ECSN Faculty

Vincenzo Di Marzo, PhD
Research Director
Endocannabinoid Research Group
Institute of Biomolecular Chemistry
Consiglio Nazionale delle Ricerche
Pozzuoli, Italy

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

Kenneth Mackie, MD
Professor of Psychology
Department of Psychological and Brain Sciences
Indiana University
Bloomington, Indiana

Olivier Manzoni, PhD
INSERM U862
Neurocentre Magendie
Physiopathology of Synaptic Plasticity
Bordeaux, France

CME Program Reviewer

Carol Maggio, PhD
Associate Research Scientist
New York Obesity Research Center
St. Luke's-Roosevelt Hospital Center

Introduction

The endocannabinoid system (ECS) is composed of endocannabinoid receptors (the cannabinoid [CB] receptors), their endogenous ligands (the endocannabinoids), and the proteins involved in endocannabinoid synthesis and inactivation.¹ All of these components are found in the central nervous system (CNS).^2-9 In 1988, Howlett and coworkers¹⁰ described the presence of high-affinity binding sites for cannabinoids in rat brain membranes and their signaling through G-proteins. Shortly afterwards, Herkenham et al¹¹ performed autoradiographic mapping studies of cannabinoid binding sites in rat, human, rhesus monkey, dog, and guinea pig brain sections.¹¹ Matsuda et al cloned the CB₁ receptor¹² and determined its distribution by in situ hybridization studies in rat brain.¹³ More recent studies showed that CB₂ receptors are expressed in the CNS, but at a much lower level than that of CB₁ receptors.^14-17

Cannabinoid Receptors

The CB₁ receptor is among the most abundant G-protein–coupled receptors in the brain, present in almost every brain region and on many different types of neurons.¹⁸ CB₂ receptors are also found within the CNS, but are expressed primarily (but not exclusively) in cells of immune and hematopoietic origin, such as microglia.^14-17 The location of CB₁ receptors in the brain has provided significant insight into their function in the CNS,¹⁹ while the expression and function of CB₂ receptors in the CNS is in the nascent phases of research.¹⁶

Studies on CB₁ and CB₂ receptor knockout mice suggest that there may be several additional CB receptors.^{20, 21} For example, several cannabinoid agonists bind to and activate the orphan G-protein–coupled receptor (GPCR) GPR55, which is expressed in brain and various peripheral tissues in humans and rodents.^{22, 23} There is also evidence that endocannabinoids can produce effects that are not mediated by GPCRs.^{24, 25} These non-CB receptor–mediated mechanisms are currently under investigation, including the capability of some endocannabinoids to activate transient receptor potential vanilloid type 1 (TRPV1) channels in the CNS as seems to be supported by the emerging literature.^{26, 27}

CB₁ Receptors

CB₁ receptors are abundant in certain parts of the brain, such as the hippocampus, cortex, cerebellum, and basal ganglia (Table 1).²⁰ In these structures, CB₁ receptors primarily exhibit a presynaptic location.² The study of the ECS in these brain areas has implicated this system in modulating motor function in the cerebellum and basal ganglia.^{28, 29} In the hippocampus, the ECS plays a role in pathways related to learning and memory,³⁰ and in the cerebral cortex the system is implicated in cognitive information processing.³¹ In the amygdala the ECS modulates emotionality and in the brain stem and spinal cord the ECS is involved in nociception.⁸⁷ Furthermore, studies of CB₁ receptors within the mesolimbic dopaminergic system indicate that the ECS plays a role in reward and motivational processes.³² Finally, it is now clear that the ECS directly modulates the activity of neuronal circuits regulating food intake, both within the hypothalamus and the limbic system.³³ In the brain areas with the highest levels of CB₁ receptors, their density is similar to levels of γ-aminobutyric acid- (GABA) and glutamate-gated ion channels, which mediate most of inhibitory and excitatory synaptic transmission,³⁴ suggesting that the CB₁ receptor plays a prominent role in neuronal function. [³⁵S]GTP_γS autoradiography studies in rat brain demonstrate that the distribution of cannabinoid-activated G proteins, in general, parallels CB₁ receptor binding.³⁴ Breivogel et al found that the relative number of G proteins activated by CB₁ receptor stimulation in rat brain (denoted as amplification factor) varies according to brain region.³⁵ The amplification factor for the hypothalamus (an area containing a low density of CB₁ receptors) was significantly higher than that of brain areas with high density of CB₁ receptors, such as the hippocampus, frontal cortex, striatum, and cerebellum.³⁵ Thus, receptor signaling efficiency cannot be predicted based on receptor density. Autoradiography studies using [³H] labeled CP 55,940 (a highly potent synthetic CB receptor agonist) demonstrate the presence of CB₁ receptors in human brain (Figure 1).¹¹

1.  CB1 receptors are abundant in

A. Cerebellum B. Hippocampus C. Basal Ganglia D. Hypothalamus E. All of the above F. A, B, and C

2.  The ECS

A. Plays a role in pathways related to learning and memory B. Is implicated in cognitive information processing C. Plays a role in reward and motivational processes D. All of the above E. A and C

3.  The ECS directly modulates the activity of neuronal circuits regulating food intake, both within the hypothalamus and the limbic system.

True False

CB₂ Receptors

CB₂ receptor mRNA and protein were reported to be present in rat, ferret, and mouse brainstem neurons.¹⁴ However, the extent of CB₂ expression in brain remains controversial and several reports have failed to find appreciable levels of CB₂ receptor expression in the brain.^36-40 CB₂ receptor expression was found in perivascular microglial cells of the human cerebellum (Figure 2).¹⁵ An interesting aspect of CB₂ receptors is that in many cases their levels strongly increase following a pathological insult. For example, CB₂ receptor expression is induced in brain microglial cells during inflammation,¹⁵ in sensory neurons following peripheral nerve injury,⁴¹ as well as in models of neuropathic and inflammatory pain.^{42, 43}

Endocannabinoids

The first-discovered and most extensively studied endocannabinoids are anandamide (N-arachidonylethanolamine) and 2-arachidonoylglycerol (2-AG). ^{1, 44} Following synthesis, anandamide and 2-AG are released into the extracellular milieu where they can bind to CB receptors.^{1, 20} Pharmacologically, 2-AG binds both CB₁ and CB₂ receptors with somewhat low affinity, but activates these receptors as full agonists, whereas anandamide has a higher affinity for CB₁ than CB₂ and is a medium- to low-efficacy agonist at both receptors, at least in vitro. Thus, anandamide can act as a partial agonist at CB₁ and CB₂ receptors, and 2-AG usually shows full agonism at both receptors.⁴⁵ 2-AG is more abundant in the CNS compared with anandamide⁴⁶ However, only a small portion of CNS 2-AG is involved in endocannabinoid signaling, with the reminder involved in lipid metabolism and other types of signaling.⁴⁷ Both molecules appear to be synthesized in response to a stimulus in a tightly regulated fashion.⁷

4.  Which of the following is true?

A. Expression of CB2 receptors is often induced following a pathological insult B. CB2 receptors are found in brain only C. Anandamide has lower affinity for CB1 than CB2 receptors D. All of the above

Because endocannabinoids are lipophilic compounds derived from membrane phospholipids, they are not stored in synaptic vesicles as are the classical neurotransmitters (eg, monoamines, glutamate, GABA, acetylcholine, peptide hormones).^{33, 44, 48} In the brain, they are produced by neurons and glia at their sites of action and, when released, generate a transient, rapid activation of CB₁ receptors before being hydrolyzed and inactivated.^{33, 48, 49} The transient activation of CB₁ receptors can lead to long-lasting modulation of synaptic transmission, and endocannabinoids are implicated in long-term synaptic plasticity throughout the CNS (see below). In all cases, endocannabinoids are considered to be local neuromodulators.³³

Endocannabinoids are rapidly cleared from the extracellular milieu (terminal half-life [t_½] is seconds to minutes).¹ Studies suggest that, following CB₁ receptor activation, anandamide and 2-AG are taken up by cells by a facilitated transport mechanism mediated by a putative anandamide membrane transporter (AMT) (reviewed in Bari et al).⁵⁰ Although data from several biochemical and pharmacological studies support the existence of an AMT, this is a controversial topic, and an AMT protein remains to be identified at the molecular level.¹ Regardless of the mechanism of entry into cells, enzymatic degradation of endocannabinoids plays a major role in the termination of their action.

5.  In the CNS, endocannabinoids

A. Are produced by neurons at their sites of action B. Are considered local neuromodulators C. Are rapidly cleared from the extracellular milieu D. All of the above E. A and C only

6.  Which of the following statements is false?

A. Anandamide and 2-AG are generated by the cleavage of cholesterol B. Fatty acid amide hydrolase (FAAH) catalyzes the hydrolysis of anandamide in vivo C. Anandamide and 2-AG are not stored in vesicles inside cells D. A and B only

Enzymes Involved with the Biosynthesis and Degradation of Endocannabinoids

As indicated above, anandamide and 2-AG are lipid signaling molecules which, unlike classical neurotransmitters, are not stored in vesicles inside the cell but are rather thought to be produced by cells, following a stimulus for their release.¹ Thus, the enzymes governing the biosynthesis and degradation of endocannabinoids ensure tight temporal and spatial control over their signaling function. Anandamide and 2-AG are generated by the cleavage of the respective cell membrane phospholipid precursors, N-arachidonoylphosphatidylethanolamine and diacylglycerol (Figure 3). Fatty acid amide hydrolase (FAAH) catalyzes the hydrolysis of anandamide (and 2-AG) in vivo ^51-55 and a monoacylglycerol lipase plays a key role in the enzymatic hydrolysis of 2-AG (Figure 3).⁷

Cannabinoid Receptor Signaling

G (guanosine triphosphate) Proteins

CB₁ and CB₂ receptors belong to the family of G-protein–coupled receptors (GPCRs), a family of membrane receptors that interact with heterotrimeric G-proteins to produce most of their biological effects. Heterotrimeric G-proteins, consist of α, β and γ subunits (Figure 4).⁵⁶ CB₁ receptors typically couple to G-proteins of the G_i/o class. Major effects of their activation include inhibition of adenylyl cyclase, modulation of ion channels, and activation of mitogen-activated protein (MAP) kinases. However, CB₁ receptors may be coupled to G_s and/or G_q/11 in addition to G_i/o proteins, although the physiological significance of this remains obscure.²⁶ Activation of CB₁ receptors coupled to G_i/o proteins results in inhibition of adenylyl cyclase activity and its downstream signaling pathways (Figure 5).⁵⁷ The β/γ subunits liberated from the α subunits following receptor activation remain together and participate in additional signaling pathways, such as the inhibition of certain voltage-gated calcium (Ca²⁺) channels or activation of some types of potassium (K⁺) channels. After a variable but highly regulated period of time, the intrinsic GTPase activity of the G protein α subunit hydrolyzes its bound GTP to GDP