Endocannabinoids in the central nervous system an overview

Transcription

1 Endocannabinoids in the central nervous system an overview E. Fride Department of Behavioral Sciences,College of Judea and Samaria, Ariel, Israel Prostaglandins, Leukotrienes and Essential FattyAcids (2002) 66(2&3),221^233 doi: /plef , available online at on Summary Many aspects of the physiology and pharmacology of anandamide (arachidonoyl ethanol amide), the first endogenous cannabinoid ligand ( endocannabinoid ) isolated from pig brain, have been studied since its discovery in1992. Ethanol amides from other fatty acids have also been identified as endocannabinoids with similar in vivo and in vitro pharmacological properties. 2-Arachidonoyl glycerol and noladin ether (2-arachidonyl glyceryl ether), isolated in1995 and 2001, respectively, so far, display pharmacological properties in the central nervous system, similar to those of anandamide.the endocannabinoids are widely distributedin brain, theyare synthesized andreleased upon neuronal stimulation, undergo reuptake and are hydrolyzed intracellularly by fatty acid amide hydrolase (FAAH). For therapeutic purposes, inhibitors of FAAH may provide more specific cannabinoid activities than direct agonists, and several such molecules have already been developed. Pharmacological effects of the endocannabinoids are very similar, yet not identical, to those of the plant-derived and synthetic cannabinoid receptor ligands.in addition to pharmacokinetic explanations, direct or indirect interactionswith other receptorshave been considered to explain some of these differences, including activities at serotonin and GABA receptors. Binding affinities for other receptors such as the vanilloid receptor, have to be taken into account in order to fully understand endocannabinoid physiology. Moreover, possible interactions with receptors for the lysophosphatidic acids deserve attention in future studies. Endocannabinoidshave beenimplicatedin avarietyof physiological functions.the areas of centralactivitiesinclude pain reduction, motor regulation, learning/memory, and reward. Finally, the role of the endocannabinoid system in appetite stimulation in the adult organism, and perhaps more importantly, its critical involvement in milk ingestion and survival of the newborn, may not only furtherourunderstanding ofthe physiologyof foodintake andgrowth, but mayalso find therapeutic applicationsinwasting disease and infant s failure to thrive. INTRODUCTION After the identification 1 and cloning 2 of the first cannabinoid (CB1) receptor and in view of the existence of an endogenous opioid opiate receptor signalling system, it was only natural to start the search for an endogenous ligand for the cannabinoid receptor. However, it took the insight that the putative ligand might be lipophilic, 3 in order to realize the discovery of the first endogenous Received 30 October 2001 Accepted 10 November 2001 Correspondence to: Ester Fride, Ph.D., Department of Behavioral Sciences, College of Judea and Samaria, Ariel, 44837,Israel.Tel./Fax: ; fride@research.yosh.ac.il This work was supported, in part, by the Israeli Ministry of Health. cannabinoid ligand ( endocannabinoid ), which turned out to be the ethanol amide of arachidonic acid (20:4, n-6) and was denoted as anandamide. 4 A second type of endocannabinoid was discovered in 1995, also a derivative of arachidonic acid, but its ester (2-arachidonoyl glycerol or 2-AG 5,6 ). Very recently, a third type has been reported, this time an ether of arachidonic acid (2-arachidonyl glyceryl ether, denoted as noladin ether 7 ). The endocannabinoid receptor signalling system, consisting of endocannabinoids, their receptor(s), uptake mechanism and hydrolyzing enzyme, is phylogenetically old, occurring across vertebrates 8 10 and invertebrate species. 11 Endocannabinoid molecules per se have been detected in mammals, including humans, 12 dogs, 5 rats 13,14 and pigs 4 and in fish. 11,15 Invertebrate species where the

2 222 Fride presence of endocannabinoids have been reported include molluscs, 16 Hydra vulgaris 17 and sea urchins. 18 However, no components of the endocannabinoid system have been detected in insects. 8,19 Brain tissue concentrations of 2-AG are approximately 200-fold higher than those of anandamide. 20 The rank order for the distribution of both endocannabinoids in different areas is similar: highest in brainstem, striatum and hippocampus and lower in cortex, diencephalon and cerebellum. No correlation was found between endocannabinoid concentrations and CB1 receptor distribution. Since receptor concentration and receptor activation were not correlated either, 21 the lack of association between endocannabinoid concentration and CB1 receptor distribution is not surprising. However, additional explanations for disparities between CB1 receptor distribution and activity have been offered and include the existence of non-cb1 receptor molecular targets for the endocannabinoids (see below). ENDOCANNABINOIDS AS SIGNALLING MOLECULES IN THE CENTRAL NERVOUS SYSTEM Anandamide is synthesized upon demand (stimulation), and released from neurons immediately afterwards Anandamide is inactivated by reuptake via a membranal transport molecule, the anandamide membrane transporter (AMT) and subsequent intracellular enzymatic degradation 23,26,27 by fatty acid amide hydroxylase (FAAH)-mediated hydrolysis. 9,28,29 2-Arachidonoylglycerol (2-AG) undergoes similar FAAH-mediated hydrolysis 9,30 and carrier-mediated transmembranal transport, 31 probably through the same anandamide membrane transporter. 32 FAAH and AMT are distributed in brain areas in a pattern corresponding to that of the CB1 receptor, i.e. high concentrations in hippocampus, cerebellum and cerebral cortex 29,30,33,34 thus further supporting the position that the endocannabinoids are true neurotransmitters. 35 It has been argued, based on structure activity relationships, that 2-AG is the natural ligand at the CB1 receptor. 14,36 On the other hand, the observation that anandamide, but not 2-AG was released upon depolarization in the rat striatum, 25 would suggest that anandamide rather than 2-AG is the primary ligand for the CB1 receptors, at least in the rat striatum controlling motor activity. The nature of endocannabinoid (anandamide and 2- AG) neurotransmission has been greatly clarified in a recent series of papers These new sets of data have been summarized by Christie and Vaughan: 39 endocannabinoids are released from a postsynaptic neuron upon stimulation, diffuse back to presynaptic neurons, where they act on CB1 receptors resulting in a reduced probability of neurotransmitters (such as glutamate and GABA) to be released. The removal of the endocannabinoids is accomplished by the uptake into neuronal or glial cells which enter via endocannabinoid transporters. Once inside the presynaptic cells, the endocannnabinoids are broken down by FAAH. It is too early to generalize these principles to noladin ether. Endocannabinoids in the central nervous system bind G i/o -coupled CB1 receptors which modulate adenylyl cyclase, ion channels and extracellular signal-regulated kinases. 21,24,35 Recently, it was determined that the CB1 receptor is coupled to ceramide, a lipid second messenger, which in turn, mediates cannabinoid-induced apoptosis. Such mechanism opens up new avenues of investigation for the ways by which the endocannabinoids control cell function. 40 DIFFERENCES BETWEEN THE PHARMACOLOGY OF EXO- AND ENDOCANNABINOIDS IN THE CENTRAL NERVOUS SYSTEM In vivo activity of cannabinoids is assessed in mice based on a battery of four assays designed by Martin and colleagues (motor activity, catalepsy, body temperature and analgesia). 41 Overall, anandamide displayed similar pharmacological effects compared to THC 42. Anandamide s shorter duration of action in vivo compared to that of the plant-derived and synthetic cannabinoids (by 1 h compared to several hours at least) has been attributed to anandamide s facile degradation 23,43 by FAAH. 28 Further differences between anandamide and THC, albeit subtle, also became apparent. Thus, anandamide has partial agonist activity in vitro for inhibition of forskolin-stimulated release of adenylate cyclase 44 and for inhibition of calcium currents in N18 neuroblastoma cells 45 and in vivo for some aspects of the tetrad (body temperature and analgesia). 3,42 When different routes of administering anandamide were compared, a complex pattern of full and partial activities was observed. 46 Further, D 9 -THC but not anandamide produced conditioned place avoidance. 47 Two more ethanol amides from fatty acids have been isolated from brain tissue. Like the original anandamide (ethanol amide of arachidonic acid), these molecules bind to CB1 receptors and have THC-like activities in the tetrad. Hence, it was decided to denote all three ethanol amides anandamides, each one derived from a different fatty acid: 20:4, n-6, arachidonoyl ethanol amide; 22:4, n-6, docosatetraenyl ethanol amide and 20:3, n-6, homog-linolenyl ethanol amide. 48 The latter two were shown to have even lower efficacies compared to anandamide (20:4, n-6). 49 Further, in vivo tolerance and crosstolerance to central effects of THC were detected upon

3 Endocannabinoids in the central nervous system 223 repeated doses of anandamide, 50,51 but unlike THCinduced tolerance, no cross-tolerance to the dynorphic system was observed. 51 Low doses of anandamide Further differences between the endocannabinoids and the prototypical THC include antagonistic activity of the anandamides at the CB1 receptor at very low doses. 52 Thus, preincubation of N18TG2 neuroblastoma cells with 1 nm anandamide antagonized forskolin-stimulated inhibition of adenylate cyclase activity, while pretreatment of mice with mg/kg of anandamide antagonized THC-induced cannabimimetic effects. Interestingly, the reverse (pretreatment with low-dose THC and testing for anandamide-induced effects) did not show inhibitory activity. 52 A possibly related phenomenon is the stimulatory activity of low doses (0.01 mg/kg) of anandamide in the tetrad of cannabimimetic effects as well as in a chemiluminescence assay for phagocytic activity. 53 It was suggested that this low-dose anandamide-induced stimulation may be ascribed to activation of Gs proteins, which are known to stimulate adenylate cyclase activity 52 opposite to the neurobehavioral depression which is mediated by G i/o protein-induced inhibition of adenylate cyclase. 54 Indeed, subsequent studies have provided support for this hypothesis. 55 Non-CB1 receptor-mediated stimulation of NMDA receptors by low concentrations of anandamide has been observed. 56 It is not known whether this mechanism underlies the behavioral observations. Much less is known about the in vivo pharmacology of 2-AG compared to anandamide. However, being deactivated by similar mechanisms as anandamide (see previous section), it is not surprising that 2-AG also has a short duration of action compared to plant-derived or synthetic cannabinoids, while partial agonist properties are also apparent for 2-AG. 5,56 Entourage effects Additional natural ethanol amides and glycerols, analogs of anandamide and 2-AG, respectively, but which do not bind CB1 receptors, have been detected in several biological tissues including brains. 57,58 Oleoylethanol amide and linoleoyl ethanol amide are prominent among the anandamide analogs, found in neural tissue 23,56,58 and by themselves, have weak but significant cannabimimetic effects, presumably by enhancing the extracellular levels and half-life of anandamide Oleamide or sleep factor induces sleep, or at least sleep-like behaviors 64 and cannabimimetic effects. 65,66 Since oleamide does not bind to the CB1 receptor, these effects have been ascribed in part at least, to oleamide s ability to inhibit anandamide hydrolysis and to enhance anandamide s affinity for the CB1 receptor. 65 The two 2-AG analogs palmitoyl glycerol and linoleoyl glycerol coexist and are co-released with anandamide. They potentiate in vivo and in vitro effects of 2-AG, thus enabling low concentrations of the endocannabinoid which by themselves have no overt activity, to have potent effects. They do this by potentiating 2-AG binding to the CB1 receptor while 2-linoleoyl glycerol also inhibits the inactivation of 2-AG in neuronal cells. 57,67 These effects were called entourage effects. 24,67 Whether the concept entourage effect should be extended to effects of the (ethanol) amides, discussed above, will have to be determined. The accumulating knowledge about the endocannabinoid-deactivating mechanismsffacilitated transport by the amide transporter (AMT) and hydrolysis by the enzyme FAAHFhas prompted research into the possibilities to develop AMT- and FAAH-inhibiting drugs as a means to enhance endocannabinoid availability, i.e. as indirect agonists. 29,68,69 It has been suggested that such inhibitors may have more selective therapeutic effects since their action would only be evident at sites where endocannabinoid production and release is taking place. 70 RECEPTORS OTHER THAN CB1 In general, cannabinoid-induced pharmacological effects are effectively inhibited by the CB1 receptor antagonist SR141716A. 71 Thus, significant blockade of THC-induced effects were observed in the mouse tetrad 72,73 and in the mouse tail-flick test for pain perception, when supraspinal CB1 receptors were exposed to the antagonist. 74 Also, THC- or anandamide-induced memory impairment was attenuated by SR141716A. 75 In rats, SR141716A effectively antagonized tetrad-like central effects induced by anandamide. 76 Moreover, anandamide-increased appetite was inhibited by SR141716A. 77 More recently, 2-AG-induced effects have been included in studies on the CB1 antagonist. Thus, SR141716A blocked the antiepileptiform effect of 2-AG, similar to that of anandamide, in rat hippocampal slices. 78,79 Hence, we have evidence now that both exogenous and endogenous cannabinoid-induced effects can be blocked by the CB1 antagonist. However, contrary to expectation, a number of anandamide-induced effects, although they are similar to THCinduced effects, could not be inhibited by the CB1 receptor antagonist. For example in mice, anandamideinduced effects in the tetrad were neither antagonized by SR141716A 73,80 nor by LY (Fride et al., unpublished observations). Further, SR141716A blocked the antinociceptive effects of THC in mice much more

4 224 Fride efficiently than those of anandamide. 74 This phenomenon has been explained as a pharmacokinetic effect since inhibition of anandamide-induced effects in the tetrad was accomplished when either a (non-specific) FAAH inhibitor, phenylmethylsulfonyl fluoride (PMSF), was coadministered with anandamide in order to enhance its half-life, or when a stable analog was administered instead of anandamide 81 (see also review by Nakamura- Palacios and colleagues 82 ). It is still not clear however, why the CB1 antagonist should only reverse the effects induced by anandamide, when its half-life is sufficiently prolonged (e.g. by PMSF). Does anandamide act via (an)other receptor in addition to the CB1 receptor? Observations on CB1 receptor knockout mice support such possibility. Thus, D 9 -THC-induced hypoalgesia in the tail-flick test was present, despite the gene deletion, in both knockout strains which were developed in different laboratories and from different parent strains. 83,84 Since the tail-flick test is presumably measuring spinal pain perception, while the hot plate test assays supraspinal mechanisms of pain, 85 this observation suggests that mainly higher level pain mechanisms are affected by CB1 receptor deletion. This is compatible with the observation that SR141716A, when injected intraperitoneally or intracerebrally, fully antagonized cannabinoid-induced analgesia, but only partially, when injected at the spinal level. 74 Since cannabinoid receptor-mediated pain has a spinal component, 86 these data suggest a non-cannabinoid receptor mechanism at the spinal level in addition to the CB1 receptor-mediated transduction. More recent experiments with the CB1 receptor knockout mice showed, surprisingly, that the CB1 / mice display anandamide-induced CB1 receptor-mediated 88 response including analgesia, catalepsy and motor inhibition, despite the absence of CB1 receptors. 87 This suggests that anandamide exerts some pharmacological effects which are similar to those induced by exogenous cannabinoids but which are not CB1 receptor mediated. Consistent with such interpretation is the observation that arachidonic acid, the major metabolite of anandamide 43 induced two of the four tetrad effects of cannabinoids, hypoactivity and antinociception, although it does not bind to CB1 receptors. 81 In the next sections, evidence for endocannabinoid mechanisms of action, other than via CB1 receptor activation, will be outlined. Non-receptor-mediated mechanisms for endocannabinoid activities in the central nervous system Before the first CB1 receptor was identified, it was generally assumed thatfin accordance with their lipid characteristicsfthe cannabinoids exert their pharmacological activities by non-receptor mechanisms. 3 Since then, many cannabinoid-induced effects have been ascribed to receptor activation. Yet, non-receptormediated effects of anandamide have also been demonstrated. 87 It has been previously suggested that the sleep factor oleamide may exert at least some of its actions by its ability to inhibit anandamide s degradation and reuptake. 65 However, non-specific membrane perturbation has been posited as a possible mechanism for oleamide s pharmacological effects. 89 Is it possible that anandamide is also such endogenous fluidity transmitter? Another potential mode of action previously studied, is the inhibition of gap junctions. 90,91 Receptor-mediated mechanisms for endocannabinoid activities in the central nervous system Known receptors as potential targets for endocannabinoids 5HT 3 receptors: Fan 92 has shown that cannabinoids including anandamide, inhibit 5-HT 3 receptor-mediated currents. These data indicated that the 5-HT 3 receptor ion channel is a site of action of cannabinoids and endocannabinoids. The direction of the effect is compatible with the antiemetic potential of cannabinoid agonists 93 since 5-HT 3 receptor antagonist are well-established antiemetic medicinal drugs. Fan s observation went largely unchallenged but was also not supported further by experimental evidence. However, we have observed that the 5-HT 3 antagonists MDL72222 and granisetron, show cannabinoid-like profiles in the mouse tetrad. 73 In assays for synaptosomal receptor binding, MDL72222 did not bind CB1 receptors; and vice versa, neither anandamide nor HU210 bound to 5-HT 3 receptors (Fride et al., in preparation). Thus, the nature of the interaction between CB1 and 5-HT 3 receptors needs to be clarified further. 5-HT 2 receptors: The current state of knowledge is complex. Mice injected with the 5-HT 2 receptor antagonist ketanserin displayed cannabimimetic effects on the tetrad, with potencies at least as high as those of anandamide. 73 Anandamide 94 and oleamide 95 have been found to bind to 5-HT 2 receptors, thus raising the possibility that endocannabinoids (and oleamide) may act by 5-HT 2 receptor blockade. In another study, however, no 3 H-ketanserin displacement at the 5-HT 2 receptor by oleamide was found. 96 Thus, the nature and physiological significance of endocannabinoid 5-HT 2 receptor interaction needs to be further investigated. NMDA receptors: Anandamide (but not THC) was found to have dual effects on NMDA receptor activity. 56 First, like THC, anandamide reduced calcium flux via CB1 receptors, this effect being reversed by SR141716A. Second, at low concentrations anandamide, but not THC, stimulated calcium influx by directly modulating the NMDA receptor. More recently, inhibition of

5 Endocannabinoids in the central nervous system 225 glutamatergic neurotransmission by the synthetic cannabinoid agonist WIN55,212-2 was reported in CB1 knockout mice. 97 It remains to be determined whether this finding has relevance to the observations at the NMDA receptor. Lysophosphatidic acid (LPA): LPA bears structural similarities to 2-AG. Contos et al. 98 have demonstrated that targeted deletion of the receptor gene for LPA, resulted in a defective suckling response in the knockout mice. This phenomenon is strikingly similar to the mortality of SR141716A-treated pups, which also die within days after birth due to a lack of milk ingestion from birth. 99 Therefore, it is possible that LPA and cannabinoids cross react with their respective receptors. The sparse data available so far do not support such hypothesis. Thus, SR141716A-induced inhibition of cannabinoid-stimulated p38 mitogen-activated protein kinases (MAPK), did not alter the effects of LPA on p38- MAPK phosphorylation. 100 Moreover, whereas THC completely reversed the effects of neonatally applied SR141716A, LPA did not. 99 In both these reports, oleoyl sn-glycero-3-phosphate were used. Thus, it remains to be seen whether other LPA species, notably LPA from arachidonic acid, will display cross-activity with cannabinoids. Vanilloid receptors: Vanilloid type 1 (VR1) receptors are not only found on sensory neurons, where they are partly coexpressed with CB1 receptors, 101 but also in several central nuclei including hypothalamus and basal ganglia, hippocampus and cerebellum. 102,103 In all these brain areas, CB1 receptors are found as well. 104 Anandamide is a full agonist at VR1 receptors. 101,105,106 Although still somewhat controversial, it appears now that sufficient amounts of anandamide are available in vivo to stimulate VR1 receptors under physiological conditions. 103,108,109 Summing up the evidence available at present, Di Marzo et al. 102 have suggested that anandamide interacts with both receptors at binding sites that are situated extra- or intra-cellularly for CB1 and VR1, respectively. 110 The specific dominant interaction depends on a number of factors such as ATP acting to enhance anandamide s effects at the VR1 receptor, levels of anandamide, tissue receptor distribution and accessibility to the receptor. 103,110 These observations suggest that anandamide may not only be an endocannabinoid but also an endovanilloid. 103 Unknown (novel) receptors for endocannabinoids in the central nervous system Several recent reports present evidence suggesting the existence of a new, unknown CB receptor in the brain. One report describes a reduction in amplitudes of excitatory postsynaptic currents by cannabinoids responsible for glutamatergic neurotransmisson in the hippocampus of wild-type mice as well as CB1 / knockout mice. 97 Further, Di Marzo and colleagues 87 showed that anandamide effectively produced major aspects of the tetrad and stimulated GTPgS binding in CB / mice; these effects were not inhibited by SR141716A. These findings were elaborated by Breivogel et al., 111 who observed that the putative receptor is not distributed in the brain in a fashion similar to that of CB1 receptors. Thus, anandamide and WIN bound to some brain regions of CB1 / knockout mice such as cortex, hippocampus and brainstem but not in the basal ganglia and cerebella of these mice. Future studies will have to determine whether these putative CB receptors are the same or different entities. It should be noted that the evidence in each of these papers relies to a great extent on experiments performed in CB1 knockout mice. The usefulness of this model for unraveling functions of the endocannabinoid system is indisputable. However, the repercussions of gene deletions for the organism start at conception. Therefore by the time the animal is born, let alone matured, as of yet undefined compensatory mechanisms may have developed. 112 Fortunately, the observation, however, that the expression of the putative receptor in wild-type and knockout mice is similar, 111 suggests that the gene deletion did not affect expression of the putative mechanisms. Interestingly, increasing levels of FAAH and the anandamide membrane transporter with age, were observed in knockout but not wild-type mice. 113 The authors proposed that this may represent a compensatory increase in anandamide degradation mechanisms which becomes more critical for the brain over time in order to reduce endocannabinoid-induced cell death. 113 This attractive hypothesis, however, will have to be reconciled with a decrease in CB1 receptors which was found in forebrain structures of wild-type aging rats Thus, as the concentrations of CB1 receptors decrease, a concomitant increase in FAAH and AMT would also be expected in wild-type animals. A recent study uncovering neuroprotective effects of 2- AG 117 would also argue against a beneficial effect of increasing levels of FAAH and AMT in aging CB1 / knockout mice. Summing up, it is possible that due to unknown compensatory mechanisms and/or other changes in the knockout mice, receptor types may become overexpressed which are physiologically irrelevant in the normal organism. Hence, evidence for new CB receptors would greatly be strengthened with experiments using normal tissue or animals. It is of benefit, however, that the two CB1 receptor knockout models are of different genetic backgrounds, 83,84 thereby allowing for some degree of generalization. Indeed, very recently, WIN55,212-2 was also shown to stimulate GTPgS binding

6 226 Fride in Ledent et al. s knockout mice. The regional distribution where this was observed, however (cerebellum, not hippocampus), 118 was different from Breivogel et al. s findings where for example, WIN55,212-2 stimulated GTPgS binding in hippocampal, but not cerebellar tissue of CB / mice. 111 PHYSIOLOGICAL FUNCTIONS OF ENDOCANNABINOIDS IN CENTRAL NERVOUS SYSTEM Pain Evidence for the use of cannabis as an analgesic medicine during childbirth as early as 1500 years ago has been described. 119 All three types of endocannabinoids (anandamide, 2-AG and noladin ether) have been shown to inhibit central pain perception (on a hot plate ) albeit not as efficaciously as THC. 5,7,42 As has been elegantly shown by Walker and colleagues 120 using in vivo microdialysis, anandamide is released in the periaqueductal gray (PAG), a midbrain area playing a pivotal role in pain perception, 121,122 in response to pain stimuli and to electrical stimulation of PAG. Moreover, hyperalgesia was observed after administration of the CB1 receptor antagonist SR141716A. 123 These findings strongly suggest that endocannabinoids maintain a tonic inhibition of pain. It should be noted, however, that in CB1 receptor knockout mice, the pain response to exposure to a hot plate was either unaffected 81 or reduced. 82 As noted above, compensatory or other changes in these knockout mice may explain this discrepancy. Opiate receptors are also richly distributed in PAG as well as in other areas, where CB1 receptors are found and thought to mediate pain. 124 Interactions between anandamide and opiates in the pain response have been demonstrated. 51 Motor functions, dopamine and schizophrenia CB1 receptors are richly distributed in the basal ganglia and cerebral cortex, regions which play a pivotal role in motor control Cannabinoids and endocannabinoids affect motor behavior in a biphasic or even triphasic fashion. 52,127 An intimate interactivity between the dopamine system and the endocannabinoids has been uncovered. Thus, earlier studies include cannabinoid-induced increases in dopamine release in the frontal brain regions 128 while chronic treatment with dopamine D 2 receptor antagonists resulted in increased CB1 receptor expression in the striatum. 129 Furthermore, localized application of cannabinoids into the nigrostriatal system in the rat counteracted the motor response to dopamine D 2 receptor agonists. 130,131 Direct studies on the endocannabinoid system indicated that stimulation of D 2 receptors enhances anandamide in the rat striatum, 132 while the anandamide transport blocker AM404 counteracted D 2 receptor-mediated responses such as apomorphine-induced yawning. 133 Based on the above, it is not surprising that investigations are conducted into the possibility to use cannabinoid-based medicines for the treatment of pathological conditions in which the dopamine system is thought to play a role. 126 These include Parkinson s 134 and Huntington s disease, 135 Tourette syndrome, 136 multiple sclerosis 137 and schizophrenia. 138 However, especially in the case of schizophrenia, the complexity and chronicity of the condition and its treatments 139,140 warrant further experimental work until widespread clinical applications may be endorsed. 141 Cognitive functions Hippocampus The hippocampus, a brain area with a high CB1 receptor density 142 fulfills a central role in learning and memory formation. 143 Both anandamide 144 and 2-AG 145 interfere with long-term potentiation in hippocampal slices, a physiological model for learning and memory. In vivo, anandamide impaired performance in a non-match-toposition task testing for working memory. 146 This was reversed by SR141716A. 75 If anandamide acts in similar ways as exocannabinoids in this mnemonic task, it would seem that the memory impairment occurs by interfering with the encoding of information which takes place in the hippocampus. As a result, short-term memory cannot be formed. 147 There is also evidence, however, that anandamide impairs memory consolidation. 148 Comparisons between mouse strains for an avoidance memory task indicated that dramatic strain-specific differences exist for the effects of anandamide on memory consolidation, causing inhibition or enhancement, depending on the strain. 149 Recent studies may clarify this complex situation. High doses disrupt the development of LTP as has been thought for a long time; however, endocannabinoid release may enhance memory by triggering depolarization induced suppression of inhibition 150 (DSI). Thus, different responses to cannabinoids in memory tasks may be explained by strain-dependent differences in concentrations of components of the endocannabinoid system. Prefrontal cortex Another important brain structure in cognitive function is the prefrontal cortex (PFC). Density of CB1 receptors in the PFC is high compared to other G-protein-coupled receptors. 151 This region is thought to integrate cognitive and emotional functions and may be the primary

7 Endocannabinoids in the central nervous system 227 dysfunctional area in schizophrenia and the site of action for antischizophrenic drugs. 152 Indeed, D 9 -THC produces schizophrenia-like symptoms in humans. 138 THC increased presynaptic dopamine efflux and utilization in the PFC, 128,153 while more recently, it has been demonstrated in PFC slices that cannabinoids influence glutamatergic synaptic transmission and plasticity. 154 Preliminary observations indicated that the PFC of acutely stressed mice (30 min of noise stress) contained 4 times as much anandamide as those of unstressed mice. Such increase was not seen in the hippocampi of these mice. 155 An interesting set of observations on children of marijuana-smoking mothers indicated that these children develop impaired executive functioning, which is thought to be a cognitive deficit of the PFC. 156 Taken together these observations suggest that the endocannabinoid and dopamine systems are closely cooperating in the regulatory role of the PFC in stress, cognition, and schizophrenia. Sleep Anandamide has been shown to increase slow-wave and REM sleep in rats at the expense of wakefulness, 148 while conversely, the CB1 receptor antagonist SR141716A increased wakefulness at the expense of slow-wave and REM sleep. 157 These findings support a role for the endocannabinoids in sleep regulation. As to the pharmacological basis of such action, anandamide has been found to bind to 5-HT 2 receptors. 94 However, behaviorally, clear differences can be detected between anandamide- and 5-HT 2 receptor antagonists-injected mice. It has been suggested, therefore, that the unique profile of anandamide (different from both that of D 9 -THC, a pure CB 1 agonist and ketanserin, a 5-HT 2 receptor antagonist) results from a combination of its interactions at CB 1 and 5-HT 2 receptors. 66 Feeding and appetite Cannabinoids enhance appetite. 93,158 Indeed D 9 -THC is used clinically for this purpose, particularly in acquired immunodeficiency syndrome (AIDS) and cancer patients. 93 Anandamide increased food intake in rats, 77 while SR141716A has been reported to inhibit the intake of palatable food Evidence for a function of the endogenous cannabinoid system in the feeding response has been obtained for the primitive invertebrate H. vulgaris. 17 These data point to a very ancient history of the endocannabinoid system in the regulation of feeding. Interestingly, preliminary clinical data suggest that SR141716A is a promising weight-reducing agent for the treatment of obesity. 162 Leptin is considered to be a key signal through which the hypothalamus senses the nutritional state of the body. 163 In a recent article, experiments were reported which indicate that leptin and endocannabinoids counterbalance each other s control of food intake. For example, it was demonstrated that leptin reduced endocannabinoid levels in the hypothalamus but not in the cerebellum of rats, and that endocannabinoid levels increase in animals with defective leptin signalling. 164 The possible role of CB1 receptors in maintaing food intake after fasting was suggested by experiments using CB1 knockout mice. 164 Endocannabinoids are present in milk, with 2-AG found in human milk in higher concentrations than anandamide AG when administered orally, albeit in high doses, is active in the mouse tetrad. 57 These findings indicate that 2-AG in milk may, in part at least, reach the central nervous system. Moreover, the observation that the levels of 2-AG, but not of anandamide, in rodent pup brain peak immediately after birth 165 may indicate a role for 2-AG in suckling in the newborn. We have recently reported that administration of SR141716A to mouse pups, within 24 h after birth, completely inhibited milk intake from the dam, thereby arresting growth and resulting in death within the first week of life. Injecting SR141716A on day 2 after birth, resulted in only 50% mortality. 99 These data strongly suggest that endocannabinoids (probably 2-AG) play a critical role in the survival of the newborn mouse by displaying an absolute control over milk ingestion. The generalizability to other species and the precise mechanism by which milk intake is blocked, awaits further clarification. In conclusion, clinical application for endocannabinoids or their direct or indirect agonists for infant s failure to thrive conditions deserves investigation. DEVELOPMENTAL ASPECTS OF THE ENDOCANNABINOID SYSTEM IN THE CENTRAL NERVOUS SYSTEM Initial reports studying development of the cannabinoid receptor system during the first weeks of postnatal life in the rat, described a gradual increase in brain CB1 receptor mrna 166 and in the density of CB1 receptors. 167,168 In later studies, investigating the gestational period, CB1 receptor mrna was detected from gestational day 11 in the rat. 169 Additional studies have uncovered more complex developmental patterns. Thus, whereas the highest levels of mrna expression of the CB1 receptor are seen at adulthood in regions such as the caudate putamen and the cerebellum, other areas such as the cerebral cortex, the hippocampus and the ventromedial hypothalamus display the highest mrna CB1 receptor levels on the first postnatal day. 165,170 Endocannabinoids

8 228 Fride were also detected from the gestational period in rodents, 2-AG at 1000-fold higher concentrations than anandamide. Interestingly, while anandamide displayed a gradual increase, 2-AG displayed constant levels throughout development with a single peak on the first postnatal day. 165 Is it possible that the high levels of CB1 receptor mrna and 2-AG which have been observed on the first day of life in structures including the hypothalamic ventromedial nucleus 165 (which is associated with feeding behavior) comprise a major stimulus for the newborn to initiate milk intake? (see also Feeding and appetite ). Atypical patterns (i.e. different from those in adult) of CB1 receptor densities were also observed: a transient presence of CB1 receptors was detected in white matter regions including the corpus callosum and anterior commissure (connecting neuronal pathways between the left and right hemispheres) between gestational day 21 and postnatal day 5, suggesting a role for endocannabinoids in brain development. 171 AMT and FAAH levels were higher in the brains of 6 months old CB1 receptor knockout mice compared to the wild-type animals. 113 More data are required before the biological significance of these findings can be fully understood. CONCLUSIONS An overview of the physiology and pharmacology of endocannabinoids in the central nervous system has been presented. Based on the data reviewed in this paper, three conclusions on the significance of the endocannabinoids are proposed: 1. The endocannabinoid system is widely distributed both phylogenetically and ontogenetically. K It appears in very ancient invertebrate species and in the most evolved mammals (phylogeny). For example, endocannabinoids have been shown to regulate food intake in very primitive (Hydra vulgaris) and in highly developed species (rodents and humans). K It fulfills important physiological roles from conception into adulthood (ontogeny). For example, anandamide has been shown to play a role in spermfertilizing capacity in sea urchins, 172 during implantation 173 (see Maccarrone et al. in this special issue) and in infant and adult food intake. 2. Biphasic effects of endocannabinoids have been shown repeatedly, compatible with the low levels found especially for anandamide in the brain. It may be conjectured that under normal physiological conditions, these low levels maintain an acceptable level of pain sensitivity, appetite and information processing (memory). When the brain is flooded with cannabinoids as after marijuana consumption and maybe during stress, motor reduction, hypoalgesia, enhanced appetite ( stress-induced eating ) 174 and memory inhibition become apparent. 3. The endocannabinoids are now known to be associated with a number of physiological functions including pain modulation, psychomotor functions, appetite regulation, memory, and reproduction. For most functions, endocannabinoids seem to play a modulating role. Thus, without endocannabinoids (such as in CB1 knockout mice or CB1 antagonisttreated animals), functions such as memory and pain are altered but the organism continues to function. In contrast, there are two reproduction-related functions where endocannabinoids have been shown to be absolutely critical for mammalian species. These are embryonal implantation 173 (see Maccarrone et al. in this special issue) and newborn milk intake. 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