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Title
Role of endogenous cannabinoids in synaptic signaling.
Permalink
https://escholarship.org/uc/item/1633t2mf
Journal
Physiological reviews, 83(3)
ISSN
0031-9333
Authors
Freund, Tamas F
Katona, Istvan
Piomelli, Daniele
Publication Date
2003-07-01
DOI
10.1152/physrev.00004.2003
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This work is made available under the terms of a Creative Commons Attribution License,
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Role of Endogenous Cannabinoids
in Synaptic Signaling
TAM
´
AS F. FREUND, ISTV
´
AN KATONA, AND DANIELE PIOMELLI
Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary; Department
of Clinical Neurobiology, University Hospital of Neurology, Heidelberg, Germany; and Department
of Pharmacology, University of California Irvine, Irvine, California
I. Introduction 1018
II. The Life Cycle of Endocannabinoids 1020
A. Introduction 1020
B. Biosynthetic pathways 1020
C. Termination of endocannabinoid effects: transport and degradation 1023
III. Regional and Cellular Distribution of Neuronal CB
1
Cannabinoid Receptors 1028
A. Characteristic differences in CB
1
receptor distribution in the brain 1028
B. Selective expression of CB
1
cannabinoid receptors by identified cell types of complex networks 1030
IV. Anatomical, Physiological, and Pharmacological Evidence for the Presynaptic Localization of CB
1
Cannabinoid Receptors in the Brain 1035
A. Anatomical evidence for presynaptic cannabinoid receptors 1036
B. Physiological and pharmacological evidence for presynaptic cannabinoid receptors 1039
C. Are there postsynaptic CB
1
receptors? 1044
V. Physiological Roles of Endocannabinoids 1045
A. The cannabinoid root 1045
B. The DSI (DSE) root: control of GABAergic and glutamatergic synaptic transmission via
retrograde synaptic signaling 1048
C. Marriage of the two lines of research explains the mechanism of DSI (and DSE) while
endowing endocannabinoids with function 1049
D. Electrical activity patterns required for the release of endocannabinoids 1054
VI. Conclusions 1056
Freund, Tama´s F., Istva´n Katona, and Daniele Piomelli. Role of Endogenous Cannabinoids in Synaptic
Signaling. Physiol Rev 83: 1017–1066, 2003; 10.1152/physrev.00004.2003.—Research of cannabinoid actions was
boosted in the 1990s by remarkable discoveries including identification of endogenous compounds with cannabimi-
metic activity (endocannabinoids) and the cloning of their molecular targets, the CB
1
and CB
2
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 CB
1
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 endocannabi-
noids 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 CB
1
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.
Physiol Rev
83: 1017–1066, 2003; 10.1152/physrev.00004.2003.
www.prv.org 10170031-9333/03 $15.00 Copyright © 2003 the American Physiological Society
I. INTRODUCTION
Descriptions of the Cannabis sativa plant and its
medicinal properties were already accessible to Greek
and Roman physicians in the first century AD, when Di-
oscorides included the plant in his classic textbook of
pharmacology, entitled Materia Medica (“The Materials
of Medicine”). Ancient Indian and Chinese medical writ-
ers were even more accurate than their European col-
leagues in describing the remarkable physiological and
psychological effects of this plant (for review, see Ref.
241). We know now that these effects, which in humans
include a variable combination of euphoria, relaxation,
reflex tachycardia, and hypothermia, are primarily pro-
duced by the dibenzopyrane derivative, delta-9-tetrahy-
drocannabinol (delta-9-THC), present in the yellow resin
that covers the leaves and flower clusters of the ripe
female plant. The chemical structure of delta-9-THC was
elucidated by the pioneering studies of R. Adams (6) and
Gaoni and Mechoulam (114). Unlike morphine, cocaine,
and other alkaloids of plant origin, delta-9-THC is a highly
hydrophobic compound, a property that, curiously
enough, has slowed the progress on the mode of action of
this compound for nearly three decades. The affinity of
delta-9-THC for lipid membranes erroneously suggested,
indeed, that the drug’s main effect was to modify in a
nonselective manner the fluidity of cell membranes rather
than to activate a selective cell-surface receptor (157,
207).
Two series of events contributed to a radical change
of this view. First, motivated by the potential therapeutic
applications of cannabis-like (“cannabimimetic”) mole-
cules, laboratories in academia and the pharmaceutical
industry began to develop families of synthetic analogs of
delta-9-THC. These agents exerted pharmacological ef-
fects that were qualitatively similar to those of delta-9-
THC but displayed both greater potency and stereoselec-
tivity. The latter feature cannot be reconciled with non-
specific membrane interactions, providing the first
evidence that delta-9-THC exerts its effects by combining
with a selective receptor. Second, as a result of these
synthetic efforts, it became possible to explore directly
the existence of cannabinoid receptors by using standard
radioligand binding techniques. In 1988, Howlett and her
co-workers (84, 167) described the presence of high-affin-
ity binding sites for cannabinoid agents in brain mem-
branes and showed that these sites are coupled to inhibi-
tion of adenylyl cyclase activity. Conclusively supporting
these findings, in 1990 Matsuda et al. (236) serendipitously
came across a complementary DNA encoding for the first
G protein-coupled cannabinoid receptor, now known as
CB
1
.
In heterologous expression systems, CB
1
receptors
were found to be functionally coupled to multiple intra-
cellular signaling pathways, including inhibition of adeny-
lyl cyclase activity, inhibition of voltage-activated calcium
channels, and activation of potassium channels (56, 148,
221, 222, 236, 239). In situ hybridization and immunohis-
tochemical studies have demonstrated that CB
1
receptors
are abundantly expressed in discrete regions and cell
types of the central nervous system (CNS) (see also sect.
III) but are also present at significant densities in a variety
of peripheral organs and tissues (41, 225, 226, 235, 345).
The selective distribution of CB
1
receptors in the CNS
provides a clear anatomical correlate for the cognitive,
affective, and motor effects of cannabimimetic drugs.
The cloning and characterization of CB
1
receptors
left several important problems unsolved. Since antiquity,
it has been known that the actions of Cannabis and
delta-9-THC are not restricted to the CNS, but include
effects on nonneural tissues such as reduction of inflam-
mation, lowering of intraocular pressure associated with
glaucoma, and relief of muscle spasms. Are these periph-
eral effects all produced by activation of CB
1
receptors?
An initial answer to this question was provided by the
discovery of a second cannabinoid receptor exquisitely
expressed in cells of immune origin (260). This receptor,
called CB
2
, only shares ⬃44% sequence identity with its
brain counterpart, implying that the two subtypes di-
verged long ago in evolution. The intracellular coupling of
the CB
2
receptor resembles, however, that of the CB
1
receptor; for example, in transfected cells, CB
2
receptor
activation is linked to the inhibition of adenylyl cyclase
activity (113).
The experience with opioid receptors and the en-
kephalins has accustomed scientists to the idea that
whenever a receptor is present in the body, endogenous
factor(s) that activate this receptor also exist. Not sur-
prisingly, therefore, as soon as cannabinoid receptors
were described, a search began to identify their naturally
occurring ligand(s). One way to tackle this problem was
based on the premise that, like other neurotransmitters
and neuromodulators, an endogenous cannabinoid sub-
stance should be released from brain tissue in a calcium-
dependent manner. Taking this route, Howlett and co-
workers incubated rat brain slices in the presence of a
calcium ionophore and determined whether the media
from these incubations contained a factor that displaced
the binding of labeled CP-55940, a cannabinoid agonist, to
brain membranes. These studies demonstrated that a can-
nabinoid-like activity was indeed released from stimu-
lated slices, but the minute amounts of this factor did not
allow the elucidation of its chemical structure (97, 98).
Devane, Mechoulam, and co-workers (85, 243), at the
Hebrew University in Jerusalem, adopted a different strat-
egy. Reasoning that endogenous cannabinoids may be as
hydrophobic as delta-9-THC, they subjected porcine
brains to organic solvent extraction and fractionated the
lipid extract by chromatographic techniques while mea-
suring cannabinoid binding activity. This approach turned
1018
FREUND, KATONA, AND PIOMELLI
Physiol Rev
• VOL 83 • JULY 2003 • www.prv.org
out to be highly successful, and the researchers were able
to isolate a lipid cannabinoid-like component, which they
characterized by mass spectrometry and nuclear mag-
netic resonance spectroscopy as the ethanolamide of ar-
achidonic acid. They named this novel compound “anan-
damide” after the sanskrit “ananda,” inner bliss.
The chemical synthesis of anandamide confirmed
this structural identification and allowed the characteriza-
tion of its pharmacological properties (112). In vitro and
in vivo tests showed a great similarity of actions between
anandamide and cannabinoid drugs. Anandamide reduced
the electrogenic contraction of mouse vas deferens and
closely mimicked the behavioral responses produced by
delta-9-THC in vivo; in the rat, the compound was found
to produce analgesia, hypothermia, and hypomotility.
However, these effects may not be exclusively due to
cannabinoid receptor activation, as anandamide is readily
metabolized to arachidonic acid, which can be converted
in turn to a variety of biologically active eicosanoid com-
pounds. Subsequent studies demonstrated that anandam-
ide is released from brain neurons in an activity-depen-
dent manner (89, 126) and elucidated the unique biochem-
ical routes of anandamide formation and inactivation in
the CNS (25, 44, 45, 69, 89). Thus anandamide fulfills all
key criteria that define an endogenous cannabinoid (en-
docannabinoid) substance.
In their 1992 study, Devane, Mechoulam, and co-
workers (242) reported that several lipid fractions from
the rat brain contained cannabinoid-binding activity, in
addition to anandamide’s. In characterizing these frac-
tions, they discovered that some of them were composed
of polyunsaturated fatty acid ethanolamides similar to
anandamide (e.g., eicosatrienoylethanolamide), but oth-
ers were instead constituted of a distinct lipid component,
sn-2-arachidonoyl-glycerol (2-AG) (242). Sugiura et al.
(330) arrived independently to the same conclusion. That
polyunsaturated fatty acid ethanolamides should mimic
anandamide, to which they are structurally very similar,
does not come as a great surprise. Moreover, the pharma-
cological properties of these fatty acid ethanolamides,
essentially indistinguishable from those of anandamide,
and their scarcity in brain relegate them, at least for the
moment, to a position secondary to anandamide’s. We
cannot say the same for 2-AG. This lipid, considered until
now a mere intermediate in glycerophospholipid turnover
(see sect.
II), is present in the brain at concentrations that
are ⬃170-fold greater than those of anandamide and pos-
sesses two pharmacological properties that make it cru-
cially different from the latter: it binds to both CB
1
and
CB
2
cannabinoid receptors with similar affinities, and it
activates CB
1
receptors as a full agonist, whereas anand-
amide acts as a partial agonist.
Research of endocannabinoids begs for a conjunc-
tion of in situ biochemistry and physiology. We have
learned much over the past 10 years on the behavioral
effects of these molecules, on how these lipid mediators
are produced physiologically, and on the functional roles
that they may serve. A major step was the discovery that
depolarization-induced suppression of inhibition (DSI; or
excitation, DSE), a type of short-term synaptic plasticity
originally discovered in the cerebellum and the hippocam-
pus (214, 288), is mediated by endocannabinoids (199,
200, 271, 375). This discovery allowed the results of over
a decade of research on retrograde synaptic signaling in
these networks to be considered as functional character-
istics of endocannabinoid signaling. The substrate of ret-
rograde signaling and DSI is the predominantly presynap-
tic distribution of CB
1
receptors on axon terminals in the
hippocampus (188), as well as throughout the brain,
where activation of CB
1
by endocannabinoids can effi-
ciently veto neurotransmitter release in many distinct
types of synapses (see sect.
IV). The conditions of synthe-
sis, release, distance of diffusion, duration of effect, and
site of action were all extensively characterized for the
mediator of DSI (for review, see Ref. 10) that turned out
to be an endocannabinoid (271, 375). The fact that neu-
rons are able to control the efficacy of their own synaptic
input in an activity-dependent manner (a phenomenon
called retrograde synaptic signaling) is functionally very
important, since this mechanism may subserve several
functions in information processing by neuronal net-
works from temporal coding and oscillations to group
selection and the fine tuning of signal-to-noise ratio. The
crucial involvement of endocannabinoids in these func-
tions just began to emerge from recent studies, which are
reviewed in section
V. Due to the exceptionally rapid
expansion of this field in recent years (and to our special
interest in neuronal signaling in complex integrative cen-
tres of the brain), we decided to focus the present review
on questions related to the composition of the endocan-
nabinoid system and its physiological roles in controlling
brain activity at the regional and cellular levels as synap-
tic signal molecules. We did not aim to provide detailed
accounts of studies dealing with other, similarly impor-
tant, aspects of cannabinoid research, which have been
dealt with in excellent recent reviews, e.g., about the
relation of the endocannabinoid system to pain modula-
tion (281, 366), the immune system (194), neuroprotection
(136), and addiction (228).
The final message of the present review is that to
understand the possible physiological functions of the
endogenous cannabinoids, their roles in normal and
pathological brain activity, pharmacological agents target-
ing the cascade of anandamide and 2-AG formation, re-
lease, uptake, and degradation will have to be developed.
Such drugs, which undoubtedly will become invaluable
research tools to study the potential functions listed
above, may also provide novel therapeutic approaches to
diseases whose clinical, biochemical, and pharmacologi-
ENDOCANNABINOIDS IN SYNAPTIC SIGNALING 1019
Physiol Rev • VOL 83 • JULY 2003 • www.prv.org
cal features suggest a link with the endogenous cannabi-
noid system.
II. THE LIFE CYCLE OF THE
ENDOCANNABINOIDS
A. Introduction
A basic principle that has emerged from the last two
decades of research on cellular signaling is that simple
phospholipids such as phosphatidylcholine or phosphati-
dylinositol should be regarded not only as structural com-
ponents of the cell membrane, but also as precursors for
transmembrane signaling molecules. Intracellular second
messengers like 1,2-diacylglycerol (DAG) and ceramide
are familiar examples of this concept. Along with their
intracellular roles, however, lipid compounds may also
serve important functions in the exchange of information
between cells. Indeed, biochemical mechanisms analo-
gous to those involved in the generation of DAG or cer-
amide give rise to biologically active lipids that leave their
cell of origin to activate G protein-coupled receptors lo-
cated on the surface of neighboring cells. Traditionally
overshadowed by amino acid, amine, and peptide trans-
mitters, biologically active lipids are now emerging as
essential mediators of cell-to-cell communication within
the CNS, where G protein-coupled receptors for multiple
families of such compounds, including lysophosphatidic
acid and eicosanoids, have been identified (67, 285).
In this section, we discuss the biochemical properties
of endogenous lipids that activate brain cannabinoid re-
ceptors. These compounds share two common structural
motifs: a polyunsaturated fatty acid moiety (e.g., arachi-
donic acid) and a polar head group consisting of ethanol-
amine or glycerol (Fig. 1). Because of these features,
endocannabinoid substances seemingly resemble the ei-
cosanoids, ubiquitous bioactive lipids generated through
the enzymatic oxygenation of arachidonic acid. However,
the endocannabinoids are clearly distinguished from the
eicosanoids by their different biosynthetic routes, which
do not involve oxidative metabolism. The two best char-
acterized endocannabinoids, anandamide (arachido-
noylethanolamide) (85) and 2-AG (242, 330), may be pro-
duced instead through cleavage of phospholipid precur-
sors present in the membranes of neurons, glia, and other
cells. In the following sections, we will first focus on the
biochemical pathways that lead to the formation of endo-
cannabinoids in neurons and then turn to the mechanism
by which these compounds are deactivated.
B. Biosynthetic Pathways
1. Anandamide biosynthesis
Anandamide formation via energy-independent con-
densation of arachidonic acid and ethanolamine was de-
scribed in brain tissue homogenates soon after the dis-
covery of anandamide and was attributed to an enzymatic
activity that was termed “anandamide synthase” (81, 83,
201). Subsequent work has demonstrated, however, that
this reaction is in fact catalyzed by fatty acid amide
hydrolase (FAAH), the primary enzyme of anandamide
hydrolysis, acting in reverse (203). Since FAAH requires
high concentrations of arachidonate and ethanolamine to
synthesize anandamide, higher than those normally found
in cells, this enzyme is unlikely to play a role in the
physiological formation of anandamide (for further dis-
cussion, see sect.
IIC6).
Another model for anandamide biosynthesis is illus-
trated schematically in Figure 2. According to this model,
anandamide may be produced via hydrolysis of the phos-
pholipid precursor N-arachidonoyl phosphatidylethanol-
amine (PE), catalyzed by a phospholipase D (PLD)-type
activity (89, 331, 332). The precursor consumed in this
reaction may be resynthesized by a separate enzyme ac-
tivity, N-acyltransferase (NAT), which may transfer an
arachidonate group from the sn-1 glycerol ester position
of phospholipids to the primary amino group of PE (89).
The validity of this model was initially questioned, be-
FIG.
1. Molecular structure of endogenous lipids
that activate brain cannabinoid receptors. These endo-
cannabinoid compounds share two common structural
motifs: a polyunsaturated fatty acid moiety (e.g., arachi-
donic acid) and a polar head group consisting of etha-
nolamine or glycerol. For details, see section
II, A and B4.
1020 FREUND, KATONA, AND PIOMELLI
Physiol Rev
• VOL 83 • JULY 2003 • www.prv.org
![FIG. 12. The cannabinoid receptor agonist WIN55,212–2 (WIN) inhibits glutamatergic but not GABAergic synaptic transmission in CB1 receptor knock-out mice. A: in CA1 pyramidal neurons of both CB1 / and CB1 / mice, the amplitudes of monosynaptically evoked excitatory postsynaptic currents (EPSCs) were reduced in a similar manner by bath application of 1 M WIN, the effects of which could be readily reversed by 1 M SR141716A (SR), a cannabinoid receptor antagonist. B: 1 M WIN decreased the amplitudes of evoked IPSCs in CB1 / mice but had no effect in CB1 / animals. [Modified from Hájos et al. (139); figure kindly prepared by Dr. Nobert Hájos.]](/figures/fig-12-the-cannabinoid-receptor-agonist-win55212-2-win-1sxa39tf.webp)
![FIG. 13. Vanilloid receptor ligands regulate glutamatergic, but not GABAergic, neurotransmission in the rat hippocampus. A: bath coapplication of the synthetic cannabinoid agonist WIN55,212–2 (1–5 M, WIN), with the vanilloid receptor antagonist capsazepine (10 M; CZ), prevented the suppression of monosynaptically evoked EPSC amplitude, but not the amplitude of evoked IPSCs in CA1 pyramidal cells. B: 10 M capsaicin, a vanilloid receptor agonist, suppressed the amplitude of evoked EPSCs, but not of evoked IPSCs. [Modified from Hájos and Freund (137); figure kindly prepared by Dr. Nobert Hájos.]](/figures/fig-13-vanilloid-receptor-ligands-regulate-glutamatergic-but-31hed8y0.webp)
![FIG. 6. Light micrographs of hippocampal sections (A and B from mouse, C from rat) immunostained for CB1 receptor using an antibody raised against a COOH-terminal intracellular epitope (A and B, showing the CA1 region), and another recognizing the NH2-terminal extracellular epitope (C, showing the CA3 region). The COOH-terminal antibody is more sensitive and provides somewhat stronger labeling, particularly in the dendritic layers, but the general staining pattern is similar. CB1positive axon terminals are seen in high density particularly in stratum pyramidale, where they surround the negative cell bodies of pyramidal cells in all subfields. Somatic staining appears only in interneurons mostly in strata radiatum and oriens, occasionally in stratum pyramidale (arrows). No immunostaining is visible in the CB1 receptor knock-out mouse (B). s.o., Stratum oriens; s.p., stratum pyramidale; s.r., stratum radiatum; CB1 / , wild-type mouse; CB1 / , CB1 receptor knock-out mouse. Scale bars, 50 m. [Modified from Katona et al. (188) and Hájos et al. (138).]](/figures/fig-6-light-micrographs-of-hippocampal-sections-a-and-b-from-3d12n23x.webp)
![FIG. 10. Subcellular localization of CB1 receptors in the human hippocampal CA1 region using an antibody raised against a COOH-terminal intracellular epitope (A and B) and another recognizing an NH2-terminal extracellular epitope (C–E). Silver-enhanced gold particles (small arrows) represent CB1-immunoreactive sites on the inner (A and B) and outer (C–E) surface of axon terminal membranes, corresponding to the subcellular localization of the respective epitopes. Only boutons forming symmetrical synapses (large arrows) were labeled, as in the rat, which is characteristic of GABAergic, but not of glutamatergic (asterisk) axons. Scale bars: A–E, 0.2 m. [From Katona et al. (187), copyright 2000 with permission from Elsevier Science.]](/figures/fig-10-subcellular-localization-of-cb1-receptors-in-the-zl5s6orb.webp)