Help   Site Map   Contact
Cannabinoid Primer

ECSN Faculty

Allyn Howlett, PhD
Professor of Physiology and Pharmacology
Department of Physiology and Pharmacology
Wake Forest University Health Sciences
Winston-Salem, North Carolina

Aron H. Lichtman, PhD
Associate Professor
Department of Pharmacology and Toxicology
Virginia Commonwealth University Medical Campus
Richmond, Virginia

Roger Pertwee, MA, PhD
Professor of Neuropharmacology
School of Medical Sciences
Institute of Medical Sciences
University of Aberdeen Foresterhill
Aberdeen, Scotland, UK

PART I

Components of the ECS

Endocannabinoid System (ECS)

The ECS is a complex physiological system composed of cannabinoid receptors, their endogenous ligands (the endocannabinoids) and the enzymes and processes involved in endocannabinoid synthesis, cellular uptake, and degradation.1 An important role of the ECS is to maintain homeostasis in both health and disease. Thus, there are certain disorders in which the ECS is upregulated in a manner that suppresses unwanted signs and symptoms or slows disease progression. This opens up the possibility of developing new medicines that mimic this “autoprotective” upregulation by directly activating cannabinoid receptors or that augment it by, for example, inhibiting endocannabinoid cellular uptake or metabolism.1

There are also disorders such as obesity in which the ECS is upregulated in a manner that exacerbates undesirable effects.1 Indeed, blockade of this system seems to represent an effective therapeutic strategy for the treatment of obesity and related metabolic disorders. Excess high-fat food intake or other metabolic dysfunctions can drive the ECS activity, and can contribute to cardiovascular and metabolic risk factors.3, 4 Because the activity of the ECS may be increased in obese states, pharmacologic blockade of this system represents a promising approach for the treatment of obesity and related metabolic disorders.

Cannabinoids

Cannabinoids are compounds that act on the cannabinoid receptors. They include the herbal cannabinoids that occur uniquely in the plant Cannabis sativa, synthetic analogs produced by drug companies and research chemists, and the endocannabinoids produced in the brain and peripheral tissues via calcium- and phospholipid-dependent pathways. The highly hydrophobic nature of cannabinoids presents a great challenge in working with these compounds in the research setting.5, 6

Endocannabinoids
Figure 1
Click to Enlarge Figure 1

Endocannabinoids are derivatives of arachidonic acid.7, 8 Membrane depolarization of neurons or activation of certain receptors in many cells leads to the formation of N-arachidonylethanolamide (anandamide) and 2-arachidonoylglycerol (2-AG) from phospholipid precursors.9, 10 Unlike peptide or aminergic neurotransmitters, endocannabinoids are lipophilic neuromodulators and are not stored in synaptic vesicles.7, 11, 12 Endocannabinoids appear to be produced “on demand” and act on cells in a paracrine or autocrine manner by binding to cannabinoid receptors.13 Anandamide and 2-AG are the most-studied endocannabinoids (Figure 1).8, 9 There are other newly proposed endocannabinoids, for which physiological roles have not yet been established.7

Endocannabinoid Biosynthesis and Inactivation

The enzymes involved in endocannabinoid synthesis include sn-1-selective-diacylglycerol lipase (DAGL) and N-acylphosphatidylethanolamine-selective phospholipase D (NAPE-PLD).2 Anandamide is generated through phosphodiesterase-mediated cleavage of a cell membrane phospholipid precursor, N-arachidonoyl-phosphatidylethanolamine (NAPE).14 NAPE is generated by the enzymatic transfer of arachidonic acid in the sn-1 position in phosphatidylcholine to the amine group of phosphatidylethanolamine.15, 16 NAPE cleavage can be catalyzed by NAPE-PLD,17 although NAPE-PLD is not required for anandamide biosynthesis in vivo.18 2-AG biosynthesis occurs by phospholipase C (PLC) mediated hydrolysis of membrane phospholipids resulting in formation of sn-1-Acyl-2-arachidonoylglycerol, which is converted to 2-AG by DAGL.2, 19 The enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) hydrolyze anandamide and 2-AG, respectively.20 In addition, the enzymes ABHD6 and ABHD12 play an important role in 2-AG metabolism.21

Cannabinoid Receptors

The cannabinoid receptor type 1 (CB1 receptor) was the first cannabinoid receptor to be characterized, followed by the CB2 receptor. The CB1 and CB2 receptors are G protein-coupled receptors (GPCRs). The family of G proteins that interact with GPCRs are predominantly the heterotrimeric G proteins consisting of α, β and γ subunits. When the G protein complex interacts with an active receptor, the α subunit exchanges its bound GDP (guanosine diphosphate) for GTP (guanosine triphosphate) and dissociates from the β/γ subunit. The subunits inhibit voltage-dependent calcium channels and activate inwardly rectifying potassium channels.

Gs proteins (stimulatory G proteins) activate adenylyl cyclase, thereby increasing cyclic adenosine monophosphate (cAMP) levels. Gi proteins (inhibitory G proteins) inhibit adenylyl cyclase and directly regulate ion channels. CB1 and CB2 receptors primarily couple to G proteins of the inhibitory G protein (Gi/o) class. For additional information on the pharmacology of cannabinoid receptors, visit http://www.iuphar-db.org/GPCR/ChapterMenuForward?chapterID=1279.

Tissue and Cellular Localization of Cannabinoid Receptors

The CB1 receptor was first cloned from rat cerebral cortex, then from human brain and testis, and then from mouse brain.22 In fact, CB1 receptors are among the most abundant GPCRs in the brain. CB1 receptors are also expressed in a wide range of tissues, including peripheral tissues and organs involved in metabolism, such as adipose tissue, skeletal muscle, liver, and pancreas.7,22 The CB1 receptor is localized at the plasma membrane, as well as in intracellular vesicles. Recently, it was shown that intracellular CB1 receptors are functional and that their spatial segregation is likely to significantly affect receptor function.23

In the brain, CB1 receptors are expressed predominantly in brain regions associated with higher cognitive functions, such as the cortex, amygdala, and basal ganglia, and are localized presynaptically on the plasma membranes of axons and axon terminals. Although the level of CB1 receptor expression varies among neuronal subpopulations and brain regions, there is little correlation between levels of expression and receptor activity.7 Growing evidence suggests that there is an endocannabinoid glial system. CB1 receptor expression was detected in astrocytes and oligodendrocytes.24-26 Glial cells have been shown to produce and release endocannabinoids in vitro.27,28

The CB2 receptor is expressed in the spleen and tonsils as well as in immune cells (B cells, monocytes, and T cells), indicating a role in immune function, and may also be expressed in the central nervous system (CNS).22 Microglial cells have been shown to express both CB1 and CB2 receptors.24,25 CB2 receptors were also shown to be expressed in perivascular microglial cells of the human cerebellum.29 CB2 receptor mRNA expression and immunoreactivity was shown in glial and neuronal patterns in a number of rat brain regions30; and CB2 receptor mRNA and protein was localized to rat, ferret, and mouse brainstem neurons.31

TRPV1

The transient receptor potential vanilloid type 1 (TRPV1) receptor is a 6-transmembrane-domain nonselective ion cation channel activated by either physical or chemical stimuli. Stimuli include thermosensation, sensory transduction, taste, and detection of irritant compounds. TRPV1 is targeted by anandamide, but it is not regarded as a cannabinoid receptor.

GPR55

A range of plant, synthetic, and endogenous cannabinoids bind to and activate the orphan Gprotein-coupled receptor 55 (GPR55), which is expressed in brain and various peripheral tissues in humans and rodents.32-34 Two recent studies concluded that GPR55 may be a cannabinoid receptor.33,34 However, additional data are needed before any definite conclusion can be drawn.

Cannabinoid Receptor Signaling

Cannabinoid Receptor Signaling in Peripheral Tissues

Endocannabinoids may act on cells in a paracrine, autocrine or endocrine manner. CB1 receptor activation leads to inhibition of adenylyl cyclase with corresponding inactivation of the protein kinase A (PKA) phosphorylation pathway, and stimulation of mitogen-activated protein kinase (MAPK).7,13 Stimulation of these cytoplasmic kinases could lead to changes in the expression of target genes.7,13 The type of signaling pathway affected by CB1 receptor activation depends on the type of agonist used and the tissue or organ involved.35

Cannabinoid Receptor Signaling in the Brain

As in peripheral tissues, CB1 receptor activation leads to inhibition of adenylyl cyclase throughout the brain. On nerve terminals, activation of CB1 receptors inhibits both excitatory and inhibitory neurotransmission.36 CB1 receptor stimulation appears to activate inwardly rectifying potassium channels, which decreases neuronal excitability. CB1 receptor stimulation is directly coupled to inhibition of voltage-activated calcium channels35 and mediates retrograde signaling.10 Retrograde signaling is a mechanism whereby a chemical signal is released from the postsynaptic neuron, traverses the synaptic space, and activates presynaptic receptors to modulate the release of neurotransmitters, thereby influencing synaptic plasticity (the ability of nerve cells to change their properties by making new synapses or altering the strength of ongoing synaptic transmission).

Comparison Between CB1 and CB2 Receptor Signaling

Activation of both CB1 and CB2 receptors stimulates Gi/o proteins, which leads to the inhibition of adenylyl cyclase-mediated conversion of adenosine triphosphate (ATP) to cAMP. In addition, activation of the CB1 and CB2 receptors stimulates several intracellular kinases, such as the p42/p44 MAPK. CB1 receptor stimulation is associated with activation of inwardly rectifying potassium channels and inhibition of voltage-activated calcium channels.35 In contrast to CB1 receptors, CB2 receptor stimulation does not appear to modulate ion channel function.22

PART II

Pharmacology concepts

General Terms Used to Describe Drug Action

Agonists are ligands that bind to a receptor and alter the receptor state, resulting in a biological response. Conventional agonists increase receptor activity.37,38

Affinity is the attraction of a particular class of receptor to a drug or endogenous ligand, at a level sufficient to give an observable degree of activation or antagonism.38

Efficacy expresses the degree to which different agonists or inverse agonists produce biological responses, even when occupying the same proportion of receptors.38 For example, Δ9-tetrahydrocannabinol (THC) has relatively low cannabinoid receptor efficacy and it displays partial agonism at CB1 and CB2 receptors in in vitro assays, though it acts as a full receptor agonist in vivo.39 Partial agonists and antagonists (see below) have reduced efficacy and no efficacy, respectively. A low-efficacy agonist (partial agonist) can compete like an antagonist against a high-efficacy agonist.

Partial agonists do not always elicit as large a maximal effect (even when applied at high concentrations) as certain other full agonists acting through the same receptors in the same tissue.

Antagonists are compounds that reduce the action of an agonist. Many antagonists act at the same receptor and binding sites as the agonists. Naturally occurring (endogenous) antagonists can act by binding to and sequestering a ligand or by binding to a receptor to prevent its response to another molecule.37,38

Competitive antagonism occurs when the binding of an agonist and antagonist are mutually exclusive. This may be because the agonist and antagonist compete for the same binding site or combine with adjacent sites that overlap. A competitive antagonist that prevents an agonist from binding, and has no effect on the receptor’s activity, is referred to as a “neutral antagonist.”

Noncompetitive antagonism occurs when an agonist and antagonist can bind to a receptor simultaneously. The antagonist binding reduces or prevents the action of the agonist with or without any effect on the binding of the agonist.

Allosteric modulators are ligands that increase or decrease the action of an agonist (primary or orthostatic) or antagonist by combining with a distinct (allosteric) site on the receptor (Figure 2).

Figure 2
Click to Enlarge Figure 2

Recognition sites are the regions of the receptor to which ligands bind. Those sites at which the endogenous agonist binds are termed primary or orthostatic sites, whereas other ligands may act through allosteric sites. A receptor can have more than one allosteric and orthostatic site.

Constitutive activity of a GPCR refers to the ability of the receptor to activate signal transduction in an agonist-independent manner. Constitutive activity can be observed at low or high GPCR expression levels. CB1 receptors exist in both an inactive state and a constitutively active state.40,41 Evidence for different receptor-G protein binding states based upon the G proteins to which the receptor is coupled is presented in Mukhopadhyay and Howlett 2005.42

Inverse agonists are ligands that, by binding to receptors, reduce the fraction of receptors that are in an active conformation. This can occur if some of the receptors are in the active form in the absence of a conventional agonist. The biological response to inverse agonists may be the “inverse” of the response expected of stimulation of the receptor with an agonist.37,38

Tolerance refers to a decrease in the response to a drug after prior exposure to an agonist. As a result, increased dose of the drug is required to achieve the same physiological effect.43 Diverse mechanisms may underlie tolerance to cannabinoid receptor agonists, including phosphorylation, internalization (endocytosis) of GPCRs with or without subsequent degradation; decreased synthesis of CB1 receptor protein; and reduced efficiency of CB1 receptor signaling.39 Martini et al43 showed that the GPCR-associated sorting protein GASP1 plays a major role in the CB1 receptor degradation after endocytosis. With regard to antagonism and tolerance, if an antagonist has suppressed a response, then the cell might adjust the number of receptors to increase the probability of a response to lower concentrations of agonists.

Cannabinoid Receptor Agonists, Antagonists, and Inverse Agonists

Cannabinoid receptor agonists are molecules that stimulate a cellular response by inducing a conformational change in a cannabinoid receptor. Agonists may be naturally occurring signaling molecules or synthetic compounds and are classified into 4 groups (Figure 3).37,44


  1. Classical cannabinoid agonists represent compounds that either occur naturally in the plant Cannabis sativa, or are synthetic analogs of these compounds. The most investigated of the naturally occurring compounds are Δ9-THC and Δ8-THC. The main psychotropic constituent of cannabis is Δ9-THC. 11-hydroxy-Δ8-THC-dimethylheptyl (HU-210) is a synthetic analog of Δ8-THC and is a potent cannabinoid receptor agonist. Most classical cannabinoids that bind to CB1 receptors have affinity for the CB2 receptor as well, without major selectivity for either receptor.


  2. Nonclassical cannabinoid agonists were developed by synthesizing new classical cannabinoid analogs that lack the dihydropyran ring of THC. The bicyclic analog CP55940 is a potent, full agonist in signal transduction, and is less lipophilic than THC, and is widely used for experimental purposes. CP55940 binds to CB1 receptors and CB2 receptors with similar affinity and is 4 to 25 times more potent than Δ9-THC in the mouse tetrad model (see below).45 The radiolabeled cannabinoid ligand [3H]CP55940 was used to demonstrate the presence of CB1 receptors in human brain,46 and has been extensively used to characterize and map cannabinoid receptors in rat brain.


  3. Aminoalkylindole cannabinoid agonists are structurally related to pravadoline. R-(+)-WIN55212 is the most highly studied and has high affinity for both CB1 receptors and CB2 receptors. [3H]R-(+)-WIN55212 has been used for ligand binding studies of cannabinoid receptors.47 JWH-015 and L-768242 are recently developed aminoalkylindoles that are selective for the CB2 receptor.48


  4. Eicosanoid cannabinoid agonists include the endocannabinoids anandamide and 2-AG. Anandamide is the prototypic member of this group. Structural modification of the anandamide molecule, which itself displays marginally higher affinity for CB1, rather than CB2 receptors, has led to the development of the first generation of CB1 receptor-selective agonists, such as arachidonyl-2-chloroethylamide (ACEA).49

Figure 3
Click to Enlarge Figure 3

Cannabinoid receptor indirect agonists include selective and potent inhibitors of the enzymes FAAH and MAGL and inhibitors of endocannabinoid cellular reuptake. These compounds are under study in preclinical models of conditions such as anxiety, depression, pain, and emesis.1,50

Cannabinoid Receptor Antagonists and Inverse Agonists

The biological response to a cannabinoid receptor inverse agonist is the “inverse” of the response expected of stimulation by a cannabinoid receptor agonist. In contrast, a neutral antagonist only affects receptor activation when this is being induced by an agonist. For example, only when food intake is being stimulated by an exogenously administered or endogenously released CB1 receptor agonist will this effect be reduced by a neutral CB1 antagonist.

Selectivity of Cannabinoid Receptor Antagonists and Inverse Agonists

Antagonists that display high affinity and significant selectivity for the CB1 receptor include SR141716 (rimonabant), AM251, AM281, and MK-0364 (taranabant).51,52 These compounds are characterized by a high affinity (Ki = low to subnanomolar range) for CB1 receptors, and much lower affinity for other receptors. Increased expression of CB1 receptors may improve the selectivity and effectiveness of a CB1 receptor agonist as a therapeutic agent.39

All well characterized, high-affinity CB1 receptor antagonists are inverse agonists in signal transduction studies in vitro. Examples include SR141716, CP-945598, AM251, AM281, LY320135, and MK-0364. Of these, the inverse agonism of SR141716 has been the most thoroughly characterized. It has been proposed that SR141716 induces a receptor state that inhibits the putative endocannabinoid-independent activation of the G protein by the CB1 receptor.41

The effects of a CB1 receptor antagonist may depend on the endocannabinoid tone of the system (ie, the level of basal ECS activity). When the endocannabinoid tone of a particular system is low, the physiologic effects of a CB1 receptor inverse agonist may be observed as the “inverse” of those produced by CB1 receptor agonists. To demonstrate that an antagonist behaves as an inverse agonist in whole animal physiology, one would have to demonstrate that no endocannabinoid agonists were being produced at the site of activity (autocrine or paracrine regulation). This demonstration will require identification of all endocannabinoid synthetic pathways and the development of effective blockers of the synthetic enzymes before one can be certain that a biological response can be attributed to constitutive activity of the CB1 receptor.

Part III

experimental models used in ECS Research

In Vivo Behavioral Methods

The medicinal potential of cannabinoids can be assessed by performing behavioral tests in laboratory animals.37,53,54 Unless specified otherwise, most tests are performed using rats and/or mice.

Drug Discrimination (Rats, Mice, and Monkeys)

Drug discrimination is considered one of the most reliable means of predicting whether test drugs produce subjective effects similar to those of a known drug. An animal is trained to press a lever for a food reward and then subsequently trained to press a specific lever for this reward when under the influence of THC, and another lever when any other drug that produces different subjective effects than THC is administered. On test days, the lever that the animal chooses indicates whether or not the test compound is perceived as THC-like.

Mouse Tetrad Model

The mouse tetrad consists of four evaluations: motor activity, catalepsy, rectal temperature, and analgesic (pain reduction) effects.54 Intravenous administration of CB1 receptor agonists to mice produces hypomotility, hypothermia, analgesia, and catalepsy (relative immobility). All four of these behaviors can be measured in sequence in the same animal for each injection.

Catalepsy is assessed in Pertwee's ring immobility test or in the bar test. In the ring immobility test a mouse is placed on a ring attached to a stand. The amount of time the mouse spends motionless on the ring is measured, with the criteria of immobility being defined as the absence of all voluntary movements, including whisker movement, but excluding respiration. In the bar test, a bar is fixed horizontally above a cage floor. The animal’s forepaws are gently placed on the bar, and the length of time during which the mouse maintains the initial position is measured.

Static Ataxia (Dog)

In dogs, cannabinoids produce sedation, catalepsy, motor incoordination, and hyperexcitability. “Static ataxia” is the combination of these effects that causes dogs to weave to and fro while remaining fixed in one spot. The primary advantage of this model is that these behaviors are highly specific for cannabinoids and are distinct from those produced by other behaviorally-active compounds. The development of the rodent tetrad assay has essentially replaced the dog static ataxia test.

Anticipatory Nausea (Rats)

Anticipatory nausea and vomiting can be associated with previously neutral stimuli. For example, cancer chemotherapy may be “paired” with neutral, environmental stimuli (eg, chemotherapy room) that then elicit anticipatory nausea and vomiting in future chemotherapy cycles.55 Although mice and rats do not vomit, a rat model of anticipatory nausea has been developed in which a gaping reaction is induced during exposure to a context previously paired with lithium chloride-induced illness.56 The ability of a treatment (such as a cannabinoid agonist) to suppress the lithium-induced gaping reaction is then determined. The taste aversion paradigm essentially uses the same procedure as the gaping model. Conditioned taste aversions produced by emetic drugs can be used as a model for evaluating potential antiemetics.57 For example, Landauer et al57 showed that THC and other cannabinoids attenuated taste aversions in mice that were induced by the cancer chemotherapeutic drug cyclophosphamide.

Analgesia

The ECS has a well-established role in modulating pain, and both CB1 and CB2 receptor agonists have been shown to be effective in animal models of acute and chronic pain.50,58,59 The analgesic (pain-reducing) effects of cannabinoids are measured by several methods.

Thermal Hyperalgesia

A hot-plate analgesia meter is used to measure analgesia levels in small laboratory animals. A mobile radiant heat source is located under a plate that is surrounded by a clear cage. The animal is habituated to the transparent plastic chamber and the plate is preheated to a constant temperature (eg, 50°C-58°C, which is low enough to avoid harm to the animal, yet high enough to be uncomfortable). When placed on the hot surface of the plate, animals will lift their paws and lick them (hind paw lick, hind paw shake, or jumping) due to attainment of pain threshold, and the paw-withdrawal latency (PWL, defined as the time for the animal to remove its hind paw from the heat source) is recorded. Hyperalgesia is determined as a decrease in PWL to the noxious thermal stimulus.60 A cut-off time may be set to prevent tissue damage.

Tail-Flick

In the radiant heat tail-flick test, the animal's tail is placed over a window on a platform while the animal is restrained. After an intense light beam to the tail is activated to a temperature range of 60°C-170°C the animal will flick its tail out of the beam. The time from onset of thermal stimulation to the animal's response is recorded electronically. Note that the tail-flick test is a spinally mediated reflex; a rat or mouse with a severed spinal cord will still flick its tail in this assay. As in the hot-plate test, a cut-off time (eg, 10 seconds) is used to prevent tissue damage.

von Frey Filaments (Hairs)

The von Frey assay is generally used to measure allodynia (ie, non-noxious mechanical stimulation eliciting a painful response) in response to nerve injury or inflammation. In the 19th century, Maximilian von Frey showed that the threshold for touch-evoked sensations can be determined by sequentially applying “hairs” of different diameters until the hair that creates a particular sensation is found. von Frey hairs (or filaments) are fine-gauge synthetic or natural filaments that are used for quantitative mechanical stimulation of skin receptors. An animal is placed in a clear cage on an elevated mesh floor and allowed to acclimate. Paw withdrawal thresholds are determined by applying calibrated von Frey filaments. The filaments exert an increasing force to the plantar surface of the animal’s paw, starting below the threshold of detection and increasing until the animal removes its paw. At the retraction reflex movement when the paw is withdrawn, the actual force at which paw withdrawal occurred is recorded. Pain sensitivity is inversely correlated with the animal’s threshold to the force applied with a von Frey filament.

Hargreave’s (Plantar Stimulation) Test

In the Hargreave’s (plantar stimulation) test, a low-intensity radiant heat stimulus is applied to the bottom of a rodent’s paw and the latency for the animal to lift its paw is recorded. Hypersensitivity to a thermal stimulus is used in a fashion similar to the von Frey model.

Behavioral Screening Models for Anxiolytic and Antidepressant Agents

Animal models of anxiety and depression are widely used in behavioral neuroscience to explore stress-evoked brain abnormalities and to screen anxiolytic/antidepressant drugs.61 Specific tests for mice are used in the domains of learning and memory, feeding, nociception, and behaviors relevant to discrete symptoms of human anxiety, depression, schizophrenia, and drug addiction.62 Tests include observations of home cage behaviors, acoustic startle, eye blink, open-field locomotion, motor coordination, and pain threshold.62 Examples of some of these behavioral animal models are described below. For further reading see Kalueff et al 200761 and Crawley 1999.62

Elevated Plus-Maze Test

The elevated plus-maze test is used to assess anxiety-like behavior in laboratory animals.63 The maze exploits the conflict between the innate fear that rodents have of open areas vs their desire to explore novel environments. The basic measure is the animal's preference for dark, enclosed places over bright, exposed places. Confinement to the open arms of the maze is associated with the observation of more anxiety-related behaviors than confinement to the closed arms. A significant increase in the percentage of time spent on the open arms and the number of entries into the open arms is observed only with clinically effective anxiolytics, while compounds that cause anxiety in humans (eg, caffeine, amphetamine) reduce the percentage of entries into, and time spent on, the open arms.

Zero Maze

A Zero Maze test is designed to monitor the levels of anxiety of mice.64 The Zero Maze apparatus consists of a circular platform that is equally divided into four quadrants. Two quadrants on opposite sides of the platform are enclosed by walls and the other two quadrants are open and bordered by a lip. The maze is elevated above the floor and there is an overhead camera and tracking system to monitor activity of the mouse. Mice are placed on the maze and the time spent in the open and closed sections is measured. Additional measures include section entrances, closed head dips (the number of times the mouse looks over the edge of the maze while a portion of the body is in the closed sections), and open head dips (the number of times the mouse looks over the edge of the maze while the mouse is completely in the open sections).

Tail-Suspension Test

When suspended by the tail, mice alternate between active attempts to escape and immobility. In the tail-suspension test (TST), animals are suspended by their tails and the amount of "immobility" is measured.65 During the test, the following measures are recorded: the number of times (events) each animal enters into an escape behavior (struggle), the duration of the event, and the average strength of each event. Longer periods of immobility are associated with higher depressive scores and immobility is reversed with antidepressant treatment.

Forced Swim Test

In the forced swim test (FST) a depressed state is induced in mice and rats by forcing them to swim in a narrow cylinder from which they cannot escape.66 After a brief period of vigorous activity the mice adopt a characteristic immobility that is reduced by antidepressant treatment. The FST is selectively sensitive to antidepressant treatments. For example, psychostimulants reduce immobility but also cause marked motor stimulation. The FST is the most widely used tool for assessing antidepressant activity.

Vogel Test

In the Vogel test, an animal is water deprived and then given access to a water spigot. The animal will occasionally receive a low-intensity electric shock when drinking. A “normal” animal will repress drinking, but an animal treated with an anxiolytic will continue to drink.

Memory Models

Both natural and synthetic cannabinoids have been shown to impair learning and memory in rodents. Several paradigms of laboratory tests are used to assess learning and memory in rodents. Maze learning and avoidance tasks are among the oldest learning and memory models used in rodents.62

Lashley III Maze
Figure 4
Click to Enlarge Figure 4

The Lashley mazes were first developed as a test for neurological damage in rats. The Lashley III consists of a start box, four alleyways, and a goal box. The animal is required to make a series of alternating left and right turns to obtain a food reward. The maze contains cul-de-sacs (C) that must be avoided and T-choices (T) at which an animal must learn whether to turn right or left (Figure 4).67 The amount time spent reaching the goal box from the start box is used as an assessment of learning and memory.

Eight-Arm Radial Maze
Figure 5
Click to Enlarge Figure 5

In this maze, each of 8 arms radiates from a central starting platform like the spokes of a wheel. Identical food wells with pellet sensors are placed at the distal end of each arm, to which the animals are pretrained to go to find pellets. The design ensures that after checking for food at the end of an arm the animal is forced to return to the central platform before making another choice. As a result, the animal always has 8 possible options. Spatial memory is assessed in acquisition trials, where animals are placed on the central platform and allowed to get pellets from all 8 locations within a prespecified time interval.

Morris Water Maze
Figure 6
Click to Enlarge Figure 6

The Morris water maze tests learning ability and memory. A pool is divided into quadrants and a transparent platform is submerged below the water surface at the center of one of the quadrants. For pretraining, animals swim freely in the pool without the platform. Animals then swim in the water maze with the platform. Learning ability is expressed as the rate of decrease in swimming time and distance from the start point to the platform from their values in the first trial. For memory evaluation, the platform is then removed and animals are placed opposite the quadrant where the platform had been located. The percentage of time spent in the quadrant where the platform had been located is used as an assessment of memory retention.68

Operant Tasks

There are also operant models of learning and memory.62 Food- or water-restricted animals are habituated to an operant chamber and trained to press a lever for the food or water reinforcer, respectively. The number of trials to acquire the lever press task is a simple measure of learning in mice or rats. The non-match to sample task is more complex. This task requires the animal to receive reinforcement for pressing the opposite lever to the lever, for example, illuminated on the previous trial by a cue lamp above the lever. Recently, Deadwyler et al69 investigated the role of endocannabinoids in the encoding of task-relevant information during performance of a short-term memory delayed non-match to sample task in rats.

Fear Conditioning

Fear conditioning is a type of Pavlovian learning task in which mice are presented with a neutral conditioning stimulus that is paired with an aversive unconditioned stimulus. The mice learn that the conditioning stimulus predicts the unconditioned stimulus and will exhibit specific behavioral responses (such as freezing) when the conditioning stimulus is presented alone. Fear-conditioning models can be designed to assess many types of conditioning sensitive to either the hippocampal system, the amygdalar system, or both. Contextual conditioning (hippocampal dependent) tests the ability of mice to associate the training environment with the aversive event.

Knockout Mice

Knockout mice have been genetically engineered to exhibit mutations in specific genes. For example, CB1 receptor knockout mice do not express functional CB1 receptor mRNA and protein. CB1 receptor knockout mice are widely used as a means to understand the role of the ECS and to assess the selectivity of pharmacological agents. In homozygote knockout mice, functional receptors are not expressed, a situation that is similar to irreversible pharmacologic blockade of 100% of receptors. In contrast, antagonism with CB1 receptor antagonists/inverse agonists may not saturate all receptors, even at doses that are maximally efficacious in clinical studies.71 For example, when the CB1 receptor inverse agonist MK-034 (taranabant) is dosed in a therapeutic fashion, the compound occupies approximately 40% of brain CB1 receptors.71 Moreover, since all commonly used cannabinoid receptor antagonists/inverse agonists act in a reversible manner, the antagonism they produce can be overcome by applying cannabinoid receptor agonists at high concentrations.

In addition to CB1 receptor knockout mice, there are other knockout mice used to study the ECS.72,73 These include CB2 receptor knockout mice,74,75 FAAH knockout mice,76 and the conditional and tissue-specific CB1 receptor knockout mice.74 In addition, knockout mice are generated that lack more than one gene. For example, Wise et al77 generated mice that lacked both FAAH and CB1 receptors.

The Cre/loxP-mediated recombination method of gene targeting allows the inducible inactivation of a target gene in mice (see Kuhn et al 1995).78 Recently, Agarawal et al59 generated tissue-specific CB1 receptor knockout mice. The CB1 receptor knockout was specifically in nociceptive neurons localized in the peripheral nervous system of mice, thereby preserving its expression in other peripheral tissues and in the CNS.59 The tissue specificity was generated via Cre/loxP-mediated recombination by mating homozygous mice carrying the loxP-flanked Cnr1 allele with a mouse line expressing Cre recombinase under the control of the promoter for the Nav1.8 gene, which is expressed selectively in nociceptive sensory neurons.

Pair-feeding

Pair-feeding refers to feeding an animal the same amount and composition of food as an animal in a test group. Pair-feeding studies are important, because they can determine whether or not factors other than reduced food intake contribute to body weight changes.

In Vitro

cAMP Production

Cyclic adenosine monophosphate (cAMP) is a nucleotide generated from ATP by adenylyl cyclase in response to stimulation of many types of cell-surface receptors. cAMP activates cyclic-AMP-dependent kinase (protein kinase A, PKA).The ability of cannabinoid receptor agonists to inhibit basal or drug-induced cAMP production at CB1 and CB2 receptors is widely used for quantitative, functional bioassays of cannabinoids.

GTPγS Binding Assay

The GTPγS binding assay exploits the coupling of CB1 and CB2 receptors to G proteins. This bioassay measures agonist-stimulated [35S]GTPγS binding to G proteins and is used in cultured cells transfected with CB1 or CB2 receptors and in CB1-receptor-containing membrane preparations from the brain.

Vas Deferens Assay (Inhibition of electrically evoked contractions)

The mouse vas deferens is a tissue in which prejunctional CB1 receptors mediate inhibition of electrically evoked contractions via inhibition of neurotransmitter release. The CB1 receptors are located on prejunctional neurons and mediate inhibition of electrically evoked contractile transmitter release. Vasa deferentia are obtained from adult mice and mounted in an organ bath. Stimuli are generated and isometric contractions are monitored by computer.79

Part IV

Therapeutic Applications

Cannabinoid Receptor Agonists

Actual Applications

In large-scale clinical trials, attenuation of ECS activity with the CB1 receptor antagonist SR141716 (rimonabant) results in clinically significant weight loss and reduced waist circumference among obese and overweight adults.96-99 Additional effects of rimonabant treatment include improvements in serum lipids and glycemic control in patients with type 2 diabetes. Some of these effects may be independent of weight loss, suggesting direct peripheral metabolic effects of CB1 receptor blockade. Rimonabant was approved by the European Medicines Agency (EMEA) in June 2006;100 however, the EMEA recommended that rimonabant is contraindicated in patients with ongoing major depression and in patients being treated with antidepressants.100,101 Future studies, such as determining the phenotype of tissue-specific CB1 receptor knockout mice, and clinical trials with a pair-feeding paradigm, will provide important details about how ECS biology is linked to the clinical effects of CB1 receptor antagonism/inverse agonism.

Potential Applications

Increases in ECS activity within or outside the CNS caused by an elevation in endocannabinoid levels and/or in the expression of cannabinoid receptors can give rise to both protective82,83 and impairing effects.84 There is evidence, for example, that endocannabinoids can take on a protective role in inflammatory neurodegeneration85 but promote obesity. Both CB1 and CB2 receptors may play a role in the pathophysiology of the ECS.86

Changes in endocannabinoid signaling have been documented both in experimental models of depression and in depressed human subjects.87 Animal studies have shown that pharmacological enhancement of ECS activity induced antidepressant-like effects.88-90

Drugs that inhibit the enzymes FAAH and MAGL are indirect cannabinoid receptor agonists. Studies support a potential role for inhibitors of FAAH and MAGL in the treatment of chronic inflammatory pain, neuropathic pain, glaucoma, emesis, inflammatory bowel disease, colon cancer, and inflammation.1,91-94 It may also prove possible to develop medicines from compounds that target allosteric sites on CB1 receptors in a manner that enhances the activation of these receptors by endocannabinoids.95

Selective CB1 receptor agonists hold potential therapeutic applications for disorders that include inflammatory bowel disease and irritable bowel syndrome.94 Selective CB2 receptor agonists hold potential therapeutic applications for disorders such as inflammatory bowel disease, irritable bowel syndrome, hepatic ischemia, chronic liver disease, and pancreatic cancer.94

Cannabinoid Receptor Antagonists

Actual Applications

In large-scale clinical trials, attenuation of ECS activity with the CB1 receptor antagonist SR141716 (rimonabant) results in clinically significant weight loss and reduced waist circumference among obese and overweight adults.96-99 Additional effects of rimonabant treatment include improvements in serum lipids and glycemic control in patients with type 2 diabetes. Some of these effects may be independent of weight loss, suggesting direct peripheral metabolic effects of CB1 receptor blockade. Rimonabant was approved by the European Medicines Agency (EMEA) in June 2006;100 however, the EMEA recommended that rimonabant is contraindicated in patients with ongoing major depression and in patients being treated with antidepressants.100,101 Future studies, such as determining the phenotype of tissue-specific CB1 receptor knockout mice, and clinical trials with a pair-feeding paradigm, will provide important details about how ECS biology is linked to the clinical effects of CB1 receptor antagonism/inverse agonism.

Potential Applications

A number of high-affinity CB1 receptor antagonists have been identified and tested for their effects on food intake and weight loss. These include SR141716, AM251, AM281, CP-945598, and MK-0364.51,52

The compounds O-2050 and AM4113 reduce food intake in rats and have been proposed to behave in vitro as competitive neutral CB1 receptor antagonists.102,103 CB1 receptor neutral antagonists are expected to reduce feeding by blocking actions of endogenously released endocannabinoids, whereas CB1 receptor antagonists/inverse agonists may in addition affect feeding by reducing the constitutive activity of CB1 receptors. There is also evidence that food intake in rats can be reduced by CB1 receptor allosteric antagonism.104

The ECS appears to be involved in the reinforcing properties of several drugs of abuse. Stimulation of nucleus accumbens CB1 receptors may suppress glutamatergic activity, with consequent inhibition of γ-aminobutyric acid (GABA)-ergic neurons that normally inhibit ventral tegmental area dopamine neurons.105 Several CB1 receptor antagonists are under study in Phase III trials for the treatment of nicotine dependence.1 A broader therapeutic potential of CB1 receptor antagonists (such as SR141716) in the treatment of drug addiction is also under study.8,81

Results from one preclinical study suggest an antidepressant effect of CB1 receptor blockade.106 Preclinical studies support a potential therapeutic role for CB2 receptor antagonists/inverse agonists in experimental models of dermatitis, arthritis, and autoimmune encephalomyelitis.1 Clinical trials of these compounds for dermatitis or autoimmune disorders do appear to be forthcoming.1

CB1 receptor antagonists also hold potential therapeutic applications for paralytic ileus, diabetic gastroparesis, and chronic liver disease.94

Figures

Figure 1. Structures of the endocannabinoids anandamide and 2-AG.



Figure 2. Allosteric vs orthosteric sites.



Figure 3. Structures of prototypical cannabinoid receptor agonists.



Figure 4. A Lashley III maze.



Figure 5. A radial maze.



Figure 6. Morris Water maze.

REFERENCES

  1. Di Marzo V: Targeting the endocannabinoid system: to enhance or reduce? Nat Rev Drug Discov. 2008;7:438-455.
  2. Basavarajappa BS: Critical enzymes involved in endocannabinoid metabolism. Protein Pept Lett. 2007;14:237-246.
  3. Eckel RH, Grundy SM, Zimmet PZ: The metabolic syndrome. Lancet. 2005;365:1415-1428.
  4. Grundy SM: Metabolic syndrome: connecting and reconciling cardiovascular and diabetes worlds. J Am Coll Cardiol. 2006;47:1093-1100.
  5. Martin BR, Wiley JL, Beletskaya I, et al: Pharmacological characterization of novel water-soluble cannabinoids. J Pharmacol Exp Ther. 2006;318:1230-1239.
  6. Pertwee RG, Gibson TM, Stevenson LA, et al: O-1057, a potent water-soluble cannabinoid receptor agonist with antinociceptive properties. Br J Pharmacol. 2000;129:1577-1584.
  7. Pagotto U, Marsicano G, Cota D, Lutz B, Pasquali R: The emerging role of the endocannabinoid system in endocrine regulation and energy balance. Endocr Rev. 2006;27:73-100.
  8. Howlett AC, Breivogel CS, Childers SR, Deadwyler SA, Hampson RE, Porrino LJ: Cannabinoid physiology and pharmacology: 30 years of progress. Neuropharmacology. 2004;47(suppl 1):345-358.
  9. De Petrocellis L, Cascio MG, Di Marzo V: The endocannabinoid system: a general view and latest additions. Br J Pharmacol. 2004;141:765-774.
  10. Wilson RI, Nicoll RA: Endocannabinoid signaling in the brain. Science. 2002;296:678-682.
  11. Cota D, Woods S: The role of the endocannabinoid system in the regulation of energy homeostasis. Curr Opin Endocrinol Diabetes. 2005;12:338-351.
  12. Bracey MH, Hanson MA, Masuda KR, Stevens RC, Cravatt BF: Structural adaptations in a membrane enzyme that terminates endocannabinoid signaling. Science. 2002;298:1793-1796.
  13. Piomelli D: The molecular logic of endocannabinoid signalling. Nat Rev Neurosci. 2003;4:873-884.
  14. Di Marzo V, Fontana A, Cadas H, et al: Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature. 1994;372:686-691.
  15. Di Marzo V, Petrosino S: Endocannabinoids and the regulation of their levels in health and disease. Curr Opin Lipidol. 2007;18:129-140.
  16. Pacher P, Batkai S, Kunos G: The endocannabinoid system as an emerging target of pharmacotherapy. Pharmacol Rev. 2006;58:389-462.
  17. Okamoto Y, Morishita J, Tsuboi K, Tonai T, Ueda N: Molecular characterization of a phospholipase D generating anandamide and its congeners. J Biol Chem. 2004;279:5298-5305.
  18. Leung D, Saghatelian A, Simon GM, Cravatt BF: Inactivation of N-acyl phosphatidylethanolamine phospholipase D reveals multiple mechanisms for the biosynthesis of endocannabinoids. Biochemistry. 2006;45:4720-4726.
  19. Stella N, Schweitzer P, Piomelli D: A second endogenous cannabinoid that modulates long-term potentiation. Nature. 1997;388:773-778.
  20. Dinh TP, Carpenter D, Leslie FM, et al: Brain monoglyceride lipase participating in endocannabinoid inactivation. Proc Natl Acad Sci USA. 2002;99:10819-10824.
  21. Blankman JL, Simon GM, Cravatt BF: A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol. Chem Biol. 2007;14:1347-1356.
  22. Demuth DG, Molleman A: Cannabinoid signalling. Life Sci. 2006;78:549-563.
  23. Rozenfeld R, Devi LA: Regulation of CB1 cannabinoid receptor trafficking by the adaptor protein AP-3. FASEB J. 2008.
  24. Carlisle SJ, Marciano-Cabral F, Staab A, Ludwick C, Cabral GA: Differential expression of the CB2 cannabinoid receptor by rodent macrophages and macrophage-like cells in relation to cell activation. Int Immunopharmacol. 2002;2:69-82.
  25. Mukhopadhyay S, Das S, Williams EA, et al: Lipopolysaccharide and cyclic AMP regulation of CB(2) cannabinoid receptor levels in rat brain and mouse RAW 264.7 macrophages. J Neuroimmunol. 2006;181:82-92.
  26. Pazos MR, Nunez E, Benito C, Tolon RM, Romero J: Functional neuroanatomy of the endocannabinoid system. Pharmacol Biochem Behav. 2005.
  27. Carrier EJ, Kearn CS, Barkmeier AJ, et al: Cultured rat microglial cells synthesize the endocannabinoid 2-arachidonylglycerol, which increases proliferation via a CB2 receptor-dependent mechanism. Mol Pharmacol. 2004;65:999-1007.
  28. Walter L, Franklin A, Witting A, Moller T, Stella N: Astrocytes in culture produce anandamide and other acylethanolamides. J Biol Chem. 2002;277:20869-20876.
  29. Nunez E, Benito C, Pazos MR, et al: Cannabinoid CB2 receptors are expressed by perivascular microglial cells in the human brain: an immunohistochemical study. Synapse. 2004;53:208-213.
  30. Gong JP, Onaivi ES, Ishiguro H, et al: Cannabinoid CB2 receptors: immunohistochemical localization in rat brain. Brain Res. 2006;1071:10-23.
  31. Van Sickle MD, Duncan M, Kingsley PJ, et al: Identification and functional characterization of brainstem cannabinoid CB2 receptors. Science. 2005;310:329-332.
  32. Baker D, Pryce G, Davies WL, Hiley CR: In silico patent searching reveals a new cannabinoid receptor. Trends Pharmacol Sci. 2006;27:1-4.
  33. Lauckner JE, Jensen JB, Chen HY, Lu HC, Hille B, Mackie K: GPR55 is a cannabinoid receptor that increases intracellular calcium and inhibits M current. Proc Natl Acad Sci USA. 2008;105:2699-2704.
  34. Ryberg E, Larsson N, Sjögren S, et al: The orphan receptor GPR55 is a novel cannabinoid receptor. Br J Pharmacol. 2007;152:1092-1101.
  35. Di Marzo V, Bifulco M, De Petrocellis L: The endocannabinoid system and its therapeutic exploitation. Nat Rev Drug Discov. 2004;3:771-784.
  36. Kawamura Y, Fukaya M, Maejima T, et al: The CB1 cannabinoid receptor is the major cannabinoid receptor at excitatory presynaptic sites in the hippocampus and cerebellum. J Neurosci. 2006;26:2991-3001.
  37. Howlett AC, Barth F, Bonner TI, et al: International Union of Pharmacology. XXVII. Classification of cannabinoid receptors. Pharmacol Rev. 2002;54:161-202.
  38. Neubig RR, Spedding M, Kenakin T, Christopoulos A: International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. XXXVIII. Update on terms and symbols in quantitative pharmacology. Pharmacol Rev. 2003;55:597-606.
  39. Pertwee RG: The diverse CB1 and CB2 receptor pharmacology of three plant cannabinoids: delta9-tetrahydrocannabinol, cannabidiol and delta9-tetrahydrocannabivarin. Br J Pharmacol. 2008;153:199-215.
  40. Pan X, Ikeda SR, Lewis DL: SR 141716A acts as an inverse agonist to increase neuronal voltage-dependent Ca2+ currents by reversal of tonic CB1 cannabinoid receptor activity. Mol Pharmacol. 1998;54:1064-1072.
  41. Bouaboula M, Perrachon S, Milligan L, et al: A selective inverse agonist for central cannabinoid receptor inhibits mitogen-activated protein kinase activation stimulated by insulin or insulin-like growth factor 1. Evidence for a new model of receptor/ligand interactions. J Biol Chem. 1997;272:22330-22339.
  42. Mukhopadhyay S, Howlett AC: Chemically distinct ligands promote differential CB1 cannabinoid receptor-Gi protein interactions. Mol Pharmacol.2005;67:2016-20140.
  43. Martini L, Waldhoer M, Pusch M, et al: Ligand-induced down-regulation of the cannabinoid 1 receptor is mediated by the G-protein-coupled receptor-associated sorting protein GASP1. FASEB J. 2007;21:802-811.
  44. Pertwee RG: Inverse agonism and neutral antagonism at cannabinoid CB1 receptors. Life Sci. 2005;76:1307-1324.
  45. Compton DR, Johnson MR, Melvin LS, Martin BR: Pharmacological profile of a series of bicyclic cannabinoid analogs: classification as cannabimimetic agents. J Pharmacol Exp Ther. 1992;260:201-209.
  46. Devane WA, Dysarz FA, 3rd, Johnson MR, Melvin LS, Howlett AC: Determination and characterization of a cannabinoid receptor in rat brain. Mol Pharmacol. 1988;34:605-613.
  47. Kuster JE, Stevenson JI, Ward SJ, D'Ambra TE, Haycock DA: Aminoalkylindole binding in rat cerebellum: selective displacement by natural and synthetic cannabinoids. J Pharmacol Exp Ther. 1993;264:1352-1363.
  48. Marriott KS, Huffman JW: Recent advances in the development of selective ligands for the cannabinoid CB(2) receptor. Curr Top Med Chem. 2008;8:187-204.
  49. Hillard CJ, Manna S, Greenberg MJ, et al: Synthesis and characterization of potent and selective agonists of the neuronal cannabinoid receptor (CB1). J Pharmacol Exp Ther. 1999;289:1427-1433.
  50. Jhaveri MD, Richardson D, Chapman V: Endocannabinoid metabolism and uptake: novel targets for neuropathic and inflammatory pain. Br J Pharmacol. 2007;152:624-632.
  51. Pertwee RG: Cannabinoid pharmacology: the first 66 years. Br J Pharmacol. 2006;147(Suppl 1):S163-S171.
  52. Lin LS, Lanza TJ, Jr., Jewell JP, et al: Discovery of N-[(1S,2S)-3-(4-Chlorophenyl)-2- (3-cyanophenyl)-1-methylpropyl]-2-methyl-2- {[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide (MK-0364), a novel, acyclic cannabinoid-1 receptor inverse agonist for the treatment of obesity. J Med Chem. 2006;49:7584-7587.
  53. Pertwee RG: Pharmacology of cannabinoid CB1 and CB2 receptors. Pharmacol Ther. 1997;74:129-180.
  54. Fride E, Perchuk A, Hall FS, Uhl GR, Onaivi ES: Behavioral methods in cannabinoid research. In: Onaivi ES (ed): Methods in Molecular Medicine: Marijuana and Cannabinoid Research: Methods and Protocols. Totowa, NJ: Humana Press Inc., 2006, 269-290.
  55. Weddington WW, Jr., Miller NJ, Sweet DL: Anticipatory nausea and vomiting associated with cancer chemotherapy. N Engl J Med. 1982;307:825-826.
  56. Limebeer CL, Hall G, Parker LA: Exposure to a lithium-paired context elicits gaping in rats: a model of anticipatory nausea. Physiol Behav. 2006;88:398-403.
  57. Landauer MR, Balster RL, Harris LS: Attenuation of cyclophosphamide-induced taste aversions in mice by prochlorperazine, delta 9-tetrahydrocannabinol, nabilone and levonantradol. Pharmacol Biochem Behav. 1985;23:259-266.
  58. Howlett AC, Bidaut-Russell M, Devane WA, Melvin LS, Johnson MR, Herkenham M: The cannabinoid receptor: biochemical, anatomical and behavioral characterization. Trends Neurosci. 1990;13:420-423.
  59. Agarwal N, Pacher P, Tegeder I, et al: Cannabinoids mediate analgesia largely via peripheral type 1 cannabinoid receptors in nociceptors. Nat Neurosci. 2007;10:870-879.
  60. Cold/Hot Plate Analgesia Meter. Columbus Instruments. http://www.colinst.com/brief.php?id=58. Accessed May 15, 2008.
  61. Kalueff AV, Wheaton M, Murphy DL: What's wrong with my mouse model? Advances and strategies in animal modeling of anxiety and depression. Behav Brain Res. 2007;179:1-18.
  62. Crawley JN: Behavioral phenotyping of transgenic and knockout mice: experimental design and evaluation of general health, sensory functions, motor abilities, and specific behavioral tests. Brain Res. 1999;835:18-26.
  63. Pellow S, Chopin P, File SE, Briley M: Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Methods. 1985;14:149-167.
  64. Zero maze protocol. Neurogenetics & Behavior Center, Department of Psychological and Brain Sciences, Johns Hopkins University. http://nbc.jhu.edu/protocols/ZeroMazeProtocol.aspx. Accessed June 4, 2008.
  65. Steru L, Chermat R, Thierry B, et al: The automated Tail Suspension Test: a computerized device which differentiates psychotropic drugs. Prog Neuropsychopharmacol Biol Psychiatry. 1987;11:659-671.
  66. Porsolt RD, Bertin A, Jalfre M: Behavioral despair in mice: a primary screening test for antidepressants. Arch Int Pharmacodyn Ther. 1977;229:327-336.
  67. Balogh SA, Kwon YT, Denenberg VH: Varying intertrial interval reveals temporally defined memory deficits and enhancements in NTAN1-deficient mice. Learn Mem. 2000;7:279-286.
  68. Morris R: Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods. 1984;11:47-60.
  69. Deadwyler SA, Hampson RE: Endocannabinoids modulate encoding of sequential memory in the rat hippocampus. Psychopharmacology (Berl). 2008;198:577-586.
  70. Fear conditioning. Neurogenetics & Behavior Center, Department of Psychological and Brain Sciences, Johns Hopkins University. http://nbc.jhu.edu/beh/fearcond.aspx. Accessed June 4, 2008.
  71. Burns HD, Van Laere K, Sanabria-Bohorquez S, et al: [18F]MK-9470, a positron emission tomography (PET) tracer for in vivo human PET brain imaging of the cannabinoid-1 receptor. Proc Natl Acad Sci USA. 2007;104:9800-9805.
  72. Kunos G, Batkai S: Novel physiologic functions of endocannabinoids as revealed through the use of mutant mice. Neurochem Res. 2001;26:1015-1021.
  73. Di Marzo V, Breivogel CS, Tao Q, et al: Levels, metabolism, and pharmacological activity of anandamide in CB(1) cannabinoid receptor knockout mice: evidence for non-CB(1), non-CB(2) receptor-mediated actions of anandamide in mouse brain. J Neurochem. 2000;75:2434-2444.
  74. Buckley NE: The peripheral cannabinoid receptor knockout mice: an update. Br J Pharmacol. 2008;153:309-318.
  75. Batkai S, Osei-Hyiaman D, Pan H, et al: Cannabinoid-2 receptor mediates protection against hepatic ischemia/reperfusion injury. FASEB J. 2007;21:1788-1800.
  76. Naidu PS, Varvel SA, Ahn K, Cravatt BF, Martin BR, Lichtman AH: Evaluation of fatty acid amide hydrolase inhibition in murine models of emotionality. Psychopharmacology (Berl). 2007;192:61-70.
  77. Wise LE, Shelton CC, Cravatt BF, Martin BR, Lichtman AH: Assessment of anandamide's pharmacological effects in mice deficient of both fatty acid amide hydrolase and cannabinoid CB1 receptors. Eur J Pharmacol. 2007;557:44-48.
  78. Kuhn R, Schwenk F, Aguet M, Rajewsky K: Inducible gene targeting in mice. Science. 1995;269:1427-1429.
  79. Pertwee RG, Ross RA, Craib SJ, Thomas A: (-)-Cannabidiol antagonizes cannabinoid receptor agonists and noradrenaline in the mouse vas deferens. Eur J Pharmacol. 2002;456:99-106.
  80. Plasse TF, Gorter RW, Krasnow SH, Lane M, Shepard KV, Wadleigh RG: Recent clinical experience with dronabinol. Pharmacol Biochem Behav. 1991;40:695-700.
  81. Mackie K: Cannabinoid receptors as therapeutic targets. Annu Rev Pharmacol Toxicol. 2006;46:101-122.
  82. Esposito G, De Filippis D, Carnuccio R, Izzo AA, Iuvone T: The marijuana component cannabidiol inhibits beta-amyloid-induced tau protein hyperphosphorylation through Wnt/beta-catenin pathway rescue in PC12 cells. J Mol Med. 2006;84:253-258.
  83. Ramirez BG, Blazquez C, Gomez del Pulgar T, Guzman M, de Ceballos ML: Prevention of Alzheimer's disease pathology by cannabinoids: neuroprotection mediated by blockade of microglial activation. J Neurosci. 2005;25:1904-1913.
  84. Sarne Y, Mechoulam R: Cannabinoids: between neuroprotection and neurotoxicity. Curr Drug Targets CNS Neurol Disord. 2005;4:677-684.
  85. van der Stelt M, Mazzola C, Esposito G, et al: Endocannabinoids and beta-amyloid-induced neurotoxicity in vivo: effect of pharmacological elevation of endocannabinoid levels. Cell Mol Life Sci. 2006;63:1410-1424.
  86. Howlett AC, Mukhopadhyay S, Norford DC: Endocannabinoids and reactive nitrogen and oxygen species in neuropathologies. J Neuroimmune Pharmacol. 2006;1:305-316.
  87. Mangieri RA, Piomelli D: Enhancement of endocannabinoid signaling and the pharmacotherapy of depression. Pharmacol Res. 2007;56:360-366.
  88. Hill MN, Gorzalka BB: Is there a role for the endocannabinoid system in the etiology and treatment of melancholic depression? Behav Pharmacol. 2005;16:333-352.
  89. Gobbi G, Bambico FR, Mangieri R, et al: Antidepressant-like activity and modulation of brain monoaminergic transmission by blockade of anandamide hydrolysis. Proc Natl Acad Sci USA. 2005;102:18620-18625.
  90. Bortolato M, Mangieri RA, Fu J, et al: Antidepressant-like activity of the fatty acid amide hydrolase inhibitor URB597 in a rat model of chronic mild stress. Biol Psychiatry. 2007;62:1103-1110.
  91. Rog DJ, Nurmikko TJ, Young CA: Oromucosal delta9-tetrahydrocannabinol/cannabidiol for neuropathic pain associated with multiple sclerosis: an uncontrolled, open-label, 2-year extension trial. Clin Ther. 2007;29:2068-2079.
  92. Nurmikko TJ, Serpell MG, Hoggart B, Toomey PJ, Morlion BJ, Haines D: Sativex successfully treats neuropathic pain characterised by allodynia: a randomised, double-blind, placebo-controlled clinical trial. Pain. 2007;133:210-220.
  93. Berman JS, Symonds C, Birch R: Efficacy of two cannabis based medicinal extracts for relief of central neuropathic pain from brachial plexus avulsion: results of a randomised controlled trial. Pain. 2004;112:299-306.
  94. Izzo AA, Camilleri M: Emerging role of cannabinoids in gastrointestinal and liver diseases: basic and clinical aspects. Gut. 2008 (e-pub).
  95. Pertwee RG: Ligands that target cannabinoid receptors in the brain: from THC to anandamide and beyond. Addict Biol. 2008;13:147-159.
  96. Van Gaal LF, Rissanen AM, Scheen AJ, Ziegler O, Rossner S: Effects of the cannabinoid-1 receptor blocker rimonabant on weight reduction and cardiovascular risk factors in overweight patients: 1-year experience from the RIO-Europe study. Lancet. 2005;365:1389-1397.
  97. Després JP, Golay A, Sjöström L: Effects of rimonabant on metabolic risk factors in overweight patients with dyslipidemia. N Engl J Med. 2005;353:2121-2134.
  98. Pi-Sunyer FX, Aronne LJ, Heshmati HM, Devin J, Rosenstock J: Effect of rimonabant, a cannabinoid-1 receptor blocker, on weight and cardiometabolic risk factors in overweight or obese patients: RIO-North America: a randomized controlled trial. JAMA. 2006;295:761-775.
  99. Scheen AJ, Finer N, Hollander P, Jensen MD, Van Gaal LF: Efficacy and tolerability of rimonabant in overweight or obese patients with type 2 diabetes: a randomised controlled study. Lancet. 2006;368:1660-1672.
  100. European medicines agency recommends Accomplia must not be used in patients on antidepressants or with major depression. Medical News Today, 21 July 2007. www.medicalnewstoday.com.
  101. The Committee for Medicinal Products for Human Use: Summary of product characteristics (Accomplia). European Medicines Agency. Approved July 19, 2007.
  102. Gardner A, Mallet PE: Suppression of feeding, drinking, and locomotion by a putative cannabinoid receptor 'silent antagonist'. Eur J Pharmacol. 2006;530:103-106.
  103. Chambers AP, Vemuri VK, Peng Y, et al: A neutral CB1 receptor antagonist reduces weight gain in rat. Am J Physiol Regul Integr Comp Physiol. 2007;293:R218