty acid sulfonyl fluorides, such as palmitylsulfonylfluoride (AM374). AM374 irreversibly inhibits FAAH activity with an IC50 of 10 nM and displays a 50-fold preference for FAAH inhibition versus CB1 receptor binding (82). Systemic administration of AM374 enhances the operant lever-pressing response evoked by anandamide administration, but exerts no overt behavioral effect per se (310), raising the possibility that AM374 may protect anandamide from peripheral metabolism but may not have access to the brain. The second group of FAAH inhibitors is represented by a series of substituted -keto-oxazolopyridines, which are both reversible and extremely potent (30), but whose pharmacological selectivity and in vivo properties are not yet known. The third group is constituted by a class of aryl-substituted carbamate derivatives (185). The most potent member of this class, the compound URB597, inhibits FAAH activity with an IC50 value of 4 nM in brain membranes and an ID50
value of 0.1 mg/kg in live rats. This compound has 25,000-fold greater selectivity for FAAH than cannabinoid receptors, which is matched by an apparent lack of cannabimimetic effects in vivo (185). The pharmacological profile of URB597, which is currently under investigation, includes profound anti-anxiety effects accompanied by modest analgesia (185).
10. Physiological roles of FAAH
The generation of mutant mice in which the faah gene was disrupted by homologous recombination has shed much light on the role of FAAH in anandamide inactivation (68). FAAH -/- mice cannot metabolize anandamide and are therefore extremely sensitive to the pharmacological effects of this compound: doses of anandamide that are inactive in wild-type mice exert profound cannabimimetic effects in these mutants. FAAH -/- mice also have markedly elevated brain anandamide levels and reduced nociception (68). This finding is consistent with the roles of anandamide in the modulation of pain sensation (see, for review, Refs. 49, 173) and is supported by the analgesic activity of FAAH inhibitors (185).
Recently, a single nucleotide polymorphism in the human gene encoding for FAAH, which produces a proteolysis-sensitive variant of the enzyme, was found to be strongly associated with street drug and alcohol abuse (324). This important observation reinforces the central role played by the endocannabinoid system in the control of motivation and reward (228).
11. 2-AG hydrolysis: the role of monoglyceride lipase
The fact that FAAH catalyzes the hydrolysis of 2-AG along with anandamide's has prompted the suggestion that this enzyme may be responsible for eliminating both endocannabinoids. There is, however, strong evidence against this hypothesis. First, inhibitors of FAAH activity have no effect on [3H]2-AG hydrolysis at concentrations that completely block anandamide degradation (24). Second, 2-AG hydrolysis is preserved in mutant FAAH -/- mice, which do not degrade either endogenous or exogenous anandamide (212).
In agreement with these results, a 2-AG hydrolase activity distinct from FAAH has been identified and partially purified from porcine brain (133). This activity likely corresponds to monoglyceride lipase (MGL), a cytosolic serine hydrolase that converts 2- and 1-monoglycerides to fatty acid and glycerol (180). Several findings support this conclusion (93). First, heterologous expression of rat brain MGL confers strong 2-AG-hydrolyzing activity and MGL immunoreactivity to HeLa cells. Second, adenovirus-mediated transfer of the MGL gene in intact neurons increases MGL expression and shortens the life span of endogenously produced 2-AG, without any effect on either 2-AG synthesis or anandamide degradation. Third, MGL mRNA and protein are discretely distributed in the rat brain, with highest levels in regions where CB1 receptors are also present (93).
The distribution of MGL in the rat hippocampus is particularly noteworthy. The high density of MGL immunoreactivity in the termination zones of the glutamatergic Schaffer collaterals suggests a presynaptic localization of this enzyme at CA3-CA1 synapses. 2-AG may be produced by CA1 pyramidal cells during Schaffer collateral stimulation (328), and the newly generated endocannabinoid may mediate depolarization-induced suppression of inhibition (271, 374, 375; see sect. VC), if able to diffuse to the nearby GABAergic boutons, or a suppression of excitation (273, but see sect. VB2). Thus MGL is exquisitely poised to terminate the actions of 2-AG at hippocampal synapses.
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III. REGIONAL AND CELLULAR DISTRIBUTION OF NEURONAL CB1 CANNABINOID RECEPTORS |
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In a landmark study published in 1990, Herkenham and co-workers took advantage of the newly developed cannabinoid agonist [3H]CP-55,940, the same highly selective ligand that had helped identify cannabinoid receptors two years earlier (84), to investigate for the first time the distribution of cannabinoid binding sites in the brain (152). Their results showed that these sites strikingly coincide with the neural substrates for cannabinoid actions predicted from behavioral experiments and started a season of intense research on the CNS distribution of cannabinoid receptors. In the following pages, we will summarize the current status of this research, highlighting the correspondence between cannabinoid receptor distribution and behavioral effects of cannabimimetic agents. In the next sections, we focus on the cellular and subcellular localization of cannabinoid receptors and on the consequences of their physiological or pharmacological activation.
Various radioactive ligands (both agonists and antagonists) have been used to identify the sites of action of cannabimimetic drugs at the regional and cellular level (129, 149–152, 225, 299, 370). One surprising observation stemming from these binding experiments, and confirmed later with other neuroanatomical techniques, is that cannabinoid receptors are much more densely expressed in the rat brain than are any other G protein-coupled receptors (Fig. 5, A and C) (152). Indeed, in several brain regions cannabinoid receptors are present in densities that are comparable to those of GABA or glutamate receptor channels, which, owing to their relatively low ligand affinities, are highly concentrated at synapses to allow fast neurotransmission to occur. This puzzling finding is still unexplained but can be conceptualized in the light of recent discoveries suggesting that the synaptic functions served by the endocannabinoid system may be much broader than previously suspected. These functions, which will be discussed in detail in section VC, appear to be primarily concerned with the short-range, activity-dependent regulation of synaptic strength and to extend to a diversity of CNS structures.
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The broad regulatory roles of the endocannabinoids also may be surmised from the diverse effects of cannabimimetic drugs on physiology and behavior. In both animals and humans, these agents elicit a wide, but very distinctive spectrum of biological responses (166), which are epitomized by a tetrad comprising rigid immobility (catalepsy), decreased motor activity, analgesia, and hypothermia. This tetrad assay, developed by Billy R. Martin and his collaborators (see for example, Ref. 65), provides a convenient early screening to identify novel cannabimimetic drugs and highlights the role of the endocannabinoid system in motor behavior. Consistent with such a role, two brain regions that are intimately involved in movement regulation, the basal ganglia and the cerebellum, stand out among others for their very high densities of cannabinoid binding sites (Fig. 5, A and C). On the other hand, the marked binding capacities observed in limbic areas of the cerebral cortex, especially the cingulate and frontal cortices, as well as the amygdala, concord with the potent analgesic and antihyperalgesic properties of cannabinoid agonists and with their impact on emotional reactivity (Fig. 5, A and C) (115, 229). Although not as dense, significant cannabinoid binding is also found in other pain-processing areas of the CNS, including the PAG and the dorsal horn of the spinal cord. An important property of cannabimimetic agents, which is not modeled by the tetrad assay, relates to the ability of these compounds to influence cognitive functions, including short-term memory and attention (142). The high densities of cannabinoid binding sites in the hippocampus and other cortical structures provide a likely neural substrate for this property (Fig. 5, A and C).
Sheer density of CNS binding sites is not sufficient to precisely account for the spectrum of cannabinoid effects. Studies on the activation of G proteins by cannabinoid agonists in acutely dissected brain slices have revealed, indeed, the existence of an uneven coupling of cannabinoid receptors with G protein activation in different brain structures (37, 38, 304–306, 322). For example, receptors in structures such as the hypothalamus and the thalamus, although relatively low in number, display very tight G protein coupling, suggesting that they may be more efficacious than receptors found elsewhere in the brain. The molecular basis for these regional variations is unclear at present, but they may help reconcile the comparatively low density of cannabinoid receptors found in the hypothalamus with the profound neuroendocrine effects of cannabinoid drugs (261).
A quantitative summary of the distribution of cannabinoid binding sites in the rat brain has been provided (see Table 1 in Ref. 151). Similar distribution patterns have been found in other mammalian and nonmammalian species (see Fig. 5C), implying that the endocannabinoid system may play conserved roles in vertebrate phylogeny (57, 152).
B. Selective Expression of CB1 Cannabinoid Receptors by Identified Cell Types of Complex Networks
The mapping of brain cannabinoid binding sites by Herkenham et al. (152) preceded by a few months the molecular identification of the first cannabinoid receptor, the G protein-coupled receptor that is now called CB1 (236). A related gene encoding a second cannabinoid-sensitive G protein-coupled receptor, the CB2, was identified soon afterward (260). The CB1 receptor is distributed throughout the body but is predominantly found in neurons of the central and peripheral nervous systems. In contrast, the CB2 receptor is highly concentrated in immune cells and appears to be absent from CNS neurons (41, 113). Genetic deletion studies have confirmed that CB1 receptors contribute in a major way to the behavioral effects of cannabimimetic drugs. Thus mutant mice lacking functional CB1 receptors do not exhibit the tetrad of behavioral responses evoked by cannabinoid agonists (208, 380). As mentioned above, the tetrad only partially illustrates the complexity of cannabinoid actions and ostensibly excludes those involving cognitive systems. It is conceivable therefore that certain responses to cannabimimetic agents may be preserved in mutant CB1-/- mice (36, 88, 253). This possibility is strongly supported by electrophysiological experiments, which show that CB1-/- mice, although impaired in their CB1-mediated regulation of GABAergic transmission, retain an intact cannabinoid modulation of glutamate transmission (Fig. 12) (139). A parsimonious interpretation of these results, which is also consistent with current morphological data (Figs. 9A and 10, A and B) (138), is that glutamatergic axon terminals contain a cannabinoid-sensitive receptor that is molecularly distinct from CB1 (see sect. IVB1B).
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