Cannabinoids mediate analgesia largely via peripheral type 1 cannabinoid receptors in nociceptors

Although endocannabinoids constitute one of the first lines of defense against pain, the anatomical locus and the precise receptor mechanisms underlying cannabinergic modulation of pain are uncertain. Clinical exploitation of the system is severely hindered by the cognitive deficits, memory impairment, motor disturbances and psychotropic effects resulting from the central actions of cannabinoids. We deleted the type 1 cannabinoid receptor (CB1) specifically in nociceptive neurons localized in the peripheral nervous system of mice, preserving its expression in the CNS, and analyzed these genetically modified mice in preclinical models of inflammatory and neuropathic pain. The nociceptor-specific loss of CB1 substantially reduced the analgesia produced by local and systemic, but not intrathecal, delivery of cannabinoids. We conclude that the contribution of CB1-type receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia is paramount, which should enable the development of peripherally acting CB1 analgesic agonists without any central side effects.     

Cannabis and cannabinoids: pharmacology and rationale for clinical use

It is now known that there are at least two types of cannabinoid receptors. These are CB1 receptors, present mainly on central and peripheral neurones, and CB2 receptors, present mainly on immune cells. Endogenous cannabinoid receptor agonists ('endocannabinoids') have also been identified. The discovery of this 'endogenous cannabinoid system' has led to the development of selective CB1 and CB2 receptor ligands and fueled renewed interest in the clinical potential of cannabinoids. Two cannabinoid CB1 receptor agonists are already used clinically, as antiemetics or as appetite stimulants. These are D 9 - tetrahydrocannabinol (THC) and nabilone. Other possible uses for CB1 receptor agonists include the suppression of muscle spasm/spasticity associated with multiple sclerosis or spinal cord injury, the relief of chronic pain and the management of glaucoma and bronchial asthma. CB1 receptor antagonists may also have clinical applications, e. g. as appetite suppressants and in the management of schizophrenia or disorders of cognition and memory. So too may CB2 receptor ligands and drugs that activate cannabinoid receptors indirectly by augmenting endocannabinoid levels at cannabinoid receptors. When taken orally, THC seems to undergo variable absorption and to have a narrow 'therapeutic window' (dose range in which it is effective without producing significant unwanted effects). This makes it difficult to predict an oral dose that will be both effective and tolerable to a patient and indicates a need for better cannabinoid formulations and modes of administration. For the therapeutic potential of cannabis or CB1 receptor agonists to be fully exploited, it will be important to establish objectively and conclusively (a) whether these agents have efficacy against selected symptoms that is of clinical significance and, if so, whether the benefits outweigh the risks, (b) whether cannabis has therapeutic advantages over individual cannabinoids, (c) whether there is a need for additional drug treatments to manage any of the disorders against which cannabinoids are effective, and (d) whether it will be possible to develop drugs that have reduced psychotropic activity and yet retain the ability to act through CB1 receptors to produce their sought-after effects.     

Cannabinoid modulation of cutaneous Adelta nociceptors during inflammation

Previous studies have demonstrated that locally administered cannabinoids attenuate allodynia and hyperalgesia through activation of peripheral cannabinoid receptors (CB(1) and CB(2)). However, it is currently unknown if cannabinoids alter the response properties of nociceptors. In the present study, correlative behavioral and in vivo electrophysiological studies were conducted to determine if peripheral administration of the cannabinoid receptor agonists arachidonyl-2'-chloroethylamide (ACEA) or (R)-(+)-methanandamide (methAEA) could attenuate mechanical allodynia and hyperalgesia, and decrease mechanically evoked responses of Adelta nociceptors. Twenty-four hours after intraplantar injection of complete Freund's adjuvant (CFA), rats exhibited allodynia (decrease in paw withdrawal threshold) and hyperalgesia (increase in paw withdrawal frequency), which were attenuated by both ACEA and methAEA. The antinociceptive effects of these cannabinoids were blocked by co-administration with the CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophen yl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) but not with the CB(2) receptor antagonist 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-y l](4-methoxyphenyl)methanone (AM630). ACEA and methAEA did not produce antinociception under control, non-inflamed conditions 24 h after intraplantar injection of saline. In parallel studies, recordings were made from cutaneous Adelta nociceptors from inflamed or control, non-inflamed skin. Both ACEA and methAEA decreased responses evoked by mechanical stimulation of Adelta nociceptors from inflamed skin but not from non-inflamed skin, and this decrease was blocked by administration of the CB(1) receptor antagonist AM251. These results suggest that attenuation of mechanically evoked responses of Adelta nociceptors contributes to the behavioral antinociception produced by activation of peripheral CB(1) receptors during inflammation.     

Mode of action of cannabinoids on nociceptive nerve endings

In recent years, cannabinoids have emerged as attractive alternatives or supplements to therapy for chronic pain states. However, in humans the activation of cannabinoid receptors in neurons of the central nervous system is associated with psychotropic side effects, temporary memory impairment and dependence, which arise via the effects of cannabinoids on forebrain circuits. For clinical exploitation of the analgesic properties of cannabinoids, a major challenge is to devise strategies that reduce or abolish their adverse effects on cognitive, affective and motor functions without attenuating their analgesic effects. The cannabinoid receptor family currently includes two cloned metabotropic receptors: CB1, CB2 and possibly GPR55 which are distributed widely across many key loci in pain-modulating pathways, including the peripheral terminals of primary afferents. Modulation of transducer ion channels expressed at nociceptive terminals occurs upon activation of metabotropic cannabinoid receptors, but direct cannabinoid action on ion channels involved in sensory transduction or regulation of neuron excitability likely contributes to the peripheral cannabinoid effects.     

The effects of cannabinoids on the brain.

Cannabinoids have a long history of consumption for recreational and medical reasons. The primary active constituent of the hemp plant Cannabis sativa is delta9-tetrahydrocannabinol (delta9-THC). In humans, psychoactive cannabinoids produce euphoria, enhancement of sensory perception, tachycardia, antinociception, difficulties in concentration and impairment of memory. The cognitive deficiencies seem to persist after withdrawal. The toxicity of marijuana has been underestimated for a long time, since recent findings revealed delta9-THC-induced cell death with shrinkage of neurons and DNA fragmentation in the hippocampus. The acute effects of cannabinoids as well as the development of tolerance are mediated by G protein-coupled cannabinoid receptors. The CB1 receptor and its splice variant CB1A, are found predominantly in the brain with highest densities in the hippocampus, cerebellum and striatum. The CB2 receptor is found predominantly in the spleen and in haemopoietic cells and has only 44% overall nucleotide sequence identity with the CB1 receptor. The existence of this receptor provided the molecular basis for the immunosuppressive actions of marijuana. The CB1 receptor mediates inhibition of adenylate cyclase, inhibition of N- and P/Q-type calcium channels, stimulation of potassium channels, and activation of mitogen-activated protein kinase. The CB2 receptor mediates inhibition of adenylate cyclase and activation of mitogen-activated protein kinase. The discovery of endogenous cannabinoid receptor ligands, anandamide (N-arachidonylethanolamine) and 2-arachidonylglycerol made the notion of a central cannabinoid neuromodulatory system plausible. Anandamide is released from neurons upon depolarization through a mechanism that requires calcium-dependent cleavage from a phospholipid precursor in neuronal membranes. The release of anandamide is followed by rapid uptake into the plasma and hydrolysis by fatty-acid amidohydrolase. The psychoactive cannabinoids increase the activity of dopaminergic neurons in the ventral tegmental area-mesolimbic pathway. Since these dopaminergic circuits are known to play a pivotal role in mediating the reinforcing (rewarding) effects of the most drugs of abuse, the enhanced dopaminergic drive elicited by the cannabinoids is thought to underlie the reinforcing and abuse properties of marijuana. Thus, cannabinoids share a final common neuronal action with other major drugs of abuse such as morphine, ethanol and nicotine in producing facilitation of the mesolimbic dopamine system.     

Behavioral effects of cannabinoid agents in animals

Two subtypes of cannabinoid receptors have been identified to date, the CB1 receptor, essentially located in the CNS, but also in peripheral tissues, and the CB2 receptor, found only at the periphery. The identification of delta9-tetrahydrocannabinol (delta9-THC) as the major active component of marijuana (Cannabis sativa), the recent emergence of potent synthetic ligands and the identification of anandamide and sn-2 arachidonylglycerol as putative endogenous ligands for cannabinoid receptors in the brain, have contributed to advancing cannabinoid pharmacology and approaching the neurobiological mechanisms involved in physiological and behavioral effects of cannabinoids. Most of the agonists exhibit nonselective affinity for CB1/CB2 receptors, and delta9-THC and anandamide probably act as partial agonists. Some recently synthesized molecules are highly selective for CB2 receptors, whereas selective agonists for the CB1 receptors are not yet available. A small number of antagonists exist that display a high selectivity for either CB1 or CB2 receptors. Cannabinomimetics produce complex pharmacological and behavioral effects that probably involve numerous neuronal substrates. Interactions with dopamine, acetylcholine, opiate, and GABAergic systems have been demonstrated in several brain structures. In animals, cannabinoid agonists such as delta9-THC, WIN 55,212-2, and CP 55,940 produce a characteristic combination of four symptoms, hypothermia, analgesia, hypoactivity, and catalepsy. They are reversed by the selective CB1 receptor antagonist, SR 141716, providing good evidence for the involvement of CB1-related mechanisms. Anandamide exhibits several differences, compared with other agonists. In particular, hypothermia, analgesia, and catalepsy induced by this endogenous ligand are not reversed by SR 141716. Cannabinoid-related processes seem also involved in cognition, memory, anxiety, control of appetite, emesis, inflammatory, and immune responses. Agonists may induce biphasic effects, for example, hyperactivity at low doses and severe motor deficits at larger doses. Intriguingly, although cannabis is widely used as recreational drug in humans, only a few studies revealed an appetitive potential of cannabimimetics in animals, and evidence for aversive effects of delta9-THC, WIN 55,212-2, and CP 55,940 is more readily obtained in a variety of tests. The selective blockade of CB1 receptors by SR 141716 impaired the perception of the appetitive value of positive reinforcers (food, cocaine, morphine) and reduced the motivation for sucrose, beer and alcohol consumption, indicating that positive incentive and/or motivational processes could be under a permissive control of CB1-related mechanisms. There is little evidence that cannabinoid systems are activated under basal conditions. However, by using SR 141716 as a tool, a tonic involvement of a CB1-mediated cannabinoid link has been demonstrated, notably in animals suffering from chronic pain, faced with anxiogenic stimuli or highly motivational reinforcers. Some effects of SR 141716 also suggest that CB1-related mechanisms exert a tonic control on cognitive processes. Extensive basic research is still needed to elucidate the roles of cannabinoid systems, both in the brain and at the periphery, in normal physiology and in diseases. Additional compounds, such as selective CB1 receptor agonists, ligands that do not cross the blood brain barrier, drugs interfering with synthesis, degradation or uptake of endogenous ligand(s) of CB receptors, are especially needed to understand when and how cannabinoid systems are activated. In turn, new therapeutic strategies would likely to emerge.     

The CB1 cannabinoid receptor mediates glutamatergic synaptic suppression in the hippocampus

Cannabinoids have profound effects on synaptic function and behavior. Of the two cloned cannabinoid receptors, cannabinoid receptor 1 (CB1) is widely distributed in the CNS and accounts for most of the neurological effects of cannabinoids, while cannabinoid receptor 2 (CB2) expression in the CNS is very limited. The presence of additional receptors [i.e. cannabinoid receptor 3 (CB3)] is suggested by growing evidence of cannabinoid effects that are not mediated by CB1 or CB2. The most direct functional evidence for a CB3 comes from a study in hippocampus where deletion of CB1 was shown to have no effect on cannabinoid-mediated suppression of the excitatory synapse between Schaffer collateral/commissural fibers and CA1 pyramidal cells [Novel cannabinoid-sensitive receptor mediates inhibition of glutamatergic synaptic transmission in the hippocampus. Neuroscience 106:1-4]. In contrast, we report here that in extracellular field recordings, the cannabinoid agonist WIN 55,212-2 (5 microM) had no effect on Schaffer collateral/commissural fiber-CA1 pyramidal cell (Sch-CA1) synaptic transmission in slices from two independently made cannabinoid receptor 1-/- lines [Zimmer et al 1999 and Ledent et al 1999] while strongly suppressing Sch-CA1 synaptic transmission in CB1+/+ mice of the background strains. Also, we observed robust cannabinoid-mediated suppression of the Sch-CA1 synapse in pure C57BL/6 mice, contradicting a recent report that cannabinoid suppression of this synapse is absent in this strain [Hoffman AF, Macgill AM, Smith D, Oz M, Lupica CR (2005) Species and strain differences in the expression of a novel glutamate-modulating cannabinoid receptor in the rodent hippocampus. Eur J Neurosci 22:2387-2391]. Our results strongly suggest that cannabinoid-induced suppression of the Sch-CA1 synapse is mediated by CB1. Non-canonical cannabinoid receptors do not seem to play a major role in inhibiting transmitter release at this synapse.     

The neuronal distribution of cannabinoid receptor type 1 in the trigeminal ganglion of the rat

Cannabinoid compounds have been shown to produce antinociception and antihyperalgesia by acting upon cannabinoid receptors located in both the CNS and the periphery. A potential mechanism by which cannabinoids could inhibit nociception in the periphery is the activation of cannabinoid receptors located on one or more classes of primary nociceptive neurons. To address this hypothesis, we evaluated the neuronal distribution of cannabinoid receptor type 1 (CB1) in the trigeminal ganglion (TG) of the adult rat through combined in situ hybridization (ISH) and immunohistochemistry (IHC). CB1 receptor mRNA was localized mainly to medium and large diameter neurons of the maxillary and mandibular branches of the TG. Consistent with this distribution, in a de facto nociceptive sensory neuron population that exhibited vanilloid receptor type 1 immunoreactivity, colocalization with CB1 mRNA was also sparse (<5%). Furthermore, very few neurons (approximately 5%) in the peptidergic (defined as calcitonin gene-related peptide- or substance P-immunoreactive) or the isolectin B4-binding sensory neuron populations contained CB1 mRNA. In contrast, and consistent with the neuron-size distribution for CB1, nearly 75% of CB1-positive neurons exhibited N52-immunoreactivity, a marker of myelinated axons. These results indicate that in the rat TG, CB1 receptors are expressed predominantly in neurons that are not thought to subserve nociceptive neurotransmission in the noninjured animal. Taken together with the absence of an above background in situ signal for CB2 mRNA in TG neurons, these findings suggest that the peripherally mediated antinociceptive effects of cannabinoids may involve either as yet unidentified receptors or interaction with afferent neuron populations that normally subserve non-nociceptive functions.     

The rodent amygdala contributes to the production of cannabinoid-induced antinociception

The amygdala is a temporal lobe region that is implicated in emotional information processing. The amygdala also is associated with the processing and modulation of pain sensation. Recently, we demonstrated that in nonhuman primates, the amygdala is necessary for the full expression of cannabinoid-induced antinociception [J Neurosci 21 (2001) 8238]. The antinociceptive effect of the cannabinoid receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo(1,2,3-de)-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,212-2) was significantly reduced in rhesus monkeys with large bilateral lesions of the amygdaloid complex. In the present study, we investigated the contribution of the amygdala to cannabinoid-induced antinociception in the rat. Using bilateral local microinjections of the GABA_A receptor agonist muscimol, we inactivated neurons originating from the central nucleus of the amygdala (CeA) or basolateral nucleus of the amygdala (BLA). In rats injected with intra-CeA saline, the cannabinoid receptor agonist WIN55,212-2 produced dose-dependent antinociception on the noxious heat-evoked tail flick assay. In rats treated with intra-CeA muscimol, however, the antinociceptive effect of WIN55,212-2 was significantly reduced. Rats treated with intra-BLA muscimol showed no deficit in WIN55,212-2-induced antinociception. The effect of CeA inactivation on WIN55,212-2-induced suppression of prolonged pain in the formalin test also was tested. In rats treated with intra-CeA saline, WIN55,212-2 reduced the incidence of formalin-induced nociceptive behaviors and also reduced formalin-evoked c-fos expression in both superficial and deep laminae of the spinal cord dorsal horn. In rats treated with intra-CeA muscimol, however, these effects of WIN55,212-2 were significantly reduced.

The results constitute the first causal data demonstrating the necessity of descending pain-modulatory circuitry (of which the CeA is a component) for the full expression of cannabinoid-induced antinociception in the rat. Furthermore, the results complement previous findings suggesting an overlap in neural circuitry activated by opioids and cannabinoids.     

Endocannabinoids and cannabinoid receptor genetics

This review presents the remarkable advances that have been achieved in marijuana (cannabinoid) research, with the discovery of specific receptors and the existence of naturally occurring cannabis-like substances in the human body and brain. The last decade has seen more rapid progress in marijuana research than any time in the thousands of years that marijuana has been used by humans, particularly in cannabinoid genomics. The cDNA and genomic sequences encoding G protein-coupled cannabinoid receptors (Cnrs) from several species have now been cloned. Endogenous cannabinoids (endocannabinoids), synthetic and hydrolyzing enzymes and transporters that define neurochemically-specific cannabinoid brain pathways have been identified. Endocannabinoid lipid signaling molecules alter activity at G protein-coupled receptors (GPCR) and possibly at anandamide-gated ion channels, such as vanilloid receptors. Availability of increasingly-specific CB1 and CB2 Cnr antagonists and of CB1 and CB2 Cnr knockout mice have increased our understanding of these cannabinoid systems and provides tantalizing evidence for even more G protein-coupled Cnrs. Initial studies of the Cnr gene structure, regulation and polymorphisms whet our appetite for more information about these interesting genes, their variants and roles in vulnerabilities to addictions and other neuropsychiatric disorders. Behavioral studies of cannabinoids document the complex interactions between rewarding and aversive effects of these drugs. Pursuing cannabinoid-related molecular, pharmacological and behavioral leads will add greatly to our understanding of endogenous brain neuromodulator systems, abused substances and potential therapeutics. This review of CB1 and CB2 Cnr genes in human and animal brain and their neurobiological effects provide a basis for many of these studies. Therefore, understanding the physiological cannabinoid control system in the human body and brain will contribute to elucidating this natural regulatory mechanism in health and disease.     

Cannabinoids modulate the P-type high-voltage-activated calcium currents in purkinje neurons

Endocannabinoids released by postsynaptic cells inhibit neurotransmitter release in many central synapses by activating presynaptic cannabinoid CB1 receptors. In particular, in the cerebellum, endocannabinoids inhibit synaptic transmission at granule cell to Purkinje cell synapses by modulating presynaptic calcium influx via N-, P/Q-, and R-type calcium channels. Using whole cell patch-clamp techniques, we show that in addition to this presynaptic action, both synthetic and endogenous cannabinoids inhibit P-type calcium currents in isolated rat Purkinje neurons independent of CB1 receptor activation. The IC50 for the anandamide (AEA)-induced inhibition of P-current peak amplitude was 1.04 +/- 0.04 microM. In addition, we demonstrate that all the tested cannabinoids in a physiologically relevant range of concentrations strongly accelerate inactivation of P currents. The effects of AEA cannot be attributed to the metabolism of AEA because a nonhydrolyzing analogue of AEA, methanandamide inhibited P-type currents with a similar efficacy. All effects of cannabinoids on P-type Ca2+ currents were insensitive to antagonists of CB1 cannabinoid or vanilloid TRPV1 receptors. In cerebellar slices, WIN 55,212-2 significantly affected spontaneous firing of Purkinje neurons in the presence of CB1 receptor antagonist, in a manner similar to that of a specific P-type channel antagonist, indicating a possible functional implication of the direct effects of cannabinoids on P current. Taken together these findings demonstrate a functionally important direct action of cannabinoids on P-type calcium currents.     

Cardiovascular actions of cannabinoids and their generation during shock

Marijuana is a widely abused recreational drug well known for its psychoactive properties. Cannabinoids, the active ingredients of marijuana, elicit their neurobehavioral effects by interacting with the CB₁ cannabinoid receptor subtype, expressed primarily in the brain but also present in some peripheral tissues. A second receptor subtype, the CB₂ receptor, is expressed on cells of the immune system and is thought to be responsible for the immunosuppressant effects of cannabinoids. Recently, endogenous lipidlike substances have been identified, including arachidonyl ethanolamide (anandamide) and 2-arachidonyl glyceride, that bind to cannabinoid receptors and mimic many of the neurobehavioral effects of plant-derived cannabinoids. Both plant-derived cannabinoids and the endogenous ligands have been shown to elicit hypotension and bradycardia via activation of peripherally located CB₁ receptors. Possible underlying mechanisms include presynaptic CB₁ receptor mediated inhibition of norepinephrine release from peripheral sympathetic nerve terminals, and/or direct vasodilation via activation of vascular cannabinoid receptors. The latter may also be the target of endocannabinoids of vascular endothelial origin. Recent studies indicate that a peripheral endogenous cannabinoid system in circulating macrophages and platelets is activated in hemorrhagic and septic shock and may contribute to the hypotension associated with these conditions via activation of vascular cannabinoid receptors. The potential role of this mechanism in human shock conditions is under investigation. Received: 20 May 1998 / Accepted: 24 August 1998     

Endogenous cannabinoid receptor agonists inhibit neurogenic inflammations in guinea pig airways

BACKGROUND: Although neurogenic inflammation via the activation of C fibers in the airway must have an important role in the pathogenesis of asthma, their regulatory mechanism remains uncertain. OBJECTIVE: The pharmacological profiles of endogenous cannabinoid receptor agonists on the activation of C fibers in airway tissues were investigated and the mechanisms how cannabinoids regulate airway inflammatory reactions were clarified. METHODS: The effects of endogenous cannabinoid receptor agonists on electrical field stimulation-induced bronchial smooth muscle contraction, capsaicin-induced bronchoconstriction and capsaicin-induced substance P release in guinea pig airway tissues were investigated. The influences of cannabinoid receptor antagonists and K+ channel blockers to the effects of cannabinoid receptor agonists on these respiratory reactions were examined. RESULTS: Both endogenous cannabinoid receptor agonists, anandamide and palmitoylethanolamide, inhibited electrical field stimulation-induced guinea pig bronchial smooth muscle contraction, but not neurokinin A-induced contraction. A cannabinoid CB2 antagonist, SR 144528, reduced the inhibitory effect of endogenous agonists, but not a cannabinoid CB1 antagonist, SR 141716A. Inhibitory effects of agonists were also reduced by the pretreatment of large conductance Ca2+ -activated K+ channel (maxi-K+ channel) blockers, iberiotoxin and charybdotoxin, but not by other K+ channel blockers, dendrotoxin or glibenclamide. Anandamide and palmitoylethanolamide blocked the capsaicin-induced release of substance P-like immunoreactivity from guinea pig airway tissues. Additionally, intravenous injection of palmitoylethanolamide dose-dependently inhibited capsaicin-induced guinea pig bronchoconstriction, but not neurokinin A-induced reaction. However, anandamide did not reduce capsaicin-induced guinea pig bronchoconstriction. CONCLUSIONS: These findings suggest that endogenous cannabinoid receptor agonists inhibit the activation of C fibers via cannabinoid CB2 receptors and maxi-K+ channels in guinea pig airways.     

Control of the cell survival/death decision by cannabinoids

Cannabinoids, the active components of Cannabis sativa (marijuana), and their derivatives produce a wide spectrum of central and peripheral effects, some of which may have clinical application. The discovery of specific cannabinoid receptors and a family of endogenous ligands of those receptors has attracted much attention to cannabinoids in recent years. One of the most exciting and promising areas of current cannabinoid research is the ability of these compounds to control the cell survival/death decision. Thus cannabinoids may induce proliferation, growth arrest, or apoptosis in a number of cells, including neurons, lymphocytes, and various transformed neural and nonneural cells. The variation in drug effects may depend on experimental factors such as drug concentration, timing of drug delivery, and type of cell examined. Regarding the central nervous system, most of the experimental evidence indicates that cannabinoids may protect neurons from toxic insults such as glutamaergic overstimulation, ischemia and oxidative damage. In contrast, cannabinoids induce apoptosis of glioma cells in culture and regression of malignant gliomas in vivo. Breast and prostate cancer cells are also sensitive to cannabinoid-induced antiproliferation. Regarding the immune system, low doses of cannabinoids may enhance cell proliferation, whereas high doses of cannabinoids usually induce growth arrest or apoptosis. The neuroprotective effect of cannabinoids may have potential clinical relevance for the treatment of neurodegenerative disorders such as multiple sclerosis, Parkinson's disease, and ischemia/stroke, whereas their growth-inhibiting action on transformed cells might be useful for the management of malignant brain tumors. Ongoing investigation is in search for cannabinoid-based therapeutic strategies devoid of nondesired psychotropic effects.     

Role of endogenous cannabinoids in synaptic signaling

Research of cannabinoid actions was boosted in the 1990s by remarkable discoveries including identification of endogenous compounds with cannabimimetic activity (endocannabinoids) and the cloning of their molecular targets, the CB1 and CB2 receptors. Although the existence of an endogenous cannabinoid signaling system has been established for a decade, its physiological roles have just begun to unfold. In addition, the behavioral effects of exogenous cannabinoids such as delta-9-tetrahydrocannabinol, the major active compound of hashish and marijuana, await explanation at the cellular and network levels. Recent physiological, pharmacological, and high-resolution anatomical studies provided evidence that the major physiological effect of cannabinoids is the regulation of neurotransmitter release via activation of presynaptic CB1 receptors located on distinct types of axon terminals throughout the brain. Subsequent discoveries shed light on the functional consequences of this localization by demonstrating the involvement of endocannabinoids in retrograde signaling at GABAergic and glutamatergic synapses. In this review, we aim to synthesize recent progress in our understanding of the physiological roles of endocannabinoids in the brain. First, the synthetic pathways of endocannabinoids are discussed, along with the putative mechanisms of their release, uptake, and degradation. The fine-grain anatomical distribution of the neuronal cannabinoid receptor CB1 is described in most brain areas, emphasizing its general presynaptic localization and role in controlling neurotransmitter release. Finally, the possible functions of endocannabinoids as retrograde synaptic signal molecules are discussed in relation to synaptic plasticity and network activity patterns.     

CB1 cannabinoid receptors in amphibian spinal cord: relationships with some nociception markers

The role of cannabinoids in spinal analgesia has so far been investigated in mammals and the interactions between cannabinoid receptors and markers involved in nociception have been described in the rat spinal cord. An endocannabinoid system is well developed also in the amphibian brain. However, the anatomical substrates of pain modulation have been scarcely investigated in anamniotes, neither is there reference to such a role for cannabinoids in lower vertebrates. In the present paper we employed multiple cytochemical approaches to study the distribution of CB1 cannabinoid receptors and their morphofunctional relationships with some nociception markers (i.e. Substance P, nitric oxide synthase, GABA and mu opioid receptors) in the spinal cord of the anuran amphibian Xenopus laevis. We found a co-distribution of CB1 receptors with the aforementioned signaling molecules, as well as a more limited cellular co-localization, in the dorsal and central fields of the spinal cord. These regions correspond to the mammalian laminae I-IV and X, respectively, areas strongly involved in spinal analgesia. Comparison of these results with those previously obtained in the mammalian spinal cord, reveals a number of similarities between the two systems and suggests that cannabinoids might participate in the control of pain sensitivity also in the amphibian spinal cord.     

Regulation of cannabinoid CB1 receptors in the central nervous system by chronic cannabinoids

Marijuana produces a number of characteristic behaviors in humans and animals, including memory impairment, antinociception, and locomotor and psychoactive effects. However, tolerance and dependence to cannabinoids develops after chronic use, as demonstrated both clinically and in animal models. The potential therapeutic benefits of certain cannabinoid-mediated effects, as well as the use of marijuana for its psychoactive properties, has raised interest in understanding the cellular adaptations produced by chronic administration of this class of drugs. The primary active constituent of marijuana, delta9-tetrahydrohydrocannabinol (THC), binds to specific G-protein-coupled receptors. The central nervous system (CNS) effects of THC are mediated by CB1 receptors, which couple primarily to inhibitory G-proteins. High levels of CB1 receptors are found in the basal ganglia, hippocampus, cortex, and cerebellum, consistent with the profile of behavioral effects. Studies over the past decade have determined that CB1 receptors undergo downregulation and desensitization following chronic administration of THC or synthetic cannabinoid agonists. In general, these adaptations are regionally widespread and of considerable magnitude, and are thought to contribute to tolerance to cannabinoid-mediated behavioral effects. Adaptation at the effector level has been more difficult to characterize, although it appears that alterations in cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) activity may be particularly important in cannabinoid dependence. A striking characteristic of CB 1 receptor adaptation is the region dependence of the magnitude and rate of development of downregulation and desensitization. These regional differences may provide interesting insights into the mechanisms of CB1 receptors receptor signaling in different brain regions. Moreover, region-specific adaptations in CB1 receptors following chronic cannabinoid administration may produce differential adaptations at the in vivo level.     

Antinociception and modulation of rostral ventromedial medulla neuronal activity by local microinfusion of a cannabinoid receptor agonist

Systemic administration of a cannabinoid agonist produces antinociception through the activation of pain modulating neurons in the rostral ventromedial medulla (RVM). The aim of the present study was to determine how a cannabinoid receptor agonist acting directly within the RVM affects neuronal activity to produce behaviorally measurable antinociception. In lightly anesthetized rats, two types of RVM neurons have been defined based on changes in tail flick-related activity. On-cells increase firing (on-cell burst), whereas off-cells cease firing (off-cell pause), just prior to a tail flick. The cannabinoid receptor agonist WIN55,212-2 was microinfused directly into the RVM while monitoring tail flick latencies and on- and off-cell activity. Microinfusion of WIN55,212-2 (2.0 microg/microl and 0.4 microg/microl) reduced the tail flick-related on-cell burst, decreased the duration of the off-cell pause, and increased off-cell ongoing activity. These changes were prevented by co-infusing the CB1 receptor antagonist, SR141716A (0.35 microg/microl), with WIN55,212-2 (0.4 microg/microl). Furthermore, 2.0 microg/microl WIN55,212-2 delayed the onset of the off-cell pause and increased tail flick latencies. Microinfusion of WIN55,212-2 to brain regions caudal or lateral to the RVM had no effect on RVM neuronal activity or tail flick latencies. These results indicate that cannabinoids act directly within the RVM to affect off-cell activity, providing one mechanism by which cannabinoids produce antinociception.     

Recent advantages in cannabinoid research

Although the active component of cannabis Delta9-THC was isolated by our group 35 years ago, until recently its mode of action remained obscure. In the last decade it was established that Delta9-THC acts through specific receptors - CB1 and CB2 - and mimics the physiological activity of endogenous cannabinoids of two types, the best known representatives being arachidonoylethanolamide (anandamide) and 2-arachidonoylglycerol (2-AG). THC is officially used against vomiting caused by cancer chemotherapy and for enhancing appetite, particularly in AIDS patients. Illegally, usually by smoking marijuana, it is used for ameliorating the symptoms of multiple sclerosis, against pain, and in a variety of other diseases. A synthetic cannabinoid, HU-211, is in advanced clinical tests against brain damage caused by closed head injury. It may prove to be valuable against stroke and other neurological diseases.     

The endocannabinoid system as a novel target for the treatment of liver fibrosis

The cannabinoid system comprises specific G protein-coupled receptors (CB1 and CB2), exogenous (marijuana-derived cannabinoids) and endogenous (endocannabinoids) ligands, and a machinery dedicated to endocannabinoid synthesis and degradation. Studies over two decades have extensively documented the crucial role of the cannabinoid system in the regulation of a variety of pathophysiological conditions. However, its role in liver pathology has only been recently unravelled, probably given the low expression of CB1 and CB2 in the normal liver. We have recently demonstrated that CB1 and CB2 receptors display opposite effects in the regulation of liver fibrogenesis during chronic liver injury. Indeed, both receptors are up-regulated in the liver of cirrhotic patients, and expressed in liver fibrogenic cells. Moreover, CB1 receptors are profibrogenic and accordingly, the CB1 antagonist rimonabant reduces fibrosis progression in three experimental models. In keeping with these results, daily cannabis smoking is a risk factor for fibrosis progression in patients with chronic hepatitis C. In contrast, CB2 display antifibrogenic effects, by a mechanism involving reduction of liver fibrogenic cell accumulation. These results may offer new perspectives for the treatment of liver fibrosis, combining CB2 agonist and CB1 antagonist therapy.