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NeuroMolecular Medicine
Copyright © 2005 Humana Press Inc.
All rights of any nature whatsoever reserved.
ISSN1535-1084/05/07:37–50/$30.00
doi: 10.1385/NMM:7:1:37

ORIGINAL ARTICLE

Cannabinoid Receptors and Their Role in Neuroprotection

Mario van der Stelt and Vincenzo Di Marzo

Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle
Ricerche, Via Campi Flegrei 34, 80078, Pozzuoli (NA), Italy
Received December 20, 2004; Accepted February 10, 2005

Abstract
Two G protein-coupled receptors for marijuana’s psychoactive component, ∆9-tetrahydro-
cannabinol, have been cloned to date, the cannabinoid CB1 and CB2 receptors. These two pro-
teins, the endogenous lipids that activate them, also known as endocannabinoids, and the proteins
for the biosynthesis and inactivation of these ligands constitute the endocannabinoid system.
Evidence has accumulated over the last few years suggesting that endocannabinoid-based drugs
may potentially be useful to reduce the effects of neurodegeneration. In fact, exogenous and
endogenous cannabinoids were shown to exert neuroprotection in a variety of in vitro and in
vivo models of neuronal injury via different mechanisms, such as prevention of excitotoxicity
by cannabinoid CB1-mediated inhibition of glutamatergic transmission, reduction of calcium
influx, anti-oxidant activity, activation of the phosphatidylinositol 3-kinase/protein kinase B
pathway, induction of phosphorylation of extracellular regulated kinases and the expression of
transcription factors and neurotrophins, lowering of cerebrovasoconstriction and induction of
hypothermia. The release of endocannabinoids during neuronal injury may constitute a pro-
tective response. If this neuroprotective function of cannabinoid receptor activation can be trans-
ferred to the clinic, it might represent an interesting target to develop neuroprotective agents.
doi: 10.1385/NMM:7:1:37

Index Entries: Endocannabinoid; anandamide; 2-arachidonoylglycerol; cannabinoid-

receptor signaling; neuromodulatory role of endocannabinoids; glutamate; excitotoxicity.

Introduction: The Endocannabinoid psychoactive compound in marijuana (Matsuda

System
To date, two G protein-coupled receptors have
been cloned that interact with high affinity with
(−)-∆ 9 -tetrahydrocannabinol (THC), the main
et al., 1990; Munro et al., 1993). They are classified
as cannabinoid CB1 and CB2 receptors. In the brain,
the cannabinoid CB1 receptor is found in areas
controlling motor, cognitive, emotional and sen-
sory functions, i.e., the hippocampus, basal ganglia,

*Author to whom all correspondence and reprint requests should be addressed. E-mail: vdimarzo@icmib.na.cnr.it

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cerebellum, cortex, and olfactory bulb (Herkenham
et al., 1990; Tsou et al., 1998). Small nuclei with high
density of cannabinoid CB1 receptors are also found
in other areas, for example those controlling pain,
body temperature, sleep–wake cycles, and hormone
function, such as the brainstem, the hypothalamus,
and the pituitary gland (Herkenham et al., 1991). The
cannabinoid CB2 receptor seems to be confined to
cells of the immune system. Although its levels in the
brain are normally undetectable, increased expres-
sion, possibly owing to infiltration of immune cells
and activation of microglia, can be observed in cer-
tain neurodegenerative disorders (Benito et al., 2003).
Endocannabinoids are endogenous substances
capable of binding to and activating at least one of
the two cannabinoid receptors (Di Marzo, 1998). To
date, the family of endocannabinoids is expanding
and all constituents are small lipids. There are at least
five different derivatives of arachidonic acid that can
activate cannabinoid receptors. Anandamide and
2-arachidonoylglycerol (2-AG) are the two best stud-
ied ones. 2-Arachidonylglyceryl ether (noladin ether)
(Hanus et al., 2001), O-arachidonoylethanolamine
(virodhamine) (Porter et al., 2002) and N-arachi-
donoyldopamine (Huang et al., 2002) have only been
recently identified as endogenous ligands for
cannabinoid receptors and their classification as true
endocannabinoids awaits further biochemical and
pharmacological characterization. It is widely rec-
ognized that at least the two best studied endo-
cannabinoids, anandamide and 2-AG, are not stored
into vescicles like other mediators but, by analogy
with other eicosanoids, are produced “on demand”
(Di Marzo et al., 1994; 1998). This means that the
biosynthesis of the endocannabinoids is immediately
followed by their release. The biosynthetic routes
underlying the formation of endocannabinoids have
been extensively studied and the responsible pro-
teins have recently been cloned or purified to homo-
geneity (Okamoto et al., 2003; Bisogno et al., 2003),
although their regulation is still far from clear (Di
Marzo et al., 2000a, for a review). It is unknown which
type of neurons express the biosynthetic enzymes,
and therefore a classification of ‘cannabinergic’ neu-
rons is not yet possible. Endocannabinoids can be
inactivated via a two-step mechanism. First, they are
proposed to be transported into the cell via one or
more selective transporter mechanisms. Second, once
inside the cell, they can be subjected to several dif-
ferent metabolic pathways such as hydrolysis by fatty
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van der Stelt and Di Marzo
acid amide hydrolase (FAAH) and monoacylglyc-
erol (MAG)-lipase or oxygenation by lipoxygenases,
cyclooxygenases, and cytochrome P450s (Di Marzo
et al., 2000a).
The existence of non-cannabinoid CB1, non-CB2
G protein-coupled receptors for endocannabinoids,
and anandamide in particular, has been suggested
based on pharmacological and biochemical data
(Sagan et al., 1999; Di Marzo et al., 2000b; Breivogel
et al., 2001). It should be noted that also several non-
G protein-coupled molecular targets for anandamide
have been proposed (Howlett and Mukhopadhyay,
2000). They are all membrane cation channels and
are usually modulated specifically by anandamide
at low or submicromolar concentrations. In particular,
TASK-1 (TWIK-related acid-sensitive K+ channel)
K+ channels (Maingret et al., 2001), and T-type Ca2+
channels (Chemin et al., 2001) are blocked by anan-
damide, whereas vanilloid type 1 (VR1) receptors
(also known as TRPV1), the sites of action of the
pungent component of “hot” red peppers, capsaicin,
are activated by this endocannabinoid (Zygmunt
et al., 1999; Smart and Jerman, 2000), as well as by
N-arachidonoyldopamine.
Transmembrane Signaling Events
Coupled to Cannabinoid Receptors
and Subsequent Effects
of Endocannabinoids
on Neurotransmission and Cell Fate
Gi/o-Mediated Regulation of Intracellular
Cyclic AMP and Protein Kinase
(PKA)-Mediated Pathways
One of the best studied intracellular events trig-
gered by activation of cannabinoid receptors is the
inhibition of cyclic AMP (cAMP) production, which
has been observed in brain tissue, neuronal cells
expressing CB1 receptors, and in host cell lines over-
expressing recombinant CB1 receptors (Felder et al.,
1993; Vogel et al., 1993; reviewed in Howlett et al.,
1995; 2002). The inhibition of cAMP production by
CB2 receptor stimulation, instead, has been observed
in human lymphocytes, mouse spleen cells express-
ing CB2 receptors, and in host cell lines expressing
CB2 receptors (Felder et al., 1995; Slipetz et al., 1995;
Gonsiorek et al., 2000). The inhibition of adenylyl
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Cannabinoid Receptors and Their Role in Neuroprotection
cyclase is pertussis toxin-sensitive, indicating the
requirement for a Gi/o protein. Anandamide, (R)-
methanandamide (a metabolically stable anan-
damide analog), and 2-AG behave as full agonists to
inhibit adenylyl cyclase activity via CB1 in N18TG2
membranes (Childers et al., 1994; Pinto et al., 1994;
Howlett & Mukhopadhyay, 2000), whereas
2-AG was a full agonist, and anandamide and
(R)-methanandamide were only partial, low-efficacy
agonists for the inhibition of forskolin-stimulated
cAMP accumulation in cells expressing recombinant
CB2 receptors (Felder et al., 1995; Slipetz et al., 1995;
Bayewitch et al., 1996; Gonsiorek et al., 2000). Inhi-
bition of adenylyl cyclase by CB1 and CB2 receptors
appears to occur in cells that express isoforms 5 and
6, and 1, 3, and 8 (Rhee et al., 1998).
CB1, but not CB2, receptors can also couple to
stimulation of adenylyl cyclase via Gs proteins when
G i/o proteins are blocked by pertussis toxin or
sequestered by other G protein-coupled receptors
(Glass & Felder, 1997). The implications of this intra-
cellular event have not been fully investigated yet.
Regulation of Ion Channels
CB1 receptors couple to the inwardly rectifying
potassium currents (Kir) in AtT-20 pituitary tumor
cells in a pertussis toxin-sensitive manner, indicating
the participation of Gi/o proteins. In this functional
assay, anandamide was a full agonist compared with
the synthetic agonist, WIN55212-2; however, it was
only a partial agonist in Xenopus laevis oocytes coex-
pressing the CB1 receptor and GIRK1 and GIRK4
channels (Mackie et al., 1995; Henry & Chavkin,
1995; McAllister et al., 1999).
The CB1 receptor, again via a Gi/o protein, inhibits
N-type voltage-gated Ca2+ channels in differenti-
ated N18 neuroblastoma, NG108-15 neuroblastoma-
glioma hybrid cells, and neuronal expression
systems (Mackie et al., 1992; 1993; Caulfield et al.,
1992; Priller et al., 1995; Pan et al., 1996). Anan-
damide in this case behaves as a partial agonist com-
pared with WIN55212-2 or CP55940. However, the
endocannabinoid acted as a full agonists to inhibit
Q-type Ca 2+ currents in AtT-20 pituitary cells
expressing recombinant CB1 receptors. Also this
response was pertussis toxin-sensitive. Anan-
damide also inhibited P/Q-type Ca 2+ fluxes as
detected by fura-2 fluorescence in rat cortical and
cerebellar brain slices (Hampson et al., 1998b), in a
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39
way sensitive to the selective CB1 receptor antago-
nist, SR141716, and to pertussis toxin, pointing to
the mediation by CB1 receptors and Gi/o proteins.
Finally, although anandamide can directly interact
with L-type Ca 2+ channels, these proteins were
inhibited by the endocannabinoid as well as by
WIN55212-2, in a way blocked by pertussis toxin
and SR141716, in cat brain arterial smooth muscle
cells which express mRNA for the CB1 receptor
(Gebremedhin, 1999).
Singal Transduction Via MAPK
and Phosphatidyl Inositol-3-Kinase
Cannabinoid receptor agonists activate mitogen
activated protein kinases (MAPK) of the p38 type
in CHO cells expressing recombinant CB1 receptors
(Rueda et al., 2000) and in human vein endothelial
cells possessing endogenous CB 1 receptors (Liu
et al., 2000), and MAPK of the p42/p44 type in WI-
38 fibroblasts, U373MG astrocytic cells, C6 glioma
cells and primary astrocytes, which express endoge-
nous CB1 receptors, as well as in host cells over-
expressing recombinant CB1 receptors (Bouaboula
et al., 1995a; Wartmann et al., 1995; Sanchez et al.,
1998). These effects were mediated by CB1 recep-
tors and Gi/o proteins as shown by their attenua-
tion by SR141716 and by pertussis toxin. Also CB2
receptors are coupled to MAPK activation in cul-
tured human promyelocytic HL60 cells possessing
endogenous CB 2 receptors, and in CHO cells
expressing recombinant CB2 receptors (Bouaboula
et al., 1996).
Mechanisms for MAPK activation by cannabi-
noid receptors have been investigated and appear
to involve sequential recruitment of phos-
phatidylinositol-3-kinase (PI3K), tyrosine phos-
phorylation and activation of raf-1, and subsequent
activation of p42/p44 MAPK. In fact, CB1 receptor
signaling via MAPK in several cell types is sensi-
tive to wortmannin or LY294002, which are inhibitors
of PI3K (Bouaboula et al., 1995a; Wartmann et al.,
1995; Gomez del Pulgar et al., 2000). Furthermore,
∆ 9 -THC, HU210 and CP55940 produced an
SR141716-sensitive stimulation of protein kinase B
(PKB/Akt) (isoform IB) in the human astrocytoma
cell line U373MG and in CHO cells expressing
recombinant CB1 receptors (Gomez del Pulgar et al.,
2000; Galve-Roperh et al., 2002), a signal that usu-
ally follows PI3K-mediated phosphorylation of
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40
inositol phospholipids. Importantly, activation of
the PI3K/PKB/Akt pathway may lead to cytopro-
tective effects such as oligodendrocyte progenitor
survival or prevention of AMPA induced neuronal
death (Molina-Holgado et al., 2002; 2004). Finally,
∆ 9-THC promotes PI3K activation and tyrosine
phosphorylation of raf-1 and its translocation to the
membrane in rat cortical astrocytes (Sanchez et al.,
1998). However, cannabinoid agonists failed to acti-
vate PKB/Akt in HL60 cells, suggesting that a PI3K
mechanism may not be regulated by CB2 receptors,
at least in this model (Gomez del Pulgar et al., 2000).
Of particular interest for the possible neuropro-
tective effects of cannabinoid receptor stimulation
(see below), MAPK activation is usually linked to
expression of immediate early genes, as has been
demonstrated for krox-24 expression mediated by
CB1 receptors in U373MG human astrocytoma cells
(Bouaboula et al., 1995b). Cannabinoid receptor ago-
nists may use the Gi/o, PI3K and ras pathway to acti-
vate c-Jun N-terminal kinase (JNK1 and JNK2)
(Rueda et al., 2000). These transcription factors
modulate the gene expression pattern of cells,
thereby altering important cellular functions such
as cell survival or differentiation. Finally, Krox-24
expression is stimulated also via CB2 receptors and
following MAPK activation, in HL60 promyelocytes
(Bouaboula et al., 1996).
Stimulation of Phospholipase C (PLC)
and Mobilization of Intracellular Ca2+
The first clue that cannabinoid CB1, but not CB2,
receptors could also be coupled to PLC, despite ear-
lier evidence that could not demonstrate this possi-
bility (Felder et al., 1993; 1995), came from the finding
of a pertussis toxin-sensitive, transient increase in
intracellular free Ca2+ in undifferentiated N18TG2
neuroblastoma and NG108-15 neuroblastoma-
glioma hybrid cells following stimulation with 2-AG
(Sugiura et al., 1996; 1997a; 1997b). This response was
blocked by SR141716, and HU210, CP55940, ∆9-THC,
anandamide and (R)-methanandamide behaved as
partial agonists in this assay (Sugiura et al., 1999).
More importantly, the response could be attenuated
by a PLC inhibitor, suggesting activation of PLCb by
G protein α or βγ subunits, and subsequent IP3 release
and intracellular calcium mobilization. Later, CB1
cannabinoid receptors were shown to activate PLC
in cultured cerebellar granule neurons, where
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van der Stelt and Di Marzo
(R)-methanandamide, WIN55212-2 and HU210
enhanced the Ca2+ signal caused by NMDA recep-
tor stimulation or K+-depolarization in a way sensi-
tive to SR141716, pertussis toxin, and the PLC
inhibitor U-73122 (Netzeband et al., 1999). The source
of the released Ca2+ appears to be a caffeine-sensi-
tive and IP3 receptor-sensitive pool.
Stimulation of Ceramide Synthesis
A novel signal transduction pathway regulated
by cannabinoid receptors, especially of the CB2
type, is the synthesis of ceramide. This pathway
appears to be particularly relevant to the neuro-
protective and neurotoxic actions of endo-
cannabinoids (see below). Long-term treatment
with ceramide, which binds to and activates raf-1,
leads to the sustained activation of p42/p44 MAPK
and apoptosis in sensitive strains of rat C6 glioma
cells (Galve-Roperh et al., 2000). Cannabinoid ago-
nists (∆9-THC, CP55940, HU210, WIN55212-2) and
the endocannabinoids anandamide and 2-AG pro-
duce antiproliferative effects in C6 cells via both
CB1 and CB2 receptors (Galve-Roperh et al., 2000;
Jacobsson et al., 2001), although stimulation of the
CB 1 receptor was instead protective against
ceramide-induced apoptosis in U373MG astrocy-
toma cells (Galve-Roperh et al., 2002). The impor-
tance of CB2 receptors in the regulation of serine
palmitoyltransferase, the rate-limiting enzyme in
the de novo synthesis of ceramide, was demon-
strated using enzyme inhibitors and selective CB2
agonists and antagonists (Jacobsson et al., 2001;
Sanchez et al., 2001).
Neuromodulatory Role of Cannabinoid CB1
Receptor Stimulation
An important functional consequence of the acti-
vation of presynaptic cannabinoid CB1 receptors is
the inhibition of neurotransmitter release (reviewed
by Schlicker and Kathmann, 2001). In fact, endo-
cannabinoids are proposed to act as retrograde mes-
sengers, thereby playing a crucial role in synaptic
plasticity (Kreitzer and Regehr, 2001; Ohno-Shosaku
et al., 2001; Wilson and Nicoll, 2001). Depolariza-
tion of the post-synaptic membrane of neurons is
thought to release endocannabinoids, which then dif-
fuse to the presynaptic cannabinoid CB1 receptors and
inhibit their glutamatergic or γ-aminobutyric acid
(GABA)-ergic input. Activation of presynaptic
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Cannabinoid Receptors and Their Role in Neuroprotection
cannabinoid CB1 receptors has been shown to be
involved in long-term depression of neurons in the
dorsal striatum and in the nucleus accumbens
(Gerdeman et al., 2002; Robbe et al., 2002). Depend-
ing on the neuronal circuit, endocannabinoids may
thus activate or inhibit neurotransmission. This
property, together with the capability of influenc-
ing intracellular Ca 2+ and ceramide levels,
PI3k/PKB/Akt pathways, the activity of MAPK and
the expression of immediate early genes involved
in neuroprotection, underlies the capability of
cannabinoid CB 1 receptors to afford protection
against cell damage.
Cannabinoid Receptor Stimulation
and Neuroprotection
Stroke and traumatic brain injury (TBI) are lead-
ing causes of death and permanent disability and
a socioeconomic factor of major importance in
developed countries. Excitotoxicity takes center
stage in the pathologic sequelae after stroke and
TBI. In excitotoxicity, cell death is initiated by the
over-stimulation of excitatory amino acid receptors
by high concentrations of extracellular glutamate
(Dirnagl et al., 1999; Kermer et al., 1999; Nicotera
et al., 1999). This leads to cytotoxic levels of calcium
and subsequent activation of destructive pathways,
involving among others caspases, calpains and the
generation of reactive oxygen species (Dirnagl
et al., 1999; Kermer et al., 1999). Excitototoxicity can
be triggered by ATP depletion after stroke, by direct
mechanic damage, lysis of neighboring cells and
edema-induced ischemia after traumatic brain
injury. Experimental evidence suggests that excito-
toxic neuronal injury is an important determinant
of cell fate in the acute phase of neurodegenerative
disorders and may also sensitize cells for delayed
injury mechanisms such as inflammatory attacks
and apoptosis. The excitotoxicity hypothesis is also
used to explain the common biochemical basis
behind many chronic neurodegenerative disorders
such as amyotrophic lateral sclerosis, Parkinson’s,
Huntington’s and Alzheimer’s diseases (Dirnagl
et al., 1999; Kermer et al., 1999; Nicotera et al., 1999),
as well as glaucoma-induced degeneration of the
optic nerve (Lipton, 2003).
Several in vitro studies have reported neuroprotec-
tionwith classical and synthetic (endo)cannabinoids
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41
in models of excitotoxicity (reviewed in van der
Stelt et al., 2002). Depending on the model used,
cannabinoids were shown to protect neurons via
CB1-mediated inhibition of glutamate exocytosis
(Shen and Thayer, 1998a), CB1 receptor-mediated clos-
ing of voltage sensitive calcium channels (Hampson
et al., 1989; Abood et al., 2001), anti-oxidant activity
(Hampson et al., 1998a; Marsicano et al., 2002), sup-
pression of the formation of tumor necrosis factor
(TNF)-α (Gallily et al., 2000), and induction of inter-
leukin-1 receptor antagonist (Molina-Holgado et al.,
2003). Anandamide and 2-AG have been shown to
rescue cerebral neurons from in vitro hypoxia and
glucose deprivation via a CB1 and CB2 receptor-
independent pathway (Sinor et al., 2000). Interest-
ingly, the protective effects of the endocannabinoid
system are not restricted to neurons, but extend also
to astrocytes and oligodentrocytes by activation of
the PI3K/PKB pathway (Gomez Del Pulgar et al.,
2002; Molina-Holgado et al., 2002)
Neuroprotective effects of (endo)cannabinoids in
in vivo models of neurodegeneration have also been
observed (for reviews: Mechoulam et al., 2002; van
der Stelt et al., 2002). Panikashvili et al. (2001) demon-
strated that 2-AG prevented neuronal damage in a
mouse model of closed head injury by acting at least
in part via the CB1 receptor. 2-AG administered in a
dose range of 0.1–10 mg/kg could reduce brain
edema and at a dose of 5 mg/kg it significantly
improved clinical recovery, reduced infarct volume
and reduced hippocampal cell death compared to
controls after 7 days. The reduction in brain edema
by 2-AG was dose-dependently attenuated by the
selective CB1 receptor antagonist SR141716A. Vari-
ous mechanisms may contribute to the neuropro-
tective actions of 2-AG, such as reduction in
excitotoxicity, inhibition of production of pro-
inflammatory TNF-α and reactive oxygen species
and lowering of cerebral vasoconstriction. Appli-
cation of SR141716A (20 mg/kg) alone did not
increase the volume of edematous tissue, which
might indicate that endogenously released 2-AG
exerts its protective actions also through non-
CB1-mediated mechanisms (Panikashvili et al., 2001).
Similar results were obtained in series of longitu-
dinal in vivo Magnetic Resonance Imaging-studies
(Veldhuis et al., 2003) (van der Stelt et al., 2001a;
van der Stelt et al., 2001b). It was demonstrated
that three different CB 1 -receptor ligands, i.e.,
∆9-THC (1 mg/kg, i.p.), anandamide (10 mg/kg, i.p.)
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42
and its more stable analog arvanil (1 mg/kg, i.p.),
could significantly reduce the lesion volume in
neonatal rats intracerebrally injected with the neu-
rotoxin ouabain to induce excitotoxicity. Pretreat-
ment with the CB1-receptor antagonist SR141716
attenuated the neuroprotective effects of the CB1
receptor ligands as measured after seven days. A
CB1 receptor-mediated reduction in calcium influx
andglutamate release were thought to be responsi-
ble for the neuroprotection in the hippocampus,
striatum and cortex. Interestingly, anandamide-
induced reduction in cellular swelling in the initial
phase was independent of cannabinoid CB1 recep-
tor. TRPV1 receptors and lipoxygenase-metabolites
of anandamide were shown not be involved in this
response (Veldhuis et al., 2003).
In glaucoma, the increased release of glutamate
is the major cause of retinal ganglion cell death. THC
and cannabidiol could attenuate retinal cell loss
induced by intravitreal injection of N-methyl-D-
aspartate (NMDA) in rats. The neuroprotective
effects were partly dependent on the cannabinoid
CB1 receptor and were suggested to be the result of
attenuation of peroxynitrite levels (El-Remessy
et al., 2003)
The protective effects of modulation of the endo-
cannabinoid system after stroke have been reported
by numerous studies. CB1 receptor expression was
enhanced in the arterial boundary zone of the cor-
tical mantle after a 20-min occlusion of the middle
cerebral artery in rats (Jin et al., 2000). Chronic ∆9-
THC administration reduces the impact of an
ischemic insult evoked by lowering blood pressure
and 12-min bilateral carotid artery occlusion (Louw
et al., 2000). The mixed CB 1 /CB 2 cannabinoid
receptor agonist WIN55,212-2 afforded protection
to hippocampal and cortical neurons in CB1 receptor-
dependent manner in rats with a permanent middle
cerebral artery occlusion (MCAO) or global ischemia
(Nagayama et al., 1999). The ultrapotent CB1-receptor
agonist HU-210 conferred robust protection against
ischemic damage in the permanent MCAO model.
This protection was mediated at least in part by
binding to CB1 receptors, and was associated with
the indirect protective effects of hypothermia (Leker
et al., 2003). WIN55,212-2 provided also a neuro-
protective effect, in part via the cannabinoid CB1
receptor, to neonatal rats subjected to severe
asphyxia (Martinez-Orgado et al., 2003). In gerbils
subjected to transient global ischemia, pretreatment
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van der Stelt and Di Marzo
with the CB1 receptor agonist CP55, 940 reduced
ischemia-induced hyperlocomotion and improved
electroencephalographic (EEG) spectral power after
24 h, which lasted for at least 7 d (Braida et al., 2000).
Interestingly, in this same model, the non-psy-
chotropic plant cannabinoid, cannabidiol, also
afforded significant protection, perhaps via TRPV1
(Braida et al., 2003), which this compound can acti-
vate in vitro (Bisogno et al., 2001). Recently, it was
demonstrated that a novel mixed CB1/CB2 recep-
tor agonist, BAY38-7271, also exhibited neuropro-
tective properties. It could reduce infarct volume in
rats when applied with a 3-h delay after the induc-
tion of a subdural hematoma and in rats with a per-
manent MCAO (Mauler et al., 2002).
The Endocannabinoid System
As an Endogenous Protection System
Because the levels of 2-arachidonoylglycerol,
anandamide, and its precursor N-arachi-
donoylphosphatidyl-ethanolamine, which are nor-
mally found in low concentrations in the brain,
increase dramatically upon neuronal injury
(Hansen et al., 1997; Sugiura et al., 2000; Hansen
et al., 2001a,b; Panikashvili et al., 2001; Franklin
et al., 2003; Berger et al., 2004; Muthian et al., 2004),
it was suggested that this represents a protective
response. Indeed, in CB 1 cannabinoid receptor
knock-out mice, mortality from permanent focal
cerebral ischemia was increased, infarct size, and
neurological deficits after transient focal cerebral
ischemia were more severe, cerebral blood flow in
the ischemic penumbra during reperfusion was
reduced, and NMDA neurotoxicity was increased
compared with wild-type littermates (Parmentier-
Batteur et al., 2002). Marsicano and colleagues
showed that, in conditional mutant mice that lack
expression of the cannabinoid CB1 receptor in prin-
cipal forebrain neurons but not in adjacent
inhibitory interneurons, the excitotoxin kainic acid
induced excessive seizures in vivo (Marsicano et al.,
2003). The threshold to kainic acid-induced neu-
ronal excitation in vitro was severely reduced in
hippocampal pyramidal neurons of mutants. Kainic
acid administration rapidly raised hippocampal
levels of anandamide and induced protective mech-
anisms, such as the phosphorylation of extracellu-
lar regulated kinases and expression of transcription
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Cannabinoid Receptors and Their Role in Neuroprotection
factors and neurotrophins, in wild-type principal
hippocampal neurons. These protective mecha-
nisms could not be triggered in mutant mice. Brain-
derived neurotrophic factor was shown to be a key
mediator in conferring protection, possibly via
phosphoinositol-3-kinase B/AKT pathways and
the pro-survival protein bcl-2 (Khaspekov et al.,
2004). Altogether, these data suggest that the endo-
cannabinoid system provides an on-demand pro-
tection against acute excitotoxicity and neuronal
damage.
Neurotoxicity by (Endo)Cannabinoids?
It should be noted that in some models
(endo)cannabinoids do not provide protection to
neurons or even produce toxic effects. Anandamide
was shown to be ineffective at protecting neurons
against prolonged exposure to toxic levels of gluta-
mate (Skaper et al., 1996; Andersson et al., 2000).
Cannabinoid CB1 receptor antagonist SR14716Apre-
treatment in neonatal rats prior to an unilateral
intrastriatal microinjection of NMDA evoked a
robust neuroprotective response by reducing the
ipsilateral infarct area and the number of degener-
ating cortical neurons (Hansen et al., 2002). This effect
was abolished by co-injection of WIN55,212-2, indi-
cating that blockade of cannabinoid receptor activ-
ity inhibited caudal propagation of the excitotoxic
response. In another study, CB1 receptor antagonists,
but not WIN55,212-2, were also protective when the
middle cerebral artery in rats was occluded (Berger
et al., 2004; Muthian et al., 2004). Furthermore, genet-
ically engineered mice lacking FAAH (the enzyme
responsible for anandamide degradation), where
brain anandamide levels are significantly and per-
manently elevated, appear to be more susceptible
towards kainate-induced seizures (Clement et al.,
2003). In addition, exogenous anandamide dramat-
ically augmented the severity of chemically induced
seizures in FAAH−/− mice but not in wild-type mice.
Several reasons may explain this apparent
dichotomy. (1) The neurodegenerative insult may
not always lead to an up-regulation of endo-
cannabinoid biosynthesis, the occurrence of which
seems to be dependent on the species and on the
type of injury (Hansen et al., 2001b; Panikashvili
et al., 2001; van der Stelt et al., 2001b; Berger et al.,
2004; Muthian et al., 2004). (2) Neuroprotective
NeuroMolecular Medicine
43
actions of endocannabinoids may result from actions
via molecular targets distinct from CB1 receptors
(see above). For example, novel yet-unidentified
cannabinoid receptors may also inhibit gluta-
matergic transmission (Breivogel et al., 2001; Hajos
et al., 2001; Franklin et al., 2003). Furthermore, neu-
roprotective actions of the CB 1 antagonist
SR141716A may also result from its interaction with
non-CB 1 cannabinoid receptor sites (Berdyshev
et al., 2001; Breivogel et al., 2001; Hajos and Freund,
2002; Berger et al., 2004). (3) Presynaptic CB1 recep-
tors are ineffective against an exogenously induced
glutamate receptor stimulation (Shen and Thayer,
1998b; van der Stelt et al., 2001b). (4) The ability of
endocannabinoids to influence downstream effects
of increased calcium concentrations is also depen-
dent on the cell type, strength, duration, and stim-
ulus type (Hampson et al., 1998b; Netzeband et al.,
1999). (5) The inflicted damage in the in vivo models
of acute neuronal injury might be too severe, which
leads to a loss of CB1 receptor mRNA expression
and ligand binding capacity (Hansen et al., 2001b).
(6) CB1receptor-induced altered GABAergic trans-
mission may be involved in the degenerative
process. (7) By acting on non-CB1/CB2 receptors
(e.g., TRPV1 channels), endocannabinoids may
exert a neurotoxic effect that partially masks the
tonic neuroprotective effect mediated by cannabi-
noid receptors. For example, anandamide has been
shown to induce cell death by activation of vanil-
loid receptors (Maccarrone et al., 2000; Grant
et al., 2002; Cernak et al., 2004). (8) Activation of
cannabinoid receptors may have a different impact
on the biochemical pathways underlying apopto-
sis and necrosis. In fact, cannabinoid receptor acti-
vation has been shown to induce apoptosis (Chan
et al., 1998). Anti-apoptotic strategies have been
identified as potentially beneficial in limiting
ischemic neuronal injury, which might imply that
CB1 receptor antagonism may also be beneficial
in neurodegenerative insults in which apoptosis
is the major cause of death. However, it is unclear
whether endocannabinoids induce apoptosis
during acute neuronal injury. (9) Finally, it has
been suggested that CB 1 receptors in the cere-
brovascular system may worsen the damage pro-
duced in middle cerebral artery occlusion models,
possibly because of disruption of metabolic
autoregulation or neuro-vascular coupling
(Muthian et al., 2004).
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44 van der Stelt and Di Marzo

Fig. 1. Cannabinoid CB1 receptor-induced intracellular events leading to both short-term and long-term neu-
roprotective effects. Whereas the short-term effects are mostly owing to modulation by pre-synaptic CB1 recep-
tors of ion channel activity, either directly or through inhibition of cAMP formation, the long-term effects are caused
by the Gi/o-mediated modulation of various intracellular messengers, followed by changes in the activities of sev-
eral protein kinases. For abbreviations see text except for NO, nitric oxide.

Conclusions G protein-mediated intracellular signalling events,

and inducing both short-term and long-term effects
The studies reported in this review indicate that (Fig. 1), might be useful to improve the outcome
agonists of CB1 receptors, by triggering a series of after acute neuronal damage. Nevertheless, many

NeuroMolecular Medicine Volume 7, 2005

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Cannabinoid Receptors and Their Role in Neuroprotection
questions have to be answered before a CB1 ago-
nists can be used as a neuroprotective agent, such
as: (a) to what extent can the findings in animal
models be translated to the human condition? (b)
to what extent do cannabinoids improve or restore
functional and neurological outcome after neuronal
damage? (c) What is the therapeutic time window?
(d) Do cannabinoid-induced vasorelaxation, psy-
chotropic side effects, and pro-apoptotic activity
pose a problem? At the moment it is not clear
whether CB2 agonists, which do not cause psy-
chotropic effects, are also able to reduce neuronal
damage in vivo, but other anti-inflammatory drugs
have been proven to reduce neuronal injury and
improve functional recovery. Because of the pres-
ence of CB2 receptors in reactive microglia and
astrocytes (Walters and Stella, 2004), it is likely that
such compounds will afford protection during
those pathological conditions where the causes of
neuronal damage are primarily the result of inflam-
matory conditions such as gliosis and reactive
microglial cells.
Importantly, cell-type specificity and dynamic
regulation of the endocannabinoid system are cru-
cial to its neuroprotective actions. Lack of spatial
and temporal activation of cannabinoid CB1 recep-
tors may disrupt its ability to serve as an endoge-
nous neuroprotection system. Therefore,
compounds that strengthens a putative tonic ben-
eficial action of endocannabinoids, by inhibiting
their degradation or uptake into the cell in the
“right” place and at the “right” time (Lutz, 2004),
might provide valid therapeutic avenues for novel
efficacious neuroprotective drugs.
References
Abood M. E., Rizvi G., Sallapudi N., and McAllister
S. D. (2001) Activation of the CB(1) cannabinoid
receptor protects cultured mouse spinal neurons
against excitotoxicity. Neurosci. Lett. 309, 197–201.
Andersson M., Jacobsson S. O., Jonsson K. O., Tiger
G., and Fowler C. J. (2000) Neurotoxicity of glu-
tamate in chick telencephalon neurons: reduction
of toxicity by preincubation with carbachol, but
not by the endogenous fatty acid amides anan-
damide and palmitoylethanolamide. Arch. Toxi-
col. 74, 161–164.
Bayewitch M., Rhee M. H., Avidor-Reiss T., Breuer A.,
Mechoulam R., and Vogel Z. (1996) (−)-Delta9-
NeuroMolecular Medicine
45
tetrahydrocannabinol antagonizes the peripheral
cannabinoid receptor-mediated inhibition of
adenylyl cyclase. J. Biol. Chem. 271, 9902–9905.
Benito C., Nunez E., Tolon R. M., et al. (2003) Cannabi-
noid CB2 receptors and fatty acid amide hydro-
lase are selectively overexpressed in neuritic
plaque-associated glia in Alzheimer ’s disease
brains. J. Neurosci. 23, 11,136–11,141.
Berdyshev E. V., Schmid P. C., Krebsbach R. J., et al. (2001)
Cannabinoid-receptor-independent cell signalling
by N-acylethanolamines. Biochem. J. 360, 67–75.
Berger C., Schmid P., Schabitz W., Wolf M., Schwab S.,
and Schmid H. (2004) Massive accumlation of N-
acylethanolamines after stroke. Cell signalling in
acute cerebral ischemia? J. Neurochem. 88, 1159–1167.
Bisogno T., Hanus L., De Petrocellis L., et al. (2001)
Molecular targets for cannabidiol and its syn-
thetic analogues: effect on vanilloid VR1 recep-
tors and on the cellular uptake and enzymatic
hydrolysis of anandamide. Br. J. Pharmacol. 134,
845–852.
Bisogno T., Howell F., Williams G., et al. (2003) Cloning
of the first sn1-DAG lipases points to the spatial
and temporal regulation of endocannabinoid sig-
naling in the brain. J. Cell. Biol. 163, 463–468.
Bouaboula M., Bourrie B., Rinaldi-Carmona M., Shire
D., Le Fur G., and Casellas P. (1995b) Stimulation
of cannabinoid receptor CB1 induces krox-24
expression in human astrocytoma cells. J. Biol.
Chem. 270, 13,973–13,980.
Bouaboula M., Poinot-Chazel C., Bourrie B., et al.
(1995a) Activation of mitogen-activated protein
kinases by stimulation of the central cannabinoid
receptor CB1. Biochem. J. 312, 637–641.
Bouaboula M., Poinot-Chazel C., Marchand J., et al. (1996)
Signaling pathway associated with stimulation of CB2
peripheral cannabinoid receptor. Involvement of both
mitogen-activated protein kinase and induction of
Krox-24 expression. Eur. J. Biochem. 237, 704–711.
Braida D., Pozzi M., and Sala M. (2000) CP 55,940 pro-
tects against ischemia-induced electroencephalo-
graphic flattening and hyperlocomotion in
Mongolian gerbils. Neurosci. Lett. 296, 69–72.
Braida D., Pegorini S., Arcidiacono M., Consalez G.,
Croci L., and Sala M. (2003) Post-ischemic treatment
with cannabidiol prevents electroencephalographic
flattening, hyperlocomotion and neuronal injury in
gerbils. Neurosci. Lett. 346, 61–64.
Breivogel C. S., Griffin G., Di Marzo V., and Martin
B. R. (2001) Evidence for a new G protein-coupled
cannabinoid receptor in mouse brain. Mol. Phar-
macol. 60, 155–163.
Volume 7, 2005
02_Di Marzo.qxd 6/15/05 11:23 AM Page 46
46
Caulfield M. P. and Brown D. A. (1992) Cannabinoid
receptor agonists inhibit Ca current in NG108-15
neuroblastoma cells via a pertussis toxin-sensitive
mechanism. Br. J. Pharmacol. 106, 231–232.
Cernak I., Vink R., Natale J., et al. (2004) The dark side
of endocannabinoids: a neurotoxic role for anan-
damide. J. Cereb. Blood Flow Metab. 24, 564–578.
Chan G. C., Hinds T. R., Impey S., and Storm D. R.
(1998) Hippocampal neurotoxicity of Delta9-
tetrahydrocannabinol. J. Neurosci. 18, 5322–5332.
Chemin J., Monteil A., Perez-Reyes E., Nargeot J., and
Lory P. (2001) Direct inhibition of T-type calcium
channels by the endogenous cannabinoid anan-
damide. Embo. J. 20, 7033–7040.
Childers S. R., Sexton T., and Roy M. B. (1994) Effects
of anandamide on cannabinoid receptors in rat
brain membranes. Biochem. Pharmacol. 47, 711–715.
Clement A., Hawkins E., Lichtman A., and Cravatt B.
(2003) Increased seizure susceptibility and pro-
convulsant activity of anandamide in mice lacking
fatty acid amide hydrolase. J. Neurosci. 23,
3916–3923.
Deadwyler S. A., Hampson R. E., Mu J., Whyte A., and
Childers S. (1995) Cannabinoids modulate volt-
age sensitive potassium A-current in hippocam-
pal neurons via a cAMP-dependent process.
J. Pharmacol. Exp. Ther. 273, 734–743.
Deadwyler S. A., Hampson R. E., Bennett B. A., et al.
(1993) Cannabinoids modulate potassium current
in cultured hippocampal neurons. Receptors Chan-
nels 1, 121–134.
Di Marzo V. (1998) “Endocannabinoids” and other
fatty acid derivatives with cannabimimetic prop-
erties: biochemistry and possible physiopatho-
logical relevance. Biochim. Biophys. Acta. 1392,
153–175.
Di Marzo V., Bisogno T., and De Petrocellis L. (2000a)
Endocannabinoids: new targets for drug devel-
opment. Curr. Pharm. Des. 6, 1361–1380.
Di Marzo V., Melck D., Bisogno T., and De Petrocellis L.
(1998) Endocannabinoids: endogenous cannabinoid
receptor ligands with neuromodulatory action.
Trends Neurosci. 21, 521–528.
Di Marzo V., Fontana A., Cadas H., Schinelli S., Cimino
G., Schwartz J. C., and Piomelli D. (1994) Forma-
tion and inactivation of endogenous cannabinoid
anandamide in central neurons. Nature 372,
686–691.
Di Marzo V., Breivogel C. S., Tao Q., et al. (2000b) Levels,
metabolism, and pharmacological activity of anan-
damide in CB(1) cannabinoid receptor knockout
NeuroMolecular Medicine
van der Stelt and Di Marzo
mice: evidence for non-CB(1), non-CB(2) receptor-
mediated actions of anandamide in mouse brain.
J. Neurochem. 75, 2434–2444.
Dirnagl U., Iadecola C., and Moskowitz M. A. (1999)
Pathobiology of ischaemic stroke: an integrated
view. Trends Neurosci. 22, 391–397.
El-Remessy A., Khalil I., Matragoon S., et al. (2003)
Neuroprotective effect of (−)Delta9-tetrahydro-
cannabinol and cannabidiol in N-methyl-D-aspar-
tate-induced retinal neurotoxicity: involvement of
peroxynitrite. Am. J. Pathol. 16, 1997–2008.
Felder C. C., Briley E. M., Axelrod J., Simpson J. T.,
Mackie K., and Devane W. A. (1993) Anandamide,
an endogenous cannabimimetic eicosanoid, binds
to the cloned human cannabinoid receptor and
stimulates receptor-mediated signal transduction.
Proc. Natl. Acad. Sci. USA 90, 7656–7660.
Felder C. C., Joyce K. E., Briley E. M., et al. (1995) Com-
parison of the pharmacology and signal trans-
duction of the human cannabinoid CB1 and CB2
receptors Mol. Pharmacol. 48, 443–450.
Franklin A., Parmentier-Batteur S., Walter M., Green-
berg D., and Stella N. (2003) Palmitoylethanolamide
increases after focal cerbral ischemia and potenti-
ates microglial cell motility. J. Neurosci. 23,
7767–7775.
Gallily R., Breuer A., and Mechoulam R. (2000) 2-Arachi-
donylglycerol, an endogenous cannabinoid, inhibits
tumor necrosis factor-alpha production in murine
macrophages, and in mice. Eur. J. Pharmacol. 406,
R5–7.
Galve-Roperh I., Rueda D., Gomez D. P., Velasco G.,
and Guzman M. (2002) Mechanism of extracellu-
lar signal-regulated kinase activation by the CB(1)
cannabinoid receptor. Mol. Pharmacol. 62,
1385–1392.
Galve-Roperh I., Sanchez C., Cortes M. L., del Pulgar
T. G., Izquierdo M., and Guzman M. (2000) Anti-
tumoral action of cannabinoids: involvement of
sustained ceramide accumulation and extracellu-
lar signal-regulated kinase activation. Nat. Med. 6,
313–319.
Gebremedhin D., Lange A. R., Campbell W. B., Hillard
C. J., and Harder D. R. (1999) Cannabinoid CB1
receptor of cat cerebral arterial muscle functions to
inhibit L-type Ca2+ channel current Am. J. Physiol.
276, H2085–H2093.
Gerdeman G. L., Ronesi J., and Lovinger D. M. (2002)
Postsynaptic endocannabinoid release is critical to
long-term depression in the striatum. Nat. Neurosci.
25, 25.
Volume 7, 2005
02_Di Marzo.qxd 6/15/05 11:23 AM Page 47
Cannabinoid Receptors and Their Role in Neuroprotection
Glass M. and Felder C. C. (1997) Concurrent stimu-
lation of cannabinoid CB1 and dopamine D2
receptors augments cAMP accumulation in stri-
atal neurons: evidence for a Gs linkage to the CB1
receptor. J. Neurosci. 17, 5327–5333.
Gomez Del Pulgar T., De Ceballos M. L., Guzman
M., and Velasco G. (2002) Cannabinoids Protect
Astrocytes from Ceramide-induced Apoptosis
through the Phosphatidylinositol 3-Kinase/Pro-
tein Kinase B Pathway. J. Biol. Chem. 277,
36,527–36,533.
Gomez del Pulgar, Velasco G., and Guzman M. (2000)
The CB1 cannabinoid receptor is coupled to the
activation of protein kinase B/Akt. Biochem. J. 347,
369–373.
Gonsiorek W., Lunn C., Fan X., Narula S., Lundell D.,
and Hipkin R. W. (2000) Endocannabinoid
2-arachidonyl glycerol is a full agonist through
human type 2 cannabinoid receptor: antagonism
by anandamide. Mol. Pharmacol. 57(5), 1045–1050.
Grant E. R., Dubin A. E., Zhang S. P., Zivin R. A., and
Zhong Z. (2002) Simultaneous intracellular calcium
and sodium flux imaging in human vanilloid recep-
tor 1 (VR1)-transfected human embryonic kidney
cells: a method to resolve ionic dependence of VR1-
mediated cell death. J. Pharmacol. Exp. Ther. 300,
9–17.
Guzman M., Galve-Roperh I., and Sanchez C. (2001)
Ceramide: a new second messenger of cannabi-
noid action. Trends Pharmacol. Sci. 22, 19–22.
Hajos N. and Freund T. (2002) Pharmacological sepa-
ration of cannabinoid sensitive receptors on hip-
pocampal excitatory and inhibitory fibers.
Neuropharmacology 43, 503.
Hajos N., Ledent C., and Freund T. F. (2001) Novel
cannabinoid-sensitive receptor mediates inhibi-
tion of glutamatergic synaptic transmission in the
hippocampus. Neuroscience 106, 1–4.
Hampson A. J., Grimaldi M., Axelrod J., and Wink D.
(1998a) Cannabidiol and (−)Delta9-tetrahydro-
cannabinol are neuroprotective antioxidants.
Proc. Natl. Acad. Sci. USA 95, 8268–8273.
Hampson A. J., Bornheim L. M., Scanziani M., et al.
(1998b) Dual effects of anandamide on NMDA
receptor-mediated responses and neurotransmis-
sion. J. Neurochem. 70, 671–676.
Hampson R. E., Foster T C., and Deadwyler S. A.
(1989) Effects of delta-9-tetrahydrocannabinol on
sensory evoked hippocampal activity in the rat:
principal components analysis and sequential
dependency. J. Pharmacol. Exp. Ther. 251, 870–877.
NeuroMolecular Medicine
47
Hansen H. H., Ikonomidou C., Bittigau P., Hansen S. H.,
and Hansen H. S. (2001a) Accumulation of the anan-
damide precursor and other N-acylethanolamine
phospholipids in infant rat models of in vivo necrotic
and apoptotic neuronal death. J. Neurochem. 76,
39–46.
Hansen H. H., Azcoitia I., Pons S., et al. (2002) Block-
ade of cannabinoid CB(1) receptor function protects
against in vivo disseminating brain damage fol-
lowing NMDA-induced excitotoxicity. J. Neurochem.
82, 154–158.
Hansen H. H., Schmid P. C., Bittigau P., et al. (2001b)
Anandamide, but not 2-arachidonoylglycerol, accu-
mulates during in vivo neurodegeneration. J. Neu-
rochem. 78, 1415–1427.
Hansen H. S., Lauritzen L., Strand A. M., Vinggaard
A. M., Frandsen A., and Schousboe A. (1997)
Characterization of glutamate-induced formation
of N-acylphosphatidylethanolamine and
N-acylethanolamine in cultured neocortical neu-
rons. J. Neurochem. 69, 753–761.
Hanus L., Abu-Lafi S., Fride E., et al. (2001) 2-arachi-
donyl glyceryl ether, an endogenous agonist of the
cannabinoid CB1 receptor. Proc. Natl. Acad. Sci.
USA 98, 3662–3665.
Henry D. J. and Chavkin C. (1995) Activation of
inwardly rectifying potassium channels (GIRK1)
by co-expressed rat brain cannabinoid receptors
in Xenopus oocytes. Neurosci. Lett. 186, 91–94.
Herkenham M., Lynn A. B., Johnson M. R., Melvin L.
S., de Costa B. R., and Rice K. C. (1991) Character-
ization and localization of cannabinoid receptors
in rat brain: a quantitative in vitro autoradiographic
study. J. Neurosci. 11, 563–583.
Herkenham M., Lynn A. B., Little M. D., et al. (1990)
Cannabinoid receptor localization in brain. Proc.
Natl. Acad. Sci. USA 87, 1932–1936.
Howlett A. C., and Mukhopadhyay S. (2000) Cellular
signal transduction by anandamide and 2-arachi-
donoylglycerol. Chem. Phys. Lipids 108, 53–70.
Howlett A. C. Barth F., Bonner T. I., et al. (2002) Inter-
national Union of Pharmacology. XXVII. Classifi-
cation of Cannabinoid Receptors Pharmacol. Rev. 54,
161–202.
Howlett A. C. (1995) Pharmacology of cannabinoid
receptors Annu. Rev. Pharmacol. Toxicol. 35, 607–634.
Huang S. M., Bisogno T., Trevisani M., et al. (2002)
An endogenous capsaicin-like substance with
high potency at recombinant and native vanil-
loid VR1 receptors. Proc. Natl. Acad. Sci. USA 99,
8400–8405.
Volume 7, 2005
02_Di Marzo.qxd 6/15/05 11:23 AM Page 48
48
Jacobsson S. O., Wallin T., and Fowler C. J. (2001) Inhi-
bition of rat C6 glioma cell proliferation by endoge-
nous and synthetic cannabinoids. Relative
involvement of cannabinoid and vanilloid recep-
tors. J. Pharmacol. Exp. Ther. 299, 951–959.
Jin K. L., Mao X. O., Goldsmith P. C., and Greenberg
D. A. (2000) CB1 cannabinoid receptor induction
in experimental stroke. Ann Neurol 48, 257–261.
Kermer P., Klocker N., and Bahr M. (1999) Neuronal
death after brain injury. Models, mechanisms, and
therapeutic strategies in vivo. Cell Tissue Res. 298,
383–395.
Khaspekov L., Brenz Verca M., Frumkina L. E., Her-
mann H., Marsicano G., and Lutz B. (2004) Involve-
ment of brain-derived neurotrophic factor in
cannabinoid receptor-dependent protection against
excitotoxicity. Eur. J. Neurosci. 19, 1691–1698.
Kreitzer A. C. and Regehr W. G. (2001) Retrograde inhi-
bition of presynaptic calcium influx by endoge-
nous cannabinoids at excitatory synapses onto
Purkinje cells. Neuron. 29, 717–727.
Leker R., Gai N., Mechoulam R., and Ovadia H. (2003)
Drug-induced hypothermia reduces ischemic
damage: effects of the cannabinoid HU-210. Stroke
34, 2000–2006.
Lipton S. (2003) Possible role for memantine in pro-
tecting retinal ganglion cells from glaucomatous
damage. Surv. Ophthalmol. Suppl. 1, S38–46.
Liu J., Gao B., Mirshahi F., et al. (2000) Functional CB1
cannabinoid receptors in human vascular endothe-
lial cells. Biochem. J. 346, 835–840.
Louw D. F., Yang F. W., and Sutherland G. R. (2000)
The effect of delta-9-tetrahydrocannabinol on
forebrain ischemia in rat. Brain Res. 857, 183–187.
Lutz B. (2004) On-demand activation of the endo-
cannabinoid system in the control of neuronal
excitability and epileptiform seizures. Biochem.
Pharmacol. 68:1691–1698.
Maccarrone M., Lorenzon T., Bari M., Melino G., and
Finazzi-Agro A. (2000) Anandamide induces apop-
tosis in human cells via vanilloid receptors. Evi-
dence for a protective role of cannabinoid
receptors. J. Biol. Chem. 275, 31,938–31,945.
Mackie K., Devane W. A., and Hille B. (1993) Anan-
damide, an endogenous cannabinoid, inhibits cal-
cium currents as a partial agonist in N18
neuroblastoma cells. Mol. Pharmacol. 44, 498–503.
Mackie K. and Hille B. (1992) Cannabinoids inhibit N-
type calcium channels in neuroblastoma-glioma
cells. Proc. Natl. Acad. Sci. USA 89, 3825–3829.
Mackie K., Lai Y., Westenbroek R., and Mitchell R. (1995)
Cannabinoids activate an inwardly rectifying
NeuroMolecular Medicine
van der Stelt and Di Marzo
potassium conductance and inhibit Q-type cal-
cium currents in AtT20 cells transfected with rat
brain cannabinoid receptor. J. Neurosci. 15(10),
6552–6561.
Maingret F., Patel A. J., Lazdunski M., and Honore E.
(2001) The endocannabinoid anandamide is a
direct and selective blocker of the background K(+)
channel TASK-1. Embo. J. 20, 47–54.
Marsicano G., Moosmann B., Hermann H., Lutz B.,
and Behl C. (2002) Neuroprotective properties of
cannabinoids against oxidative stress: role of the
cannabinoid receptor CB1. J. Neurochem. 80,
448–456.
Marsicano G., Goodenough S., Monory K., et al. (2003)
CB1 cannabinoid receptors and on-demand
defense against excitotoxicity. Science 302, 84–88.
Martinez-Orgado J., Fernandez-Frutos B., Gonzalez R.,
et al. (2003) Neuroprotection by the cannabinoid
agonist WIN-55212 in an in vivo newborn rat model
of acute severe asphyxia. Brain Res. Mol. 114,
132–139.
Matsuda L. A., Lolait S. J., Brownstein M. J., Young A.
C., and Bonner T. I. (1990) Structure of a cannabi-
noid receptor and functional expression of the
cloned cDNA. Nature 346, 561–564.
Mauler F., Mittendorf J., Horvath E., and De Vry J.
(2002) Characterization of the diarylether sul-
fonylester (−)-(R)-3-(2- hydroxymethylindanyl-4-
oxy)phenyl-4,4,4-trifluoro-1-sulfonate (BAY 38-
7271) as a potent cannabinoid receptor agonist
with neuroprotective properties. J. Pharmacol. Exp.
Ther. 302, 359–368.
McAllister S. D., Griffin G., Satin L. S., and Abood M. E.
(1999) Cannabinoid receptors can activate and
inhibit G protein-coupled inwardly rectifying
potassium channels in a xenopus oocyte expres-
sion system J. Pharmacol. Exp. Ther. 291, 618–626.
Mechoulam R., Pani kashvili D., and Shohami E. (2002)
Cannabinoids and brain injury: therapeutic impli-
cations. Trends Mol. Med. 8, 58–61.
Molina-Holgado E., Vela J., Arevalo-Martin A., et al.
(2002) Cannabinoids promote oligodendrocyte
progenitor survival: involvement of cannabinoid
receptors and phosphatidylinositol-3 kinase/
Akt signaling. J. Neurosci. 22, 9742–9753.
Molina-Holgado F., Pinteaux E., Moore J., et al. (2003)
Endogenous interleukin-1 receptor antagonist
mediates anti-inflammatory and neuroprotective
actions of cannabinoids in neurons and glia.
J. Neurosci. 23, 6470–6474.
Molina-Holgado F., Pinteaux E., Heenan L., Moore J. D.,
Rothwell N. J., and Gibson R. M. Neuroprotective
Volume 7, 2005
02_Di Marzo.qxd 6/15/05 11:23 AM Page 49
Cannabinoid Receptors and Their Role in Neuroprotection
effects of the synthetic cannabinoid HU-210 in
primary cortical neurons are mediated by phos-
phatidylinositol 3-kinase/AKT signaling Mol. Cell
Neurosci., in press.
Munro S., Thomas K. L., and Abu-Shaar M. (1993) Mol-
ecular characterization of a peripheral receptor for
cannabinoids. Nature 365, 61–65.
Muthian S., Rademacher D. J., Roelke C. T., Gross G.
J., and Hillard C. (2004) Anandamide content is
increased and Cb1 cannabinoid receptor blockade
is protective during transient, focal cerebral
ischemia. Neurosci. 129, 743–750.
Nagayama T., Sinor A. D., Simon R. P., et al. (1999)
Cannabinoids and neuroprotection in global and
focal cerebral ischemia and in neuronal cultures.
J. Neurosci. 19, 2987–2995.
Netzeband J. G., Conroy S. M., Parsons K. L., and Gruol
D. L. (1999) Cannabinoids enhance NMDA-elicited
Ca2+ signals in cerebellar granule neurons in cul-
ture. J. Neurosci. 19, 8765–8777.
Nicotera P., Leist M., and Manzo L. (1999) Neuronal
cell death: a demise with different shapes. Trends
Pharmacol. Sci. 20, 46–51.
Ohno-Shosaku T., Maejima T., and Kano M. (2001)
Endogenous cannabinoids mediate retrograde sig-
nals from depolarized postsynaptic neurons to
presynaptic terminals. Neuron. 29, 729–738.
Okamoto Y., Morishita J., Tsuboi K., Tonai T., and Ueda
N. (2004), Molecular characterization of a phos-
pholipase D generating anandamide and Its con-
geners. J. Biol. Chem. 279, 5298–5305
Pan X., Ikeda S. R., and Lewis D. L. (1996) Rat brain
cannabinoid receptor modulates N-type Ca2+
channels in a neuronal expression system Mol.
Pharmacol. 49, 707–714.
Panikashvili D., Simeonidou C., Ben-Shabat S., et al.
(2001) An endogenous cannabinoid (2-AG) is neu-
roprotective after brain injury. Nature 413, 527–531.
Parmentier-Batteur S., Jin K., Mao X., Xie L., and
Greenberg D. (2002) Increased severity of stroke
in CB1 cannabinoid receptor knock-out mice. J.
Neurosci. 22, 9771–9775.
Pinto J. C., Potie F., Rice K. C., et al. (1994) Cannabinoid
receptor binding and agonist activity of amides and
esters of arachidonic acid Mol. Pharmacol. 46,
516–522.
Porter A. C., Sauer J. M., Knierman M. D., et al. (2002)
Characterization of a novel endocannabinoid,
virodhamine, with antagonist activity at the CB1
receptor. J. Pharmacol. Exp. Ther. 301, 1020–1024.
Priller J., Briley E. M., Mansouri J., Devane W. A.,
Mackie K., and Felder C. C. (1995) Mead
NeuroMolecular Medicine
49
ethanolamide, a novel eicosanoid, is an agonist for
the central (CB1) and peripheral (CB2) cannabi-
noid receptors Mol. Pharmacol. 48, 288–292.
Rhee M. H., Bayewitch M., Avidor-Reiss T., Levy R.,
and Vogel Z. (1998) Cannabinoid receptor activa-
tion differentially regulates the various adenylyl
cyclase isozymes J. Neurochem. 71, 1525–1534.
Robbe D., Kopf M., Remaury A., Bockaert J., and
Manzoni O. J. (2002) Endogenous cannabinoids
mediate long-term synaptic depression in the
nucleus accumbens. Proc. Natl. Acad. Sci. USA
99, 8384–8388.
Rueda D., Galve-Roperh I., Haro A., and Guzman M.
(2000) The CB(1) cannabinoid receptor is coupled
to the activation of c-Jun N-terminal kinase Mol.
Pharmacol. 58, 814-820.
Sagan S., Venance L., Torrens Y., Cordier J., Glowinski
J., and Giaume C. (1999) Anandamide and WIN
55212-2 inhibit cyclic AMP formation through G-
protein-coupled receptors distinct from CB1
cannabinoid receptors in cultured astrocytes. Eur.
J. Neurosci. 11, 691–699.
Sanchez C., de Ceballos M. L., del Pulgar T. G., et al.
(2001) Inhibition of glioma growth in vivo by selec-
tive activation of the CB(2) cannabinoid receptor
Cancer Res. 61, 5784–5789.
Sanchez C., Galve-Roperh I., Rueda D., and Guzman
M. (1998) Involvement of sphingomyelin hydrol-
ysis and the mitogen-activated protein kinase cas-
cade in the Delta9-tetrahydrocannabinol-induced
stimulation of glucose metabolism in primary
astrocytes Mol. Pharmacol. 54, 834–843.
Schlicker E. and Kathmann M. (2001) Modulation of
transmitter release via presynaptic cannabinoid
receptors. Trends Pharmacol. Sci. 22, 565–572.
Shen M. and Thayer S. A. (1998a) Cannabinoid receptor
agonists protect cultured rat hippocampal neurons
from excitotoxicity. Mol. Pharmacol. 54, 459–462.
Shen M. and Thayer S. A. (1998b) The cannabinoid
agonist Win55, 212-2 inhibits calcium channels by
receptor-mediated and direct pathways in cultured
rat hippocampal neurons. Brain Res. 783, 77–84.
Sinor A. D., Irvin S. M., and Greenberg D. A. (2000)
Endocannabinoids protect cerebral cortical neurons
from in vitro ischemia in rats. Neurosci. Lett. 278,
157–160.
Skaper S. D., Buriani A., Dal Toso R., et al. (1996) The
ALIAmide palmitoylethanolamide and cannabi-
noids, but not anandamide, are protective in a
delayed postglutamate paradigm of excitotoxic
death in cerebellar granule neurons. Proc. Natl.
Acad. Sci. USA 93, 3984–3989.
Volume 7, 2005
02_Di Marzo.qxd 6/15/05 11:23 AM Page 50
50
Slipetz D. M., O’Neill G. P., Favreau L., et al. (1995)
Activation of the human peripheral cannabinoid
receptor results in inhibition of adenylyl cyclase
Mol. Pharmacol. 48, 352–361.
Smart D. and Jerman J. C. (2000) Anandamide: an
endogenous activator of the vanilloid receptor.
Trends Pharmacol. Sci. 21, 134.
Sugiura T., Kodaka T., Kondo S., et al. (1997b) Is the
cannabinoid CB1 receptor a 2-arachidonoylglyc-
erol receptor? Structural requirements for trigger-
ing a Ca2+ transient in NG108-15 cells J. Biochem.
(Tokyo) 122, 890–895.
Sugiura T., Kodaka T., Kondo S., et al. (1996) 2-
Arachidonoylglycerol, a putative endogenous
cannabinoid receptor ligand, induces rapid, tran-
sient elevation of intracellular free Ca2+ in neu-
roblastoma × glioma hybrid NG108-15 cells
Biochem. Biophys. Res. Commun. 229, 58–64.
Sugiura T., Kodaka T., Kondo S., et al. (1997a) Inhi-
bition by 2-arachidonoylglycerol, a novel type
of possible neuromodulator, of the depolariza-
tion-induced increase in intracellular free cal-
cium in neuroblastoma × glioma hybrid
NG108-15 cells Biochem. Biophys. Res. Commun.
233, 207–210.
Sugiura T., Kodaka T., Nakane S., et al. (1999) Evidence
that the cannabinoid CB1 receptor is a 2-arachi-
donoylglycerol receptor. Structure-activity rela-
tionship of 2-arachidonoylglycerol, ether-linked
analogues, and related compounds J. Biol .Chem.
274, 2794–2801.
Sugiura T., Yoshinaga N., Kondo S., Waku K., and
Ishima Y. (2000) Generation of 2-arachidonoyl-
glycerol, an endogenous cannabinoid receptor
ligand, in picrotoxinin-administered rat brain.
Biochem. Biophys. Res. Commun. 271, 654–658.
Tsou K., Brown S., Sanudo-Pena M. C., Mackie K.,
and Walker J. M. (1998) Immunohistochemical
distribution of cannabinoid CB1 receptors in the
NeuroMolecular Medicine
van der Stelt and Di Marzo
rat central nervous system. Neuroscience 83,
393–411.
van der Stelt M., Veldhuis W. B., Bar P. R., Veldink G.
A., Vliegenthart J. F., and Nicolay K. (2001a) Neu-
roprotection by Delta9-tetrahydrocannabinol, the
main active compound in marijuana, against
ouabain-induced in vivo excitotoxicity. J. Neurosci.
21, 6475–6479.
van der Stelt M., Veldhuis W. B., Maccarrone M., et al.
(2002) Acute neuronal injury, excitotoxicity, and the
endocannabinoid system. Mol. Neurobiol.26,317–346.
van der Stelt M., Veldhuis W. B., van Haaften G. W.,
et al. (2001b) Exogenous anandamide protects rat
brain against acute neuronal injury in vivo. J. Neu-
rosci. 21, 8765–8771.
Veldhuis W., Van der Stelt M., Wadman M., et al. (2003)
Neuroprotection by the endogenous cannabinoid
anandamide and arvanil against in vivo excito-
toxicity in the rat: role of vanilloid receptors and
lipoxygenases. J. Neurosci. in press.
Vogel Z., Barg J., Levy R., Saya D., Heldman E., and
Mechoulam R. (1993) Anandamide, a brain
endogenous compound, interacts specifically with
cannabinoid receptors and inhibits adenylate
cyclase J. Neurochem. 61, 352–355.
Walter L. and Stella N. (2004) Cannabinoids and neu-
roinflammation Br. J. Pharmacol. 141, 775–785.
Wartmann M., Campbell D., Subramanian A., Burstein
S. H., and Davis R. J. (1995) The MAP kinase signal
transduction pathway is activated by the endoge-
nous cannabinoid anandamide FEBS Lett 359,
133–136.
Wilson R. I. and Nicoll R. A. (2001) Endogenous
cannabinoids mediate retrograde signalling at hip-
pocampal synapses. Nature 410, 588–592.
Zygmunt P. M., Petersson J., Andersson D. A., et al.
(1999) Vanilloid receptors on sensory nerves medi-
ate the vasodilator action of anandamide. Nature
400, 452–457.
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