Download Immunohistochemical description of the endogenous cannabinoid

THE JOURNAL OF COMPARATIVE NEUROLOGY 509:400 – 421 (2008)
Immunohistochemical Description of the
Endogenous Cannabinoid System in the
Rat Cerebellum and Functionally Related
Nuclei
JUAN SUÁREZ,1* FRANCISCO JAVIER BERMÚDEZ-SILVA,1 KEN MACKIE,2
CATHERINE LEDENT,3 ANDREAS ZIMMER,4 BENJAMIN F. CRAVATT,5
1
AND FERNANDO RODRÍGUEZ DE FONSECA *
1
Laboratorio de Medicina Regenerativa, Fundación IMABIS, 29010 Málaga, Spain
2
Departments of Psychological and Brain Sciences, Indiana University, Bloomington, IN 47401
3
Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Université Libre
de Bruxelles, B-1050 Bruxelles, Belgium
4
Institute of Molecular Psychiatry, University of Bonn, 53115 Bonn, Germany
5
Chemical Biology and Cell Biology, The Scripps Research Institute,
La Jolla, California 92037
ABSTRACT
We report a detailed analysis of the distribution of relevant proteins of the endogenous
cannabinoid system in the rat cerebellum (cerebellar cortex and deep cerebellar nuclei) and
the two functionally related nuclei, the vestibular nuclei and the inferior olive. These proteins
include the two main cannabinoid receptors (CB1 and CB2), the enzymes involved in cannabinoid biosynthesis (DAGL␣, DAGL␤, and NAPE-PLD), and the endocannabinoiddegradating enzymes (FAAH and MAGL). With regard to the cerebellar cortex, these data
confirm several published reports on the distribution of cannabinoid CB1 receptors, DAGL␣,
MAGL, and FAAH, which suggests a role of endocannabinoids as retrograde messengers in
the synapses of the Purkinje cells by either parallel fibers of granule cells or climbing fibers
from the inferior olive or GABAergic interneuron. Additionally, we describe the presence of
CB2 receptors in fibers related to Purkinje somata (Pinceau formations) and dendrites
(parallel fibers), suggesting a potential role of this receptor in the retrograde cannabinoid
signaling. A remarkable finding of the present study is the description of the different
elements of the endogenous cannabinoid system in both the main afferent nuclei to the
cerebellar cortex (the inferior olive) and the efferent cerebellar pathway (the deep cerebellar
nuclei). The presence of the endogenous cannabinoid system at this level establishes the basis
for endocannabinoid-mediated synaptic plasticity as a control mechanism in motor learning,
opening new research lines for the study of the contribution of this system in gait disorders
affecting the cerebellum. J. Comp. Neurol. 509:400 – 421, 2008. © 2008 Wiley-Liss, Inc.
Indexing terms: endocannabinoid system; cerebellar cortex; deep cerebellar nuclei; vestibular
nuclei; inferior olive; CB1 receptor; CB2 receptor; immunohistochemistry
This article includes Supplementary Material available via the Internet
at http://www.interscience.wiley.com/jpages/0021-9967/suppmat.
Grant sponsor: Consejerı́a de Salud (Junta de Andalucı́a); Grant number: PI-0220; MEC; Grant number: SAF 2004/07762; Grant sponsor: Instituto de Salud Carlos III; Grant number: 07/1226; Grant number: 07/0880;
Grant sponsor: Plan Nacional Sobre Drogas; Grant sponsor: Consejeria de
Innovación Ciencia y Empresa (Junta de Andalucı́a); Grant number: REDES RTA RD06/001; Grant sponsor: 5th Framework Programme; Grant
number: TARGALC QLRT-2001-01048.
© 2008 WILEY-LISS, INC.
*Correspondence to: Juan Suárez and Fernando Rodrı́guez de Fonseca,
Laboratorio de Medicina Regenerativa, Fundación IMABIS, Avenida Carlos
Haya 82, 29010 Málaga, Spain. E-mail: [email protected];
[email protected]
Received 21 February 2007; Revised 14 August 2007; Accepted 28 April
2008
DOI 10.1002/cne.21774
Published online in Wiley InterScience (www.interscience.wiley.com).
The Journal of Comparative Neurology
ENDOCANNABINOID AND CEREBELLUM
401
Analysis of the CB1 receptor expression in the rat brain
by in situ hybridization histochemistry and immunocytochemistry has provided important insights into the functional neuroanatomy of the endocannabinoid system (Matsuda et al., 1993; Pettit et al., 1998; Egertová and Elphick,
2000; Van Sickle et al., 2005; Gong et al., 2006). Electrophysiological studies and the finding of CB1 receptor in
the cerebellar synapses suggest that endocannabinoids
act as retrograde messengers in the cerebellum. This role
of the endocannabinoid system is confirmed by the finding
of different types of endocannabinoid-mediated synaptic
plasticity, including both short-term [depolarizationinduced suppression of inhibition (DSI) and excitation
(DSE)] and the more permanent long-term depression
(LTD; for review see Wilson and Nicoll, 2002; Diana and
Marty, 2004; Safo and Regehr, 2005). The presence of the
endocannabinoid CB1 receptor in the cerebellum implies
that the endocannabinoid system plays a central role in
real-time regulation of movement and neuroadaptations
underlying motor control and motor learning. In fact, relevant pharmacological actions of exogenously administered cannabinoids are ataxia and catalepsy (Rodrı́guez de
Fonseca et al., 1998) and modulation of eye blink conditioning (Kishimoto and Kano, 2006; Skosnik et al., 2007).
CB1 receptors are located in axon terminals of parallel
fibers of cerebellar granular cells and climbing fibers of
inferior olive neurons that provide excitatory input on
Purkinje cells. In addition, CB1 receptors are located in
axon terminals of cerebellar basket and stellate cells providing inhibitory input on Purkinje cells (Mailleux and
Vanderhaeghen, 1992; Matsuda et al., 1993; Tsou et al.,
1998; Egertová and Elphick, 2000; Cristino et al., 2006;
Kawamura et al., 2006). Modulation of excitatory and
inhibitory input on Purkinje cells by the endocannabinoid
system allows Purkinje cells to refine the output of motor
responses from cerebellum.
However, so far, little information is available on the
presence and function of the cannabinoid CB2 receptor
(Skaper et al., 1996; Lu et al., 2000; Zhang et al., 2003;
Pazos et al., 2004; Benito et al., 2005; Sheng et al., 2005)
and other components of the endocannabinoid system,
such as cannabinoid biosynthesis and degradation en-
zymes, in the brain (Dihn et al., 2002; Romero et al., 2002;
Bisogno et al., 2003; Egertová et al., 2003; Okamoto et al.,
2004). Recently, immunohistochemical studies revealed
the distribution of CB2 receptor in the rat brain, particularly in cerebellum and hippocampus (Van Sickle et al.,
2005; Gong et al., 2006). The finding of CB2 receptor in the
cerebellum suggests the need to reevaluate the effects of
exogenous and endogenous cannabinoids on neurotransmission.
There are some studies demonstrating the presence of
the cannabinoid degradation enzymes FAAH (Cravatt et
al., 1995, 1996; Egertová et al., 1998; Goparaju et al.,
1998; Tsou et al., 1998) and MAGL (Dihn et al., 2002) in
the brain. Other studies report a general analysis of
FAAH expression in specific neuronal population of mouse
and human brain, including cerebellar Purkinje cells, neurons of cerebellar nuclei and inferior olive, neocortical and
hippocampal pyramidal neurons, and striatal projecting
neurons. These localizations of FAAH suggest a complementary distribution with CB1 expression in these brain
regions (Romero et al., 2002; Egertová et al., 2003). Northern blot and in situ hybridization analyses reveal that
MAGL is heterogeneously expressed in some brain areas,
including hippocampus, cortex, cerebellum, and anterior
thalamus, where CB1 receptor is also expressed, indicating a presynaptic localization of the enzyme (Dihn et al.,
2002).
Indeed, the recent identification of 2-AG and AEA biosynthesis and release enzymes, DAGL␣ and DAGL␤
(Bisogno et al., 2003), and NAPE-PLD (Okamoto et al.,
2004) has provided new insights on the endocannabinoid
signaling system in the brain. Pharmacological studies
suggest that DAGL and NAPE-PLD activity is required
for inhibition of ␥-aminobutyric acid (GABA)-ergic transmission by glutamatergic input (Chevaleyre and Castillo,
2003). Additionally, DAGL activity is related to axonal
growth and guidance during development (Brittis et al.,
1996; Williams et al., 2003). The expression of DAGL
isozymes (␣ and ␤ forms) changes during development of
the brain; that is, they are expressed in axonal tracts of
the embryo and then in dendritic fields of the adult mouse
brain (Bisogno et al., 2003). In the adult mouse cerebel-
Abbreviations
b
c
CbCx
CbN
cf
DEn
DG
g
G
GrL
Hi
icp
IntA
IntDL
IntDM
IntP
IntPPC
IO
IOD
IODM
IOM
IOPr
basket cell
collaterals
cerebellar cortex
cerebellar nuclei
climbing fibers
dorsal endopiriform nucleus
dentate gyrus
granular cell
golgi cell
granular layer
hippocampus
inferior cerebellar peduncle
interposed cerebellar nucleus, part anterior
interposed cerebellar nucleus, dorsolateral hump
interposed cerebellar nucleus, dorsomedial crest
interposed cerebellar nucleus, part posterior
interposed cerebellar nucleus, posterior parvicellular part
inferior olive
inferior olive, dorsal nucleus
inferior olive, dorsomedial cell group
inferior olive, medial nucleus
inferior olive, principal nucleus
LA
Lat
LatPC
LVe
mf
Med
MedDL
ML
MVe
MVeMC
MVePC
P
pf
pif
py
s
scp
sp5
SpVe
SuVe
VeCb
VeN
lateral amygdaloid nucleus
lateral (dentate) cerebellar nucleus
lateral cerebellar nucleus, parvicellular part
lateral vestibular nucleus
mossy fibers
medial (fastigial) cerebellar nucleus
medial cerebellar nucleus, dorsolateral protuberance
molecular layer
medial vestibular nucleus
medial vestibular nucleus, magnocellular part
medial vestibular nucleus, parvicellular part
purkinje cell
parallel fibers
pinceau formation
pyramidal tract
superficial stellate cell
superior cerebellar peduncle
spinal trigeminal tract
spinal vestibular nucleus
superior vestibular nucleus
vestibulocerebellar nucleus
vestibular nuclei
The Journal of Comparative Neurology
402
lum, the substantial down-regulation of DAGL␤ contrasts
with the strong staining of DAGL␣ in the Purkinje cell
dendritic field (Bisogno et al., 2003).
For this study, we selected NAPE-PLD as the
anandamide-synthesizing enzyme. A recent paper described the molecular characterization of NAPE-PLD; its
authors noted the presence of NAPE-PLD activity in
mouse brain, including the cerebellum (Okamoto et al.,
2004). We have also detected NAPE-PLD expression in rat
cerebellum (Ferrer et al., 2007). However, its immunohistochemical localization in the brain has not been analyzed
so far. It is important to note that NAPE-PLD is not the
only source for anandamide in the brain (Leung et al.,
2006) but is the first of a series of enzymes capable of
generating anandamide from the membrane precursor
N-arachidonyl-phosphatidyl ethanolamide (NAPE), such
as ␣/␤ hydrolase 4, lyso-PLD, lyso-PLC, and phosphatases
such as PTPN22 (Leung et al., 2006; Simon and Cravatt,
2006; Liu et al., 2007). However, despite discrepancies in
substrate specificity and the lack of specific test for the
activation of NAPE-PLD in neural circuits (Liu et al.,
2007), NAPE-PLD remains an important source of anandamide in the brain. Therefore, the molecular characterization of new synthesis pathways for anandamide in the
brain will determine this important aspect of endocannabinoid physiology.
The aim of this study was to determine the distribution
of the endocannabinoid receptors CB1 and CB2 and the
endocannabinoid biosynthesis and degradation enzymes
DAGL␣, DAGL␤, NAPE-PLD, FAAH, and MAGL by immunohistochemistry in the rat cerebellum (cerebellar cortex and cerebellar nuclei) and other functionally related
brain areas, such as vestibular nuclei and inferior olive.
The exact localization of these cannabinoid enzymes and
receptors in the rat cerebellum may facilitate a neuroanatomical framework for the analysis of the physiological
roles of the endocannabinoid signaling system.
MATERIAL AND METHODS
Generation of NAPE-PLD-, DAGL␣-, DAGL␤-,
and MAGL-specific antibodies
We have generated polyclonal rabbit antibodies against
proteins of the cannabinoid machinery. Immunizing peptides were 1) a 13-amino-acid (aa) peptide comprising part
of both the C-terminal and the N-terminal regions of
NAPE-PLD (MDENSCDKAFEET); 2) a 16-aa peptide
from the C-terminal region of DAGL␣ (CGASPTKQDDLVISAR); 3) a 16-aa peptide from an internal sequence of
DAGL␤ (SSDSPLDSPTKYPTLC); 4) a 15-aa peptide from
the N-terminal region of MAGL (SSPRRTPQNVPYQDL);
5) a 73-aa peptide (401– 473) from the C-terminal region of
CB1 receptor; and 6) a 14-aa peptide (328 –342) from the
C-terminal region of CB2 receptor. We employed a chimeric sequence peptide as immunogen for NAPE-PLD
antibody generation. The aim of this chimeric construction
was to contain two distant epitopes exposed in the native
protein because one of them belongs to the N-terminal and
the other to the C-terminal region of the protein, both
regions having random coil structures. NAPE-PLD,
DAGL␣, and DAGL␤ peptides were synthesized and coupled to keyhole limpet hemocyanin (KLH; JPT Peptide
Technologies, Berlin, Germany). The three peptides were
injected into rabbits (two animals per peptide), according
J. SUÁREZ ET AL.
to standard protocols for generation of antisera, with the
IgG fraction subsequently purified by means of a protein A
column (Sigma, St. Louis, MO). MAGL antibody was produced in the laboratory of Dr. D. Piomelli (Dihn et al.,
2002). MAGL peptide was synthesized and coupled to
KLH by addition of a cysteine at the peptide N terminus
(United Biochemical, Seattle, WA). The conjugated peptide was injected into two rabbits to generate antisera
(Strategic Biosolutions, Ramona, CA). The peptide was
then conjugated to an agarose column and the antiserum
purified according to the manufacturer’s instructions
(AminoLink; Pierce Endogen, Rockford, IL). For CB1 and
CB2 antibody generation, we injected rabbits with a fusion
protein composed of glutathione-S transferase (GST) and
CB1 residues 401– 473 or CB2 residues 328 –342, using
conventional techniques. Polyclonal antisera and purified
antibodies specific for CB1 and CB2 were collected by
affinity chromatography against both GST and immunizing fusion protein.
Immunohistochemistry
We have evaluated the presence of CB1 and CB2 receptors, FAAH, MAGL, DAGL␣, DAGL␤, and NAPE-PLD in
the adult rat cerebellum by immunohistochemistry. Manipulation of animals was in accordance with the European Communities Council Directives (86/609/EEC) on
the treatment of experimental animals.
Adult Wistar rats (n ⫽ 6; 300 g) were deeply anesthetized with 2,2,2-tribromoethanol (300 mg/kg i.p.) and
briefly transcardially perfused with 0.1 M phosphatebuffered saline (PBS; pH 7.4), followed by 4% paraformaldehyde in PBS at 4°C for 30 minutes. Brains were dissected, postfixed overnight in buffered paraformaldehyde
at 4°C, equilibrated with 30% sucrose in PBS at 4°C,
frozen, and cut into 40-␮m-thick-transverse or sagittal
sections using a sliding microtome. We then collected 19
alternate series of sections from each rat brain to process
the seven antibodies and Nissl staining.
Free-floating sections were first incubated in H2O distilled containing 50 mM sodium citrate (pH 9) for 30
minutes at 80°C, followed by several washes in PBS.
Then, we incubated sections with 3% hydrogen peroxide in
PBS for 20 minutes at room temperature to inhibit endogenous peroxidase, followed by three times in PBS.
We have used seven primary antibodies. The anti-CB1
was developed in rabbits by using a fusion protein as
immunogen containing 73 amino acid residues (401– 473)
of the rat CB1 receptor (Wager-Miller et al., 2002); the
anti-CB2 was produced in rabbits by using a fusion protein
containing 14 amino acid residues (328 –342) from rat CB2
receptor; the anti-FAAH was developed in rabbits by using a synthetic peptide corresponding to 561–579 amino
acid fragment of rat fatty acid amine hydrolase conjugated
to KLH as immunogen (Cayman Chemical; catalog No.
101600, lot. No. 157878). The anti-NAPE-PLD, antiDAGL␣, anti-DAGL␤, and anti-MAGL were developed in
rabbits as described above. Sections were incubated in the
diluted primary antibody (anti-CB1, diluted 1:500; antirat CB2, 1:500; anti-rat FAAH, 1:200; anti-MAGL, 1:200;
anti-DAGL␣, 1:500; anti-DAGL␤, 1:200; and anti-NAPEPLD, 1:400) overnight at room temperature. After three
washes in PBS, the sections were incubated in a biotinylated donkey anti-rabbit immunoglobulin (Amersham, Little Chalfont, England) diluted 1:500 for 1 hour, washed
again in PBS, and incubated in ExtrAvidin peroxidase
The Journal of Comparative Neurology
ENDOCANNABINOID AND CEREBELLUM
(Sigma) diluted 1:2,000 for 1 hour. We revealed immunolabeling with 0.05% diaminobenzidine (DAB; Sigma),
0.05% nickel ammonium sulfate, and 0.03% H2O2 in PBS.
All steps were carried out with gentle agitation at room
temperature. After the sections had been washed in PBS,
they were mounted on gelatinized slides, air dried, dehydrated in ethanol, cleared in xylene, and coverslipped with
Eukitt mounting medium (Kindler GmBH and Co.,
Freiburg, Germany).
Digital photographs were taken on an Olympus BX41
microscope equipped with an Olympus DP70 digital camera. Digital images were adjusted for brightness/contrast
in Adobe Photoshop (Adobe, San Jose, CA), and the figures
were mounted and labelled in Adobe PageMaker.
Antibody specificity and controls
We performed Western blot analyses to demonstrate
that CB1, CB2, FAAH, MAGL, DAGL␣, DAGL␤, and
NAPE-PLD antibodies recognized the corresponding antigen in the rat cerebellum. To perform Western blot analysis, we used fresh tissue from Wistar male rats. Animals
were killed by 2,2,2-tribromoethanol (Fluka, Steinheim,
Germany), and the cerebellum was immediately isolated,
snap frozen in liquid nitrogen, and stored at – 80°C until
use. Membrane extracts of rat cerebellum were prepared
in HEPES 50 mM (pH 8)-sucrose 0.32 M buffer by using a
homogenizer. The homogenate was centrifuged at 800g for
10 minutes at 4°C and the supernatant centrifuged at
40,000g for 30 minutes. We resuspended the pellets in
HEPES 50 mM buffer and potterized using a homogenizer.
For immunoblotting, equivalent amounts of membrane
proteins (45 ␮g) from rat cerebellum were separated by
10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted onto nitrocellulose
membranes, and controlled by Ponceau red staining. For
DAGL␤ immunoblotting, we used stringent conditions
with the addition of dithiothreitol 0.15 mM in the sample
buffer. We preincubated blots with a blocking buffer containing PBS, 0.1% Tween 20, and 2% albumin fraction V
from bovine serum (Merck) at room temperature for 1
hour. For protein detection, each blotted membrane lane
was incubated separately with the specific CB1 (1:250),
CB2 (1:250), FAAH (1:100), MAGL (1:200), DAGL␣ (1:
300), DAGL␤ (1:200), and NAPE-PLD (1:400) antibodies
and diluted in PBS containing 0.1% Tween 20 and 2%
albumin fraction V from bovine serum at room temperature overnight. After extensive washing in PBS containing
1% Tween 20 (PBS-T), a peroxydase-conjugated goat antirabbit antibody (Promega, Madison, WI) was added (1:
2,500) for 1 hour at room temperature. Biotinylated
marker proteins with defined molecular weights were
used for molecular weight determination in Western blots
(ECL Western Blotting Molecular Weight Markers; Amersham). We incubated the corresponding markers lane
with ExtrAvidin peroxidase (Sigma). Membranes were
then subjected to repeated washing in PBS-T and the
specific protein bands visualized by using the enhanced
chemiluminiscence technique (ECL; Amersham) and
Auto-Biochemi Imaging System (LTF Labortechnik
GmbH, Wasserburg/Bodensee, Germany). Western Blots
showed that each primary antibody detected a protein of
the expected molecular size (see Fig. 1A).
As additional controls, cerebellar and hippocampal sections from CB1 receptor knockout mice (Ledent et al.,
403
1999), CB2 receptor knockout mice (Buckley et al., 2000),
NAPE-PLD knockout mice (Cravatt et al., 2001), and wildtype controls (n ⫽ 2 pairs) were also analyzed. Immunohistochemical protocol was carried out as described above
(anti-CB1, diluted 1:500; anti-rat CB2, 1:500; anti-rat
NAPE-PLD, 1:400). We observed that immunostaining
was almost completely absent in CB1 knockout mouse
brain, but weak staining was found in the spinal trigeminal tract at cerebellar levels and in the cerebral peduncle
at hippocampal levels (Suppl. Fig. 1). With the exception
of these features, all of the staining in wild-type brain is
specifically attributable to CB1 expression. We did not
observed labeling in the CB2 receptor knockout mouse or
NAPE-PLD receptor knockout mouse, whereas the wildtype mouse showed labelling similar to that of the rat
brain (Suppl. Figs. 2, 3).
We also incubated blotted membrane lanes with the
primary antibody preadsorbed with the immunizing peptide CB1 (10 ␮g/ml), CB2 (10 ␮g/ml), FAAH (20 ␮g/ml;
Cayman, Ann Harbor, MI), MAGL (13 ␮g/ml; kindly donated by Dr. D. Piomelli), DAGL␣ (175 ␮g/ml; JPT; see
above), DAGL␤ (32 ␮g/ml; JPT; see above), and NAPEPLD (16 ␮g/ml; JPT; see above) or incubated by replacing
the primary antiserum by 2% albumin fraction V from
bovine serum (see Fig. 1A). In addition, we incubated
brain sections with the primary antibody preadsorbed
with the immunizing peptide under the same conditions
as described above. We did not detect staining under these
conditions (Suppl. Figs. 4, 5).
RESULTS
In the present study, we mapped the expression of the
cannabinoid receptors CB1 and CB2; the degradation enzymes FAAH and MAGL; and the synthesis enzymes
DAGL␣, DAGL␤, and NAPE-PLD in the rat cerebellum
and two main cerebellar-related areas, vestibular nuclei
and inferior olive. The analysis of the immunostaining
patterns was carried out at transverse and sagittal planes
of the adult rat brain by comparing it with the cytoarchitecture and with published data on its neurochemistry
and connections. The intensity of the immunoreactivity
for each antibody was very similar in all the six rat brains
used in this study. Numerous brain regions such as hippocampus, basal ganglia, substantia nigra, and cerebellum intensely expressed CB1 receptors, FAAH, MAGL,
and DAGL␣, in comparison with CB2 receptors, DAGL␤,
and NAPE-PLD expression. The nomenclature for nuclei
and subdivisions referred to in the present study is widely
accepted and used in the rat brain atlas by Paxinos and
Watson (1998). Results for this study are described in the
text and summarized in a rating scale (Table 1). Grayscale values measured in single cerebellar, vestibular, and
olive nuclei are represented on an arbitrary scale of four
labelling intensities, from “⫹,” meaning “very low”
(slightly above the density measured in sections incubated
in preadsorbed antibody; see Suppl. Figs. 4, 5) to “⫹⫹⫹⫹,”
meaning “very high” (according to the highest signal density in the specimen, i.e., parvicellular part of the medial
vestibular nucleus). Previously, we analyzed controls and
Western blot results to demonstrate that CB1, CB2,
FAAH, MAGL, DAGL␣, DAGL␤, and NAPE-PLD antibodies recognize the corresponding antigen in the rat cerebellum.
The Journal of Comparative Neurology
404
J. SUÁREZ ET AL.
TABLE 1. Immunoreactivity in Cerebellum, Vestibular Nuclei, and Inferior Olive1
CB1
c
Cerebellar cortex
Molecular layer
Granular layer
Purkinje cells
Cerebellar nuclei
IntA
IntP
IntPPC
IntDL
IntDM
Lat
LatPC
Med
MedDL
Vestibular nuclei
LVe
MVeMC
MVePC
SpVe
SuVe
VeCb
Inferior olive
–
–
CB2
f
c
⫹⫹⫹⫹
⫹⫹
–
–
–
FAAH
f
c
⫹⫹
⫹⫹
–
⫹
–
MAGL
f
c
⫹⫹⫹⫹
⫹
⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹
DAGL␣
f
c
⫹⫹⫹
⫹⫹
–
–
DAGL␤
f
c
⫹⫹⫹⫹
⫹⫹
–
⫹
⫹
–
–
–
–
–
–
–
–
–
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
–
–
–
–
–
–
–
–
–
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫹
–
⫹⫹
⫹
⫹
–
⫹⫹
⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
–
–
–
–
–
–
–
–
–
⫹⫹
⫹⫹
⫹⫹⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹
⫹⫹
⫹
⫹⫹
⫹
⫹⫹
⫹⫹
⫹⫹⫹
–
–
–
–
–
–
⫹⫹
⫹⫹⫹
⫹
⫹⫹⫹
⫹
⫹
⫹⫹
–
–
–
–
–
–
–
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹
⫹⫹
⫹
⫹
–
⫹⫹
⫹⫹⫹⫹
⫹
⫹
⫹
⫹⫹⫹⫹
⫹⫹
⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹
⫹
⫹
⫹⫹
⫹
⫹⫹
⫹
⫹⫹
–
–
–
–
–
–
–
⫹⫹⫹
⫹⫹
⫹⫹⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹
⫹
⫹⫹
f
⫹⫹⫹
⫹⫹
⫹⫹⫹
NAPE-PLD
c
⫹
⫹⫹
f
⫹⫹⫹
⫹⫹⫹
⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹
⫹⫹
⫹⫹
⫹⫹
⫹
⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹
⫹⫹⫹
⫹
⫹⫹
⫹⫹
⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹
⫹
⫹
⫹⫹
⫹⫹
⫹⫹⫹
⫹
⫹⫹
⫹⫹
⫹⫹
1
Rating scale of the immunoreactivity of each structure in cells (c) and fibers (f). Symbols are as follows: very high (⫹⫹⫹⫹), high (⫹⫹⫹), low (⫹⫹), very low (⫹), and without
immunoreactivity (–).
Western blot analysis
Western blot analyses of membrane extracts from rat
cerebellum revealed CB1 immunostaining as a prominent
band at about 60 kD (Fig. 1A, lane 1). CB2 immunostaining also showed a prominent band at about 55 kD (Fig. 1A,
lane 7). Immunoblots for FAAH and NAPE-PLD revealed
a single band with a molecular mass of 63 and 46 kD,
respectively (Fig. 1A, lanes 3 and 5, respectively). In addition, under more stringent conditions, DAGL␤ immunoblotting showed a prominent band of 76 kD and another,
less intense one of 97 kD (Fig. 1A, lane 11). DAGL␣ immunoblotting showed an expected band of 120 kD (Fig. 1A,
lane 13). Analysis of MAGL immunoreactivity confirmed
two bands of 37 and 35 kD, but we also observed an
additional band at about 62 kD (Fig. 1A, lane 9). In all
cases, the immunoreactive bands were abolished after
absorption with the immunizing peptides (Fig. 1A, lanes
2, 4, 6, 8, 10, 12, 14).
CB1 immunoreactivity
We observed a prominent fiber CB1 labelling surrounding Purkinje somata and proximal axons that may consist
of clustered basket cell axons, called Pinceau formations
(Fig. 2B, inset). Numerous immunostained fibers are also
widespread throughout the branches of the cerebellar
white matter and well-defined fibers (but not mossy fibers)
dispersed from the granular layer into the molecular layer
(Fig. 2B, inset). Therefore, these fibers may represent
axons from inferior olive (climbing fibers). We clearly distinguished the molecular layer of cerebellar cortex by an
intense CB1 immunoreactivity consisting of a dense network of immunostaining fibers and puncta, which may
principally correspond to climbing fiber terminals but also
parallel fibers from granular cells. The climbing fibers
represented the main external Purkinje afferent innervations originating from the inferior olive (see Fig. 10B). We
did not observed stained cell bodies in the cerebellar cortex.
Cerebellar and vestibular nuclei present a very low CB1
immunoreactivity (Fig. 2A,C–F). In general, our results
showed that all cerebellar nuclei (medial, lateral, and
interposed nuclei) contained very weak neuropil. However, it should be noted that numerous fiber tracts crossed
the cerebellar nuclei, which could be distinguished by
their position and orientation. Some of these fiber bundles
crossed unstained peduncles and formed part of the superior cerebellar peduncle (Fig. 2C,D). Therefore, part of
these fibers may represent axons from cerebellar nuclei
that projected to red nuclei, dorsal thalamus, and motor
cortex. Other peduncles of CB1⫹ fibers coursed dorsally
from the inferior cerebellar peduncle to the cerebellar
cortex. Most of these fibers constituted axons from the
inferior olive, in agreement with the presence of climbing
fibers in the molecular layer (Fig. 2E). An intense CB1
immunoreactivity consisting of a network of neuropil and
puncta characterized the dorsal portion of the principal
nucleus of the inferior olive (IOPr; see Fig. 9A). The presence of numerous CB1⫹ fibers in the rubroolivary tract
(data not shown) suggested that most of the fiber terminals in the IOPr originated from the red nucleus. The rest
of the inferior olivary complex showed very low immunoreactivity.
Most vestibular nuclei also presented very weak CB1
immunoreactivity (Fig. 2C). We distinguished spinal vestibular nucleus (SpVe) and the magnocellular part of medial vestibular nucleus (MVeMC) from its parvocellular
part (MVePC) by a stronger CB1⫹ neuropil (Fig. 2C). It is
worth noticing that typical giant neurons of the lateral
vestibular nuclei (LVe) were moderately CB1 immunostained, whereas their dendritic initial segments could be
clearly distinguished (Fig. 2E).
CB2 immunoreactivity
The distribution of CB2 immunoreactivity in the cerebellar cortex was similar to that of CB1 immunoreactivity.
As occurred with CB1 immunoreactivity, we observed
CB2-immunoreactive (CB2⫹) fiber terminals surrounding
Purkinje somata and proximal axons (Pinceau formations). Purkinje cells were not CB2 immunoreactive. The
The Journal of Comparative Neurology
ENDOCANNABINOID AND CEREBELLUM
405
Fig. 1. A: Western blots of membrane extracts from rat cerebellum
show prominent immunoreactive bands of expected molecular masses
of 60 kD for CB1; 63 kD for FAAH; 46 kD for NAPE-PLD; 55 kD for
CB2; 62, 37, and 35 kD for MAGL; 97 and 76 kD for DAGL␤; and 120
kD for DAGL␣. Positions of molecular markers (MW) are indicated at
left. B: Major biochemical pathways for endogenous cannabinoid signalling system. Anandamide (AEA) is released from a membrane lipid
precursor, N-arachidonoyl-phosphatidylethanolamine (NAPE), by the
action of a specific phospholipase D (PLD) activated by depolarization,
postsynaptic calcium increases, or G-protein-coupled receptor stimulation. The membrane enzyme N-acyltransferase (NAT) catalyses
NAPE biosynthesis, which transfers arachidonic acid from phosphatidylcholine (PhChol) to the head group of phosphatidylethanolamine
(PhEth). Postsynaptic calcium influx and the activation of metabotropic receptors coupled to phosphatidyl-inositol-specific phospholipase C (PLC) and diacylglycerol (DAG) lipase pathway lead to increases
in
2-arachidonoylglycerol
(2-AG)
production.
Endocannabinoid signalling includes uptake into cells mediated by a
transporter (AT) and hydrolysis by two specific enzymatic systems,
the fatty acid amide hydrolase (FAAH) and the monoacylglyceride
lipase (MAGL).
molecular layer also contained a dense network of immunostained fibers and neuropil, extending from the granular layer to the pial surface of the molecular layer. Most
CB2⫹ fibers of the molecular layer were disposed in parallel and presented numerous varicosities along their surface. Therefore, these fibers may principally represent
parallel fibers originating from granular cells (Fig. 3B,
inset). In contrast to CB1 immunoreactivity, the granular
layer showed moderate neuropil CB2 immunoreactivity.
These fibers showed a mossy aspect (mossy fibers) and so
probably may represent axons from pontine nuclei, vestibular nuclei, and spinal cord (Fig. 3B). In addition, all
subdivisions of the inferior olive showed a strong CB2
immunoreactivity that consisted of a dense network of
fibers (Fig. 9B). The main olivary afferent innervations
originated from the spinal cord, so these fiber terminals in
the IO may constitute collaterals of the mossy fibers in the
cerebellar cortex from the spinal cord.
The CB2 immunoreactivity in the cerebellar and vestibular nuclei was different from the CB1 immunoreactivity,
especially the prominent neuropil immunoreactivity (Fig.
3C–F). Most cerebellar nuclei are characterized by a dense
network of CB2⫹ neuropil and fiber terminals that define
numerous unstained fiber bundles and cell profiles (Fig.
3D,F). These CB2⫹ fibers may have the same origin as the
CB2⫹ mossy fibers in the granular layer of the vestibular
nuclei, pontine nuclei, and spinal cord, but they may also
constitute projections from Purkinje cells. A stronger neuropil immunostaining also characterized vestibular nuclei
such as MVePC and SpVe (Fig. 3C,E). Most fiber termi-
The Journal of Comparative Neurology
406
Fig. 2. CB1 immunoreactivity in the rat cerebellum and vestibular
nuclei. A: General view of a coronal section through the cerebellum
and vestibular nuclei. B: High-magnification photomicrographs of the
cerebellar cortex showing clustered basket cell axons (pinceau formations) surrounding unstained Purkinje somata (inset). Numerous
fibers fill the branches of the cerebellar white matter and disperse
from the granular layer into the molecular layer (arrowhead in inset).
J. SUÁREZ ET AL.
C: Low-magnification photomicrograph showing CB1 immunoreactivity throughout the cerebellar and vestibular nuclei. Note fiber tracts
that cross the cerebellar nuclei and can be distinguished by their
position and orientation into the superior (D) and inferior (E) cerebellar peduncle and the giant neurons in LVe (F). For abbreviations
see list. Scale bars ⫽ 1 mm in A; 200 ␮m in B,C; 100 ␮m in D–F; 20
␮m in inset.
The Journal of Comparative Neurology
ENDOCANNABINOID AND CEREBELLUM
Fig. 3. CB2 immunoreactivity in the rat cerebellum and vestibular
nuclei. A: General view of a coronal section through the cerebellum
and vestibular nuclei. B: Detail of a transverse section of the cerebellar cortex showing mossy fibers in the granular layer, parallel fibers in
the molecular layer (arrows), and “pinceau” formations in the Purkinje layer (arrowheads). Note the unstained Purkinje somata (in-
407
set). C: Low-magnification photomicrograph showing CB2 immunoreactivity throughout the cerebellar and vestibular nuclei. Note cell
profiles immersed in a dense fiber network in all cerebellar nuclei
(D,E) and the intense neuropil immunoreactivity in the MVe (F). For
abbreviations see list. Scale bars ⫽ 1 mm in A; 100 ␮m in B,E,F; 200
␮m in C; 50 ␮m in D; 20 ␮m in inset.
The Journal of Comparative Neurology
408
J. SUÁREZ ET AL.
nals in the vestibular nuclei may constitute projections
from Purkinje cells.
FAAH immunoreactivity
We clearly observed FAAH immunoreactivity in Purkinje neurons, which showed intensely stained cell bodies
and a dense network of fibers in the molecular layer that,
in sagittal sections, characterized the dendrite tree of the
Purkinje cells (Fig. 4B, inset). On the other hand, we
observed an evident FAAH immunoreactivity in a subpopulation of granular cells (Fig. 4B) and in numerous
neurons of IO (Fig. 9C). Therefore, we can expect that
some of the FAAH-immunoreactive (FAAH⫹) puncta of
the molecular layer probably represent climbing and parallel fiber terminals. Poor neuropil immunoreactivity in
the granular layer was similar to that of fibers in the
cerebellar white matter.
The strong FAAH immunoreactivity observed in all cerebellar nuclei was related mainly to the presence of a
dense meshwork of fibers, consisting of FAAH⫹ punctate
labelling that contained immunoreactive cell bodies (Fig.
4C,D). Most likely, these fibers represented dendritic fibers. Note the moderate density of FAAH⫹ neurons in the
IntDL, MeDL, and Med (Fig. 4C,D) and the strongly
FAAH⫹ fibers in all cerebellar nuclei (Fig. 4C).
However, we observed that most of the vestibular nuclei
presented a low immunoreactivity for FAAH (Fig. 4C,E,F),
with the exception of LVe, which showed immunoreactivity similar to that of cerebellar nuclei. LVe staining consisted of a dense network of fibers disposed between tracts
of the juxtarestiform body along with a number of giants
FAAH⫹ neurons spreading from LVe to SpVe (Fig. 4E).
Both parts (parvicellular and magnocellular) of the medial
vestibular nucleus showed a very low FAAH immunostaining consisting of a number of FAAH⫹ cells embedded
in a very poorly stained neuropil (Fig. 4F).
MAGL immunoreactivity
MAGL immunostaining of the cortex cerebellum
showed a distinct pattern in the cerebellar layers (Fig.
5A). The molecular layer was distinguished by its moderate MAGL immunoreactivity consisting of a number of
small cells that likely represented basket cells and superficial stellate cells, clearly discernable within a moderately MAGL-immunoreactive (MAGL⫹) neuropil (Fig. 5B).
Most MAGL⫹ neuropil in the molecular layer seemed to
represent parallel fiber terminals from the granular cells
(Fig. 5B) and also probably climbing fibers from IO. Purkinje cell bodies were moderately stained, whereas Purkinje dendrites were not immunoreactive (Fig. 5B, arrowheads). A remarkable feature was the MAGL
immunoreactivity of the granular layer, related mainly to
the presence of densely packed, well-stained granular
cells and possibly others cell types embedded in a moderate neuropil labelling (Fig. 5B). Part of this neuropil likely
consisted of mossy fibers from spinal, pontine, and vestibular nuclei. The outer one-third of the granular layer (with
respect to its radial dimension to the pial surface) contained dispersed cells with a stronger staining that likely
represented Golgi cells according to their unique location
under the Purkinje layer (Fig. 5B, inset).
Cerebellar and vestibular nuclei presented numerous
strongly stained MAGL⫹ neurons, showing a perikaryal
and dendritic Golgi-like labelling (Fig. 5C–F). Mediumsized neurons characterized most cerebellar nuclei based
on their morphology and orientation (Fig. 5D). However,
IntDM, IntPPC, and LatPC showed small MAGL⫹ cells
(Fig. 5C,D). LVe and SpVe also presented numerous giant
neurons (more dispersed in SpVe) showing a prominent
Golgi-like labelling (Fig. 5E). MVePC showed numerous
small MAGL⫹ neurons embedded in a moderately immunoreactive neuropil, in contrast to the large neurons in
MVeMC (Fig. 5F). All IO subdivisions presented numerous small MAGL⫹ neurons showing less staining than
those of the Golgi-like labelling of surrounding areas (Fig.
9D).
DAGL␣ immunoreactivity
In contrast to MAGL immunoreactivity, the expression
of DAGL␣ was particularly prominent in the molecular
layer that clearly corresponded to the dendritic field of the
Purkinje cells (Fig. 6A,B). Note the weak DAGL␣ immunoreactivity Purkinje cell bodies and the numerous varicosities along the dendritic fibers in transverse and sagittal views (Fig. 6B, insets b⬘,b⬘⬘, respectively). However,
the lack of DAGL␣⫹ cells in the granular layer and in all
subdivisions of IO (see Fig. 9E) suggests that parallel and
climbing fibers did not present DAGL␣ immunoreactivity.
We also observed typically DAGL␣⫹ mossy fibers that
coursed along the branches of the cerebellar white matter
and spread into the granular layer, showing a moderately
immunoreactive neuropil (Fig. 6B).
However, we did not observe DAGL␣ immunoreactivity
in cell bodies of cerebellar and vestibular nuclei (Fig.
6C–F) or in any other region with mossy fiber projections
in the granular layer such as the pontine nuclei or the
spinal cord (data not shown). Cerebellar and vestibular
nuclei presented moderate DAGL␣ immunoreactivity that
consisted of a conspicuous network of neuropil and puncta
(Fig. 6C–F). In some cerebellar and vestibular regions, the
dense neuropil defined numerous profiles of unstained cell
bodies (Fig. 6D,F). Of relevance, IntPPC, LatPC, and
MVePC were characterized by a prominent neuropil
DAGL␣ immunoreactivity (Fig. 6D,F). Additionally, IO
was also characterized by the presence of intense neuropil
immunoreactivity in contrast to that of surrounding areas
(Fig. 9E).
DAGL␤ immunoreactivity
DAGL␤ immunostaining in the cerebellar cortex was
considerably less pronounced than that of DAGL␣ (Fig.
7A,B). We also observed stained DAGL␤⫹ Purkinje cell
bodies and a moderately DAGL␤ immunoreactivity in the
molecular layer that may be consistent with the immunohistochemical description by Bisogno and collaborators
(2003) for mouse cerebellum (Fig. 7B). In the granular
layer, DAGL␤ immunoreactivity was associated mainly
with the presence of immunoreactive neuropil but was
weaker than that of the molecular layer and some dispersed granular cells (Fig. 7B). Additionally, IO contained
abundant DAGL␤⫹ neurons (Fig. 9F), so DAGL␤⫹ neuropil of the molecular layer probably contained parallel and
climbing fibers from granular cells and IO neurons, respectively, in contrast to the DAGL␣ immunoreactivity.
DAGL␤ immunoreactivity in the cerebellar and vestibular nuclei was associated mainly with cell bodies embedded in a network of fibers, in contrast to DAGL␣ immunoreactivity (Fig. 7C). Most cerebellar nuclei presented a
DAGL␤⫹ neuronal distribution similar to that of MAGL⫹
neurons, which is in medium-sized cell bodies homog-
The Journal of Comparative Neurology
ENDOCANNABINOID AND CEREBELLUM
Fig. 4. FAAH immunoreactivity in the rat cerebellum and vestibular nuclei. A: General view of a coronal section through the cerebellum and vestibular nuclei. B: High-magnification photomicrographs
of the cerebellar cortex showing intensely stained Purkinje cell bodies
and the dense network of Purkinje dendritic fibers (inset in B) and
dispersed granular cells (arrowheads). C: Low-magnification photomi-
409
crograph showing FAAH immunoreactivity throughout the cerebellar
and vestibular nuclei. Note the low density of FAAH⫹ neurons in the
MeDL (D) and the weak immunostaining of the giant neurons in the
LVe (E) and the small neurons of the MVePC (F). For abbreviations
see list. Scale bars ⫽ 1 mm in A; 100 ␮m in B,D–F; 200 ␮m in C; 20
␮m in inset.
The Journal of Comparative Neurology
410
Fig. 5. MAGL immunoreactivity in the rat cerebellum and vestibular nuclei. A: General view of a coronal section through the cerebellum and vestibular nuclei. B: High-magnification photomicrographs
of the cerebellar cortex showing the high density of granular cells,
Purkinje somata, and basket and stellate cells homogeneously distributed in the molecular layer. Purkinje dendrites are not immunostained (arrowheads). Note the prominent immunoreactivity of a neuronal subpopulation into the granular layer that, by its position under
J. SUÁREZ ET AL.
the Purkinje layer, may correspond to Golgi cells (inset in B). C: Lowmagnification photomicrograph showing MAGL immunoreactivity
throughout the cerebellar and vestibular nuclei. Note the Golgi-like
labelling of the medium-sized neurons in IntP, IntA, and Lat (D) and
the small neurons in IntPPC (D) and MVePC (F). Giant neurons of the
LVe and large neurons of the MVeMC are also intensely immunoreactive (E,F). For abbreviations see list. Scale bars ⫽ 1 mm in A; 100
␮m in B,D–F; 200 ␮m in C; 20 ␮m in inset.
The Journal of Comparative Neurology
ENDOCANNABINOID AND CEREBELLUM
Fig. 6. DAGL␣ immunoreactivity in the rat cerebellum and vestibular nuclei. A: General view of a coronal section through the cerebellum and vestibular nuclei. B: High-magnification photomicrographs of the cerebellar cortex showing the Purkinje somata and the
dense network of Purkinje dendritic fibers in the molecular layer.
Note the numerous varicosities along the dendritic fibers in trans-
411
verse (bⴕ) and sagittal (bⴕⴕ) views. C: Low-magnification photomicrograph showing an intense DAGL␣ immunoreactivity in a network of
fibers throughout the cerebellar and vestibular nuclei. Note the
higher density of fibers in the IntPPC, LatPC, and MVePC. Scale
bars ⫽ 1 mm in A; 100 ␮m in B,D–F; 200 ␮m in C; 20 ␮m in b⬘; 10 ␮m
in b⬘⬘.
The Journal of Comparative Neurology
412
Fig. 7. DAGL␤ immunoreactivity in the rat cerebellum and vestibular nuclei. A: General view of a coronal section through the cerebellum and vestibular nuclei. Stained DAGL␤⫹ Purkinje somata and
scattered DAGL␤⫹ granular cells are observed in the cerebellar cortex
(B, arrowheads). C: Low-magnification photomicrograph showing
J. SUÁREZ ET AL.
DAGL␤ immunoreactivity throughout the cerebellar and vestibular
nuclei. Note the small neurons in the IntDM (D) and MVePC (F), in
comparison with the larger neurons in the IntA (D) and MVeMC (F),
and the giant neurons in LVe (E). For abbreviations see list. Scale
bars ⫽ 1 mm in A; 100 ␮m in B,D–F; 200 ␮m in C.
The Journal of Comparative Neurology
ENDOCANNABINOID AND CEREBELLUM
enously distributed (Fig. 7C,D). However, fewer small
DAGL␤⫹ neurons were observed in IntPPC and parts of
LatPC and IntDM compared with the remaining parts of
cerebellar nuclei (Fig. 7C,D). Giant DAGL␤⫹ neurons
characterized LVe (Fig. 7E), whereas small DAGL␤⫹ neurons immersed in a dense immunoreactive neuropil characterized MVePC (Fig. 7F).
NAPE-PLD immunoreactivity
The distribution of the NAPE-PLD immunoreactivity
was quite different from that described above for CB1 in
the cerebellar cortex (Fig. 8A,B). The molecular layer was
characterized by a moderately immunoreactive neuropil,
in contrast to the considerably weaker immunostaining of
the granular layer, which was partially related to dendritic fiber from the strongly stained Purkinje cell bodies
(Fig. 8B, inset) but also possibly to parallel fibers from a
number of granular cells showing NAPE-PLD immunoreactivity (Fig. 8B). In addition, most IO subdivisions presented a number of weakly NAPE-PLD-immunoreactive
(NAPE-PLD⫹) neurons (Fig. 9G), so the molecular layer of
the cerebellar cortex could also contain NAPE-PLD⫹
climbing fibers. Note the weakly stained NAPE-PLD⫹
cells in the molecular layer, which may correspond to
basket and stellate cells (Fig. 8B, inset).
The distribution of NAPE-PLD immunoreactivity in the
cerebellar and vestibular nuclei was quite similar to that
of DAGL␤ immunoreactivity (Figs. 7C, 8C). Most cerebellar nuclei consisted of intense neuropil immunoreactivity
and a number of moderately labelled NAPE-PLD⫹ neurons (Fig. 8C,D). As with DAGL␤ immunoreactivity, IntPPC and LatPC showed considerably lower number of
NAPE-PLD⫹ neurons and a less intense neuropil immunoreactivity than in the remaining cerebellar regions (Fig.
8C).
Vestibular nuclei showed well-stained neurons consisting of abundant giant NAPE-PLD⫹ neurons in LVe and
SpVe (Fig. 8E), and large neurons in MVeMC (Fig. 8F).
MVePC also showed numerous stained cell bodies immersed in moderately NAPE-PLD⫹ neuropil (Fig. 8F).
DISCUSSION
Here we report the first detailed analysis of the presence and the comparative distribution of functionally
relevant proteins of the endogenous cannabinoid system, namely, the two main cannabinoid receptors (CB1
and CB2), the enzymes involved in cannabinoid biosynthesis (DAGL␣, DAGL␤, and NAPE-PLD), and two
endocannabinoid-degradating enzymes (FAAH and
MAGL) in the rat cerebellum (cerebellar cortex and
cerebellar nuclei) and two functionally related nuclei,
the vestibular nuclei and the inferior olive. It is important to note that additional putative endocannabinoid
receptors (i.e., orphan receptor GPR55, vanilloid VR1
receptor) and enzymes for biosynthesis and degradation
have been proposed (Leung et al., 2006; Simon and
Cravatt, 2006; Liu et al., 2007). However, their molecular characterization and their contribution to endocannabinoid physiology are still under active investigation.
Our results confirm data from previous studies on the
presence and localization of CB1 in the cerebellar cortex.
Our study also provides new insight in relation to the
localization of CB1 and CB2 receptors and FAAH, but
principally in relation to the presence of MAGL, DAGL␣,
413
DAGL␤, and NAPE-PLD in cerebellum and functionally
related nuclei that have not been described previously.
Additionally, the segregated localization of CB1 and CB2
in the cerebellum suggests a complementary distribution
of the two receptors associated with the specific distribution of the cannabinoid degradation and biosynthesis enzymes in the cerebellum.
Because of the described variability of NAPE-PLD, CB1,
and CB2 distribution with regard to the different antibodies used, we carried out careful control experiments for
specificity. Thus, we have used the NAPE-PLD knockout
mouse, CB1 knockout mouse, CB2 knockout mouse, and
Western blot analyses as additional controls for immunohistochemistry to characterize the NAPE-PLD, CB1, and
CB2 antibodies and demonstrate their antibody specificity. We observed that immunostaining was almost completely absent in CB1 knockout mouse brain. However,
weak staining was found in the spinal trigeminal tract at
cerebellar levels and in the cerebral peduncle at hippocampal levels (Suppl. Fig. 1). With the exception of
these features, all of the staining in wild-type brain is
specifically attributable to CB1 expression. We did not
observed labeling in the CB2 receptor knockout mouse or
NAPE-PLD receptor knockout mouse, whereas the wildtype mouse showed labelling similar to that of the rat
brain (Suppl. Figs. 2, 3). CB1-immunostained bands (60
kD) were similar to those described by Egertová and Elphick (2000). CB2 immunoblotting also showed a band (55
kD) similar to that described in recent reports for rat brain
(Van Sickle et al., 2005; Gong et al., 2006). The single
bands observed for FAAH (63 kD) and NAPE-PLD (46 kD)
were identical to those described previously (Giang and
Cravatt, 1997; Okamoto et al., 2004). The DAGL␣ molecular mass (120 kD) is identical to that described for COS
cells by Bisogno and collaborators (2003). Carrying out
more stringent conditions for DAGL␤ immunoblotting, we
detected a prominent DAGL␤ band (76 kD) that was similar to that described by Bisogno and collaborators (2005;
70 kD). The weaker band at 97 kD may be explained by
the presence of a glycosylated form of DAGL␤ that has yet
to be defined. Analysis of MAGL immunoreactivity confirmed two bands of 37 and 35 kD, but we also observed an
additional band at about 62 kD, similar to the weak band
observed in Figure 2 of Dihn et al. (2002). This band can
represent a post-translationally modified form of MAGL in
the rat cerebellum that still has to be characterized. We
carried out Western blots and immunohistochemistry in
the presence of specific immunizing peptides to confirm
the specificity of the labelling. The absence of labelling
under these conditions indicated that the seven antibodies
utilized in the present study were selective for the histological identification and discrimination of their protein
expression.
Distribution of CB1 and CB2 in cerebellum
Our data indicated that CB1 and CB2 immunoreactivities show partial complementary localization in the cerebellar cortex. Characterization and localization of CB1immunopositive staining throughout all the cerebellar
lobules was consistent with previous studies on the expression of CB1 mRNA, the detection of [3H]CP-55,940
binding sites, and the immunocytochemical mapping of
CB1 in rodent brain (Herkenham et al., 1991b; Matsuda et
al., 1993; Pettit et al., 1998; Egertová and Elphick, 2000;
Cristino et al., 2006). We have located the majority of CB1
The Journal of Comparative Neurology
414
Fig. 8. NAPE-PLD immunoreactivity in the rat cerebellum and
vestibular nuclei. A: General view of a coronal section through the
cerebellum and vestibular nuclei. NAPE-PLD-immunoreactive
(NAPE-PLD⫹) Purkinje somata and granular cell (arrows) are observed in the cerebellar cortex (B,D). Note the weakly stained NAPEPLD⫹ cells in the molecular layer that may correspond to basket and
J. SUÁREZ ET AL.
stellate cells (arrowheads). C: Low-magnification photomicrographs
showing NAPE-PLD immunoreactivity throughout the cerebellar and
vestibular nuclei. Note the well-labeled somata of the giant neurons in
the LVe (E) and the small neurons in the MVePC (F). For abbreviations see list. Scale bars ⫽ 1 mm in A; 100 ␮m in B,D–F; 200 ␮m in C;
20 ␮m in inset.
Fig. 9. Photomicrographs of coronal sections through the rat inferior olive (IO), showing CB1 (A), CB2 (B), FAAH (C), MAGL (D),
DAGL␣ (E), DAGL␤ (F), and NAPE-PLD (G) immunohistochemistry.
Numerous stained small cells are located in all IO subdivisions, except for CB1, CB2, and DAGL␣ immunoreactivity, whereas IO shows
a denser neuropil than the surrounding areas (A,B,E). Note the restricted location of CB1⫹ neuropil in the dorsal part of the IOPr (A).
H: Schematic representation of the IO subdivisions at Bregma –12.72
mm, described in the rat brain atlas of Paxinos and Watson (1998).
For abbreviations see list. Scale bars ⫽ 100 ␮m.
The Journal of Comparative Neurology
416
immunoreactivity in the molecular layer of the rat cerebellar cortex, largely associated with climbing and parallel
fibers that extended on the Purkinje dendrites and in
clustered basket cell axons surrounding Purkinje somata,
especially on their basal areas, which correspond to the
initial axonal segment (Herkenham, 1995; Egertová and
Elphick, 2000; Egertová et al., 2003; Cristino et al., 2006;
Kawamura et al., 2006). Previous studies have reported
dense [3H]CP-55,940 labelling in the molecular layer and
sparse binding in the granular layer, including mutant
mice deficient in Purkinje cell expression, suggesting that
Purkinje cells were not the source of CB1 expression in the
molecular layer (Herkenham et al., 1991a,b; Herkenham,
1995). As expected, cell bodies of the molecular layer (basket and stellate cells) expressed CB1 mRNA, but Purkinje
cells did not (Mailleux and Vanderhaeghen, 1992; Matsuda et al., 1993). In agreement with these studies, we did
not detect CB1 immunoreactivity in Purkinje somata and
their dendritic processes (Egertová and Elphick, 2000).
The present study showed an intense CB1 immunoreactivity in fibers of the molecular layer, which agrees with
previous studies describing the expression of CB1 mRNA
in fibers of the molecular layer and neurons of IO (Mailleux and Vanderhaeghen, 1992), which suggests the presence of CB1 immunoreactivity in climbing fibers (Pettit et
al., 1998). However, by silver-enhanced immunogold,
Kawamura et al. (2006) detected occasionally weak CB1
labelling in climbing fibers that terminated on the proximal Purkinje dendrites. On the other hand, our results
also indicated an absence of CB1 immunoreactivity in the
granular layer (as in mossy fibers), in contrast to the
detection of CB1 mRNA labelling in the deep cerebellar
nuclei (Mailleux and Vanderhaeghen, 1992; Matsuda et
al., 1993). However, we have detected CB1⫹ immunoreactivity in fiber bundles of the superior cerebellar peduncle
(possibly from the interposed and lateral cerebellar nuclei)
and in fiber terminals of the red nucleus and some nuclei
of the dorsal thalamus and motor cortex (data not shown).
Our results revealed a distribution of CB2 immunoreactivity in part similar to that of CB1 immunoreactivity. In
contrast to recent immunocytochemical studies (Ashton et
al., 2006; Gong et al., 2006; Onaivi et al., 2006), CB2
immunoreactivity was not associated with Purkinje cell
bodies and their dendritic processes. We have observed
strong CB2 immunostaining in a number of varicose fibers
(parallel fibers) in the molecular layer; most of them may
be associated with granular cells, but they could also be
associated with mossy fibers in the granular layer. These
data match the detection of granular layer cells and neurons in brainstem and spinal cord by in situ hybridization
in previous reports (Skaper et al., 1996; Van Sickle et al.,
2005).
The results obtained for CB1 and CB2 immunostaining
indicate that CB1 and CB2 receptors are in part located in
the same presynaptic structures of the cerebellar cortex,
such as clustered basket cell axons (Pinceau formation),
but they are also present in complementary presynaptic
structures. Therefore, CB1 receptors are preferably located in climbing fibers (olivary projections), whereas CB2
receptors are preferably located in mossy fibers (spinal,
pontine, and vestibular projections) and parallel fibers
(cerebellar granular cells). The presynaptic localization of
both cannabinoid receptors in the cerebellar cortex supports the hypothesis of endocannabinoids as retrograde
messengers proposed for different brain areas, including
J. SUÁREZ ET AL.
cerebellum, amygdala, basal ganglia, and hippocampus
(Stella et al., 1997; Rodrı́guez de Fonseca et al., 1998,
2005; Giufrida et al., 1999; Wilson and Nicoll, 2001; Wilson et al., 2001; Diana et al., 2002; Gerdeman et al., 2002;
Robbe et al., 2002; Chevaleyre and Castillo, 2003). At this
moment, we cannot exclude that CB2 receptors might
serve as a retrograde signalling gate controlling neuronal
depolarization or trophic maintenance of the synapses. In
any case, the finding of CB2 receptor in the cerebellum
suggests the need for reevaluating the effects of exogenous
and endogenous cannabinoids on neurotransmission.
Presence and distribution of cannabinoid
degradation enzymes in cerebellum in
relation to CB1 and CB2 receptors
FAAH and MAGL are two hydrolytic enzymes that mediate the degradation of different endocannabinoids.
FAAH mediates endocannabinoid degradation, including
AEA, but also 2-AG, whereas MAGL was found to mediate
85% of total brain membrane 2-AG hydrolase activity (Fig.
1B; Piomelli et al., 2000; Blankman et al., 2007). As described for the synthesis of endocannabinoids, additional
hydrolytic enzymes degradating anandamide and 2-AG
have been recently proposed (Wei et al., 2006; Blankman
et al., 2007; Muccioli et al., 2007), but their role in neural
circuits is still unknown. Thus we will limit the discussion
to both FAAH and MAGL. The presence of both endocannabinoid degradation enzymes in the cerebellum gives
support for the existence of multiple regulatory mechanisms terminating endocannabinoid signaling. As occurs
with CB1 and CB2 receptors, the specific localization of
FAAH and MAGL also suggests a complementary distribution of the two enzymes in the cerebellar cortex. Consistent with previous studies of the rodent and human
cerebellar cortex (Egertová et al., 1998, 2003; Romero et
al., 2002; Gulyas et al., 2004), FAAH immunoreactivity
was present in Purkinje somata. Additionally, we have
clearly detected in sagittal cerebellar slides the characteristic dendritic tree of the Purkinje cell, including the tiniest branches, which contained intense FAAH immunostaining. These data disagree with data from Egertová et
al. (2003) but agree with the dendritic staining described
in the molecular layer of human and rat cerebellum (Romero et al., 2002; Gulyas et al., 2004) and observed in
human cerebellar samples in our laboratory (Suarez et al.,
unpublished). However, we have not detected stained cells
in rat cerebellar molecular layer. In contrast to previous
studies in rat cerebellum (Egertová et al., 1998; Tsou et
al., 1998; Gulyas et al., 2004), FAAH immunoreactivity
was also evident in a small population of granular cells
that was consistent with the detection of granular cells in
rat cerebellum by in situ hybridization (Thomas et al.,
1997) and in human and mouse cerebellum by immunohistochemistry (Romero et al., 2002; Egertová et al., 2003).
All these data indicate that the localization of FAAH is
quite complementary to that of CB1 and CB2 receptors in
the cerebellar cortex, so Purkinje somata and their dendritic processes and a specific population of granular cells
expressed FAAH, whereas theirs presynaptic structures,
climbing fibers, expressed CB1 receptor, and parallel fibers and mossy fibers expressed CB2 receptor.
Concerning the presence of MAGL in the cerebellum,
and in contrast to the findings of Gulyas et al. (2004), we
found that not only the molecular layer neuropil (possibly
The Journal of Comparative Neurology
ENDOCANNABINOID AND CEREBELLUM
consisting of axon terminals) but also densely packed cells
of the granular layer showed MAGL immunoreactivity.
We have clearly distinguished two cell types according to
the intensity of MAGL immunoreactivity: a dense population of moderately immunoreactive cells disposed
throughout the granular layer, which may be granular
cells, and lower numbers of strongly immunoreactive cells
disposed near Purkinje layer, which probably are Golgi
cells. Additionally, cells of the molecular layer, as well as
Purkinje somata, showed moderate MAGL staining.
These molecular layer cells probably represented basket
cells and stellate cells.
The localization of MAGL in the cerebellar cortex could
partially overlap that of CB1 and CB2 receptors. Despite
the difficulty in defining the specific localization of MAGL
expression because of the immunoreactivity in the granular layer, the stained fibers and puncta in the molecular
and granular layers could be related to mossy and climbing and/or parallel fibers; although negative Purkinje cell
dendrites could be clearly seen (Gulyas et al., 2004). The
location of MAGL as a presynaptic enzyme, which agreed
with CB1 and CB2 receptor distribution in presynaptic
structures of the cerebellar cortex, may be related to the
retrograde messenger role of 2-AG, as described recently
for the hippocampus, which determines basal endocannabinoid tone (Hashimotodani et al., 2007).
For the present study, we have optimized the immunohistochemical protocol for MAGL antibody (kindly donated by Dr. D. Piomelli) by using a combination of immunological methods. We tested different incubations,
pretreatments, and antibody dilutions in comparison with
a commercial MAGL antibody (Cayman; catalog No.
100035; Suppl. Fig. 6). Both MAGL antibodies recognized
the same N-terminal aa sequence and revealed the same
molecular masses. Only the distinct dilutions tested for
both antibodies resulted in differences in the general intensity of the MAGL immunoreactivity. In contrast, both
MAGL antibodies resulted in the same molecular weight
and the same immunohistochemical distribution in the rat
cerebellum. Therefore, the immunohistochemical differences observed in this study and that of Gulyas et al.
(2004) can only be explained by the use of different MAGL
lots.
Additional explanations might be considered in order to
clarify the nature of this potentially nonspecific signal.
Although MAGL is thought to be the major 2-AGhydrolyzing enzyme (Blankman et al., 2007), it also acts
as an inactivator of other monoacylglycerol and prostaglandin glycerol esters (Dihn et al., 2002, 2004; Vila et al.,
2007). Thus, MAGL is also a relevant enzyme controlling
the acyl glycerol metabolism that might not be oriented
only to synaptic transmission. Furthermore, a recent
study has provided evidence of MAGL activity that controls 2-AG levels in microglia, not described previously
(Muccioli et al., 2007). The novel MAGL activity is especially rich in mitochondrial and nuclei. The findings of
Mucciolli and collaborators suggest that the cloned
MAGL, which is responsible for the majority of 2-AG hydrolysis in healthy brains (Hohmann et al., 2005), does not
play a major role in primary microglia. Possible explanations for this include cell-specific regulation of MAGL
translation and, furthermore, the differential regulation
that cytokine activation produces on the expression of
MAGL and the novel MAGL in the brain. The authors
observed an inverse regulation of both enzymes by
417
interferon-␥ (Witting et al., 2006). All these possibilities
require further clarification but may support the evidence
of a wider distribution of the enzyme in the brain.
Presence and distribution of cannabinoid
biosynthesis enzymes in the cerebellum in
relation to CB1 and CB2 receptors
We have reported here the first analysis of the presence
of DAGL␣, DAGL␤, and NAPE-PLD in the cerebellum.
The expression of these biosynthesis enzymes will determine where endocannabinoids are made and released in
the cerebellum. DAGL␣ and DAGL␤ constitute two recently identified isoforms of closely related genes correlated with 2-AG biosynthesis and release (Fig. 1B;
Mechoulam et al., 1995; Sugiura et al., 1995; Piomelli et
al., 2000; Bisogno et al., 2003). Additionally, Bisogno and
collaborators (2003) have found coexpression of DAGL␣
and DAGL␤ in a similar staining pattern in mouse brain.
They also indicated that the expression of both isozymes
changed in the developing brain from axonal tracts in
the embryonic stages to dendritic fields in the adulthood. It is important to note that, even though 2-AG is
considered a full cannabinoid receptor agonist, it is also
an important intermediate in triacyl/diacylglycerol metabolism as well as a prominent molecule linking the
cannabinoid signaling with lysophospholipids and diacycilglycerol-PKC signaling systems. Therefore, we cannot strictly consider both DAGL␣ and DAGL␤ as pure
endocannabinoid-synthesizing enzymes. Besides their
obvious role on the endocannabinoid system, it is very
possible that they also play other, additional physiological roles. For instance, pharmacological studies have
suggested that DAGL activity is required for axonal
growth and guidance in developing brain (Brittis et al.,
1996; Williams et al., 2003). However, we will focus only
on their potential role in the endocannabinoid system.
In agreement with Bisogno and collaborators (2003),
our results for the rat cerebellum showed high DAGL␣
expression in the dendritic field of the Purkinje cells,
consisting of prominent tube-like structures that contained numerous varicosities on their surface. In addition, Purkinje cells expressed the highest levels of
DAGL␣ mRNA but not cerebellar granular cells (Yoshida et al., 2006). However, our results indicated that
the staining of DAGL␤ was quite different from that of
DAGL␣ in rat cerebellum; that is, whereas Purkinje cell
dendrites strongly expressed DAGL␣, Purkinje cell bodies specifically expressed DAGL␤. Additionally, we have
detected a small population of granular cells that was
DAGL␤ immunoreactive. The distribution of DAGL␣
mRNA and DAGL␤ mRNA also differed in the cerebellar cortex. DAGL␤ mRNA was highly expressed in the
cerebellar granular layer (Yoshida et al., 2006). The
different localizations of DAGL␣ and DAGL␤ suggest
that at least three different postsynaptic locations preferably make and release 2-AG in the cerebellar cortex:
Purkinje cell dendrites postsynaptically release 2-AG by
DAGL␣ and Purkinje somata and granular somata/
dendrites by DAGL␤. Indeed, the postsynaptic localization of DAGL␣ in Purkinje cell dendrites correlates with
the presynaptic localization of CB1 receptor in climbing
fibers that terminate on distal and proximal Purkinje
cell dendrites and with the presynaptic location of CB2
receptor in mossy and parallel fibers that terminate on
The Journal of Comparative Neurology
418
J. SUÁREZ ET AL.
granular cells and Purkinje dendrites, respectively. In
agreement with our results, a previous report indicated
that DAGL␣ was essentially targeted by postsynaptic
spines in cerebellar Purkinje cells and suggested close
proximity between production sites of endocannabinoids
and their receptors (Yoshida et al., 2006). The postsynaptic localization of DAGL␤ in Purkinje somata and
granular somata also agrees with the presynaptic localization of CB1 and CB2 receptors in clustered basket cell
axons (Pinceau formation) and with the location of CB2
receptor in mossy fibers that surround granular somata.
On the other hand, NAPE-PLD is another recently characterized cannabinoid biosynthesis enzyme that mediates
the release of N-acyl ethanolamines (including AEA) from a
phospholipid precursor [N-acyl-phosphatidylethanolamide
(NAPE); Fig. 1B; Piomelli et al., 2000; Okamoto et al., 2004].
Again, N-acyl ethanolamides are not only endocannabinoid
mediators; some of them (oleoylethanolamide, palmithylethanolamide) are also activators of other receptor types, including nuclear receptors of the peroxisome proliferatorsactivated receptor family (Fu et al., 2003). However, c-fos
mapping did not reveal a substantial change in the pattern
of cellular activity in the cerebellum after exogenous
oleoylethanolamide administration, underscoring its contribution to cerebellar physiology (Rodriguez de Fonseca et al.,
2001).
Our results indicated that the staining of NAPE-PLD
was quite similar to that of DAGL␤ in the cerebellar
cortex, as indicated by the fact that Purkinje somata and
a small population of granular somata presented prominent NAPE-PLD immunoreactivity. Here, we have also
observed weak NAPE-PLD expression in cells and fibers of
the molecular layer. The appearance of these fibers in the
molecular layer possibly represents the dendritic field of
the Purkinje cells. So, NAPE-PLD and DAGL␣ may be
coexpressed in the Purkinje cell dendritic field. Similarly
to DAGL␣ and DAGL␤, the postsynaptic localization of
NAPE-PLD in Purkinje somata and granular somata correlates with the presynaptic localization of CB1 and CB2
receptor in clustered basket cell axons (Pinceau formation) and the location of CB2 receptor in mossy fibers,
whereas the postsynaptic localization of NAPE-PLD in
Purkinje dendrites and molecular layer cells correlates
with the presynaptic localization of CB1 receptor in climbing fibers and the location of CB2 receptor in parallel
fibers. The induction of retrograde signals by the biosynthesis of endocannabinoids in Purkinje dendrites
(DAGL␣⫹ and NAPE-PLD⫹) is enhanced when parallel
fiber (CB2⫹) stimulation is combined with climbing fiber
(CB1⫹) stimulation (Brenowitz and Regehr, 2005).
Presence of the endocannabinoid system in
the cerebellar nuclei, vestibular nuclei, and
inferior olive
The importance should be noted of the CB2 receptor in
fiber terminals, which homogeneously filled all cerebellar
and vestibular nuclei and suggests a Purkinje origin. In
addition, IO also showed a dense network of CB2⫹ fibers
that may originate principally from spinal projections. On
the other hand, the presence of the CB1 receptor in fibers
that coursed into the inferior cerebellar peduncles could
relate to the olivary-cerebellar projections, which also correlates with the presence of climbing fibers in the cerebellar cortex (Fig. 10).
Fig. 10. A: Schematic parasagittal section of a mammalian cerebellum redrawn from Cajal (1911). B: Schematic representation of the
inferior olive-cerebellar and vestibulocerebellar connections and intrinsic cerebellar circuits in mammals. For abbreviations see list.
These data were not consistent with previous studies
showing an intense CB1 and CB2 immunoreactivities associated with neuronal somata of the cerebellar nuclei (see
Fig. 7 in Pettit et al., 1998; Gong et al., 2006) and vestib-
The Journal of Comparative Neurology
ENDOCANNABINOID AND CEREBELLUM
ular nuclei (Ashton et al., 2004; see Figs. 5F and 13A in
Gong et al., 2006). However, our study agrees with the low
level of [3H]CP-55,940 binding and the low intensity of
CB1 mRNA signal found in the deep cerebellar nuclei,
vestibular nuclei, and IO (Herkenham et al., 1991b; Matsuda et al., 1993) but also with the low levels of CB1
radiographic labelling in the human medial and lateral
vestibular nucleus (Glass et al., 1997). In contrast, the
presence of cannabinoid degradation and biosynthesis enzymes was prominent in cerebellar nuclei, vestibular nuclei, and IO complex. Although FAAH, MAGL, DAGL␤,
and NAPE-PLD immunoreactivities were associated
mainly with cell bodies, DAGL␣ immunoreactivity was
associated exclusively with a conspicuous network of fibers and puncta in these three functionally related regions. In agreement with Bisogno and collaborators
(2003), DAGL␣ expression became restricted to synaptic
fields in the adult brain, possibly in postsynaptic dendrites, correlating with the postsynaptic requirement for
the synthesis of endocannabinoids as a retrograde messenger (Kreitzer and Regehr, 2001; Diana et al., 2002;
Bisogno et al., 2003). The presence of the proteins of the
endogenous cannabinoid system in the input and output
relays of the cerebellar cortex suggests a potential role for
endocannabinoid-mediated plasticity in motor timing and
learning mediated by these circuits (Raymond et al.,
1996).
Role for the endocannabinoid system in the
cerebellum: new targets for the study of
motor learning and the ataxias
Early studies using natural and synthetic cannabinoids
reported ataxia as one of the key features of the pharmacological profile of these compounds (Dewey, 1986; Patel
and Hillard, 2001). The effects on ataxia are mediated by
cannabinoid CB1 receptors, and apparently there are no
CB2-mediated gait alterations in experimental animals
(Patel and Hillard, 2001; Valenzano et al., 2005). These
results indicated that, at least, motor timing and coordination require an intact endogenous cannabinoid receptor
signalling system that, despite the activation of CB1 receptors, may regulate sensorimotor integration. However,
the cerebellum is an additional motor learning device
needed for many different forms of motor learning (Raymond et al., 1996). It has been suggested that the endogenous cannabinoid system modulates motor learning (i.e.,
eye blink conditioning) in the cerebellum (Kishimoto and
Kano, 2006; Skosnik et al., 2007). The truth is that there
is a lack of information on the role of CB1 and CB2 receptors in these learning processes involving cerebellar circuits. Most of the work so far has been performed in the
cerebellar cortex, using synaptic plasticity paradigms. Recent studies have clearly established a role for the endogenous cannabinoid system in short-term and long-term
forms of synaptic plasticity in the cerebellar cortex. Longterm depression, depolarization-induced suppression of
inhibition, or depolarization-induced suppression of excitation is modulated by endogenous cannabinoid release
(Diana and Marty, 2004; Kreitzer and Regehr, 2001; Safo
and Regehr, 2005). These effects are mediated through
cannabinoid CB1 receptors. However, little is known on
the role of cannabinoid CB2 receptors, which, as described
here, are present in clustered basket cell axons (Pinceau
formations) and other relevant relays of the cerebellar
419
circuitry. Whether they affect postsynaptic endocannabinoid production, downstream intracellular signalling, or
survival/remodelling processes still requires further investigation. This is relevant not only for motor learning
but also for the neuroadaptions associated with cerebellar
insults or chronic degenerative disorders such as the ataxias.
Moreover, we lack crucial information on the role of the
endogenous cannabinoid system in deep cerebellar nuclei
and the IO complex. Because of the crucial role of these
structures in motor timing and learning, especially in
sensorymotor integration and conditioning, it is possible
to anticipate a role for cannabinoid receptors in modulating the input and output information streams of the cerebellar cortex, beyond the Purkinje cell (as an example, it
is worth remembering that the IO is the sole source for an
entire afferent system to the cerebellum, the climbing
fibers). More research is thus needed to establish this
hypothesis by analyzing the effects of cannabinoid CB1
and CB2 receptors as well as FAAH and MAGL inhibitors
in simple learning paradigms, such as the vestibuloocular
reflex and the eyelid conditioning responses. Such studies
may also help to establish the potential pharmacological
utility of cannabinoids in cerebellar disorders.
ACKNOWLEDGMENTS
The authors are indebted to Dr. Daniele Piomellli for
kindly providing MAGL antibody.
LITERATURE CITED
Ashton JC, Zheng Y, Liu P, Darlington CL, Smith PF. 2004. Immunohistochemical characterisation and localisation of cannabinoid CB1 receptor protein in the rat vestibular nucleus complex and the effects of
unilateral vestibular deafferentation. Brain Res 1021:264 –271.
Ashton JC, Friberg D, Darlington CL, Smith PF. 2006. Expression of the
cannabinoid CB2 receptor in the rat cerebellum: an immunohistochemical study. Neurosci Lett 396:113–116.
Benito C, Kim WK, Chavarria I, Hillard CJ, Mackie K, Tolon RM, Williams
K, Romero J. 2005. A glial endogenous cannabinoid system is upregulated in the brains of macaques with simian immunodeficiency virusinduced encephalitis. J Neurosci 25:2530 –2536.
Bisogno T, Howell F, Williams G, Minassi A, Cascio MG, Ligresti A, Matias
I, Schiano-Moriello A, Paul P, Williams EJ, Gangadharan U, Hobbs C,
Di Marzo V, Doherty P. 2003. Cloning of the first sn1-dag lipases points
to the spatial and temporal regulation of endocannabinoid signaling in
the brain. J Cell Biol 163:463– 468.
Bisogno T, Ligresti A, Di Marzo V. 2005. The endocannabinoid signalling
system: biochemical aspects. Pharmacol Biochem Behav 81:224 –238.
Blankman JL, Simon GM, Cravatt BF. 2007. A comprehensive profile of brain
enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol.
Chem Biol 14:1347–1356.
Brenowitz SD, Regehr WG. 2005. Associative short-term synaptic plasticity mediated by endocannabinoids. Neuron 45:419 –31.
Brittis PA, Silver J, Walsh FS, Doherty P. 1996. Fibroblast growth factor
receptor function is required for the orderly projection of ganglion cell
axons in the developing mammalian retina. Mol Cell Neurosci 8:120 –
128.
Buckley NE, McCoy KL, Mezey E, Bonner T, Zimmer AM, Felder CC, Glass
M, Zimmer A. 2000. Immunomodulation by cannabinoids is absent in
mice deficient for the CB2 cannabinoid receptor. Eur J Pharmacol
396:141–149.
Chevaleyre V, Castillo PE. 2003. Heterosynaptic LTD of hippocampal
gabaergic synapses: a novel role of endocannabinoids in regulating
excitability. Neuron 38:461– 472.
Cravatt BF, Prospero-Garcia O, Siuzdak G, Gilula NB, Henriksen SJ,
Boger DL, Lerner RA. 1995. Chemical characterization of a family of
brain lipids that induce sleep. Science 268:1506 –1509.
Cravatt BF, Giang DK, Mayfield SP, Boger DL, Lerner RA, Guilula NB.
The Journal of Comparative Neurology
420
1996. Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides. Nature 384:83– 87.
Cravatt BF, Demarest K, Patricelli MP, Bracey MH, Giang DK, Martin BR,
Lichtman AH. 2001. Supersensitivity to anandamide and enhanced
endogenous cannabinoid signaling in mice lacking fatty acid amide
hydrolase. Proc Natl Acad Sci U S A 98:9371–9376.
Cristino L, De Petrocellis L, Pryce G, Baker D, Guglielmotti V, Di Marzo D.
2006. Immunohistochemical localization of cannabinoid type 1 and
vanilloid transient receptor potential vanilloid type 1 receptor in the
mouse brain. Neuroscience 139:1405–1415.
Dewey WL. 1986. Cannabinoid pharmacology. Pharmacol Rev 38:151–178.
Diana MA, Marty A. 2004. Endocannabinoid-mediated short-term synaptic
plasticity: depolarization-induced suppression of inhibition (DSI) and
depolarization-induced suppression of excitation (DSE). Br J Pharmacol 142:9 –19.
Diana MA, Levenes C, Mackie K, Marty A. 2002. Short-term retrograde
inhibition of GABAergic synaptic currents in rat Purkinje cells is
mediated by endogenous cannabinoids. J Neurosci 22:200 –208.
Dinh TP, Carpenter D, Leslie FM, Freund TF, Katona I, Sensi SL, Kathuria S, Piomelli D. 2002. Brain monoglyceride lipase participating in
endocannabinoid inactivation. Proc Natl Acad Sci U S A 99:10819 –
10824.
Dinh TP, Kathuria S, Piomelli D. 2004. RNA interference suggests a
primary role for monoacylglycerol lipase in the degradation of the
endocannabinoid 2 arachidonoylglycerol. Mol Pharmacol 66:1260 –
1264.
Egertová M, Elphick MR. 2000. Localisation of cannabinoid receptors in
the rat brain using antibodies to the intracellular C-terminal tail of
CB1. J Comp Neurol 422:159 –171.
Egertová M, Giang DK, Cravatt BF, Elphick MR. 1998. A new perspective
on cannabinoid signalling: complementary localization of fatty acid
amide hydrolase and the CB1 receptor in rat brain. Proc R Soc Lond B
Biol Sci 265:2081–2085.
Egertová M, Cravatt BF, Elphick MR. 2003. Comparative analysis of fatty
acid amide hydrolase and CB(1) cannabinoid receptor expression in the
mouse brain: evidence of a widespread role for fatty acid amide hydrolase in regulation of endocannabinoid signaling. Neuroscience 119:
481– 496.
Ferrer B, Bermudez-Silva FJ, Bilbao A, Alvarez-Jaimes L, Sanchez-Vera I,
Giuffrida A, Serrano A, Baixeras E, Khaturia S, Navarro M, Parsons
LH, Piomelli D, Rodriguez de Fonseca F. 2007. Regulation of brain
anandamide by acute administration of ethanol. Biochem J 404:97–
104.
Fu J, Gaetani S, Oveisi F, Lo Verme J, Serrano A, Rodriguez De Fonseca
F, Rosengarth A, Luecke H, Di Giacomo B, Tarzia G, Piomelli D. 2003.
Oleylethanolamide regulates feeding and body weight through activation of the nuclear receptor PPAR-alpha. Nature 425:90 –93.
Gerdeman GL, Ronesi J, Lovinger DM. 2002. Postsynaptic endocannabinoid release is critical to long-term depression in the striatum. Nat
Neurosci 5:446 – 451.
Giang DK, Cravatt BF. 1997. Molecular characterization of human and
mouse fatty acid amide hydrolases. Proc Natl Acad Sci U S A 94:2238 –
2242.
Giuffrida A, Parsons LH, Kerr TM, Rodriguez de Fonseca F, Navarro M,
Piomelli D. 1999. Dopamine activation of endogenous cannabinoid signaling in dorsal striatum. Nat Neurosci 2:358 –363.
Glass M, Dragunow M, Faull RL. 1997. Cannabinoid receptors in the
human brain: a detailed anatomical and quantitative autoradiographic
study in the fetal, neonatal and adult human brain. Neuroscience
77:299 –318.
Gong JP, Onaivi ES, Ishiguro H, Liu QR, Tagliaferro PA, Brusco A, Uhl
GR. 2006. Cannabinoid CB2 receptors: immunohistochemical localization in rat brain. Brain Res 1071:10 –23.
Goparaju SK, Ueda N, Yamaguchi H, Yamamoto S. 1998. Anandamide
amidohydrolase reacting with 2-arachidonoylglycerol, another cannabinoid receptor ligand. FEBS Lett 422:69 –73.
Gulyas AI, Cravatt BF, Bracey MH, Dihn TP, Piomelli D, Boscia F, Freund
TF. 2004. Segregation of two endocannabinoid-hydrolyzing enzymes
into pre- and postsynaptic compartments in the rat hippocampus,
cerebellum and amygdala. Eur J Neurosci 20:441– 458.
Hashimotodani Y, Ohno-Shosaku T, Kano M. 2007. Presynaptic monoacylglycerol lipase activity determines basal endocannabinoid signaling in
the hippocampus. J Neurosci 27:1211–1219.
Herkenham M. 1995. Localization of cannabinoid receptors in brain and
J. SUÁREZ ET AL.
periphery. In: Pertwee R, editor. Cannabinoid receptors. London: Academic Press. p 145–166.
Herkenham M, Groen BGS, Lynn AB, deCosta BR, Richfield EK. 1991a.
Neuronal localization of cannabinoid receptors and second messengers
in mutant mouse cerebellum. Brain Res 552:301–310.
Herkenham M, Lynn A, Johnson MR, Melvin LS, de Costa BR, Rice KC.
1991b. Characterization and localization of cannabinoid receptors in
rat brain: a quantitative in vitro autoradiographic study. J Neurosci
11:563–583.
Hohmann AG, Suplita RL, Bolton NM, Neely MH, Fegley D, Mangieri R,
Krey JF, Walker JM, Holmes PV, Crystal JD, Duranti A, Tontini A,
Mor M, Tarzia G, Piomelli D. 2005. An endocannabinoid mechanism for
stress-induced analgesia. Nature 435:1108 –1112.
Kawamura Y, Fukaya M, Maejima T, Yoshida T, Miura E, Watanabe M,
Ohno-Shosaku T, Kano M. 2006. The CB1 cannabinoid receptor is the
major cannabinoid receptor at excitatory presynaptic sites in the hippocampus and cerebellum. J Neurosci 26:2991–3001.
Kishimoto Y, Kano M. 2006. Endogenous cannabinoid signaling through
the CB1 receptor is essential for cerebellum-dependent discrete motor
learning. J Neurosci 26:8829 – 8837.
Kreitzer AC, Regehr WG. 2001. Cerebellar depolarization-induced suppression of inhibition is mediated by endogenous cannabinoids. J Neurosci 21:1–5RC174.
Ledent C, Valverde O, Cossu G, Petitet F, Aubert JF, Beslot F, Böhme GA,
Imperato A, Pedrazzini T, Roques BP, Vassart G, Fratta W, Parmentier M. 1999. Unresponsiveness to cannabinoids and reduced addictive
effects of opiates in CB1 receptor knockout mice. Science 283:401– 404.
Leung D, Saghatelian A, Simon GM, Cravatt BF. 2006. Inactivation of
N-acyl phosphatidylethanolamine phospholipase D reveals multiple
mechanisms for the biosynthesis of endocannabinoids. Biochemistry
45:4720 – 4726.
Liu J, Wang L, Harvey-White J, Huang BX, Kim HY, Luquet S, Palmiter
RD, Krystal G, Rai R, Mahadevan A, Razdan RK, Kunos G. 2007.
Multiple pathways involved in the biosynthesis of anandamide. Neuropharmacology [June 6 E-pub ahead of print].
Lu Q, Straiker A, Lu Q, Maguire G. 2000. Expression of CB2 cannabinoid
receptor mRNA in adult rat retina. Vis Neurosci 17:91–95.
Mailleux P, Vanderhaeghen JJ. 1992. Distribution of neuronal cannabinoid receptor in the adult rat brain: a comparative receptor binding
radioautography and in situ hybridization histochemistry. Neuroscience 48:655– 668.
Matsuda LA, Bonner TI, Lolait SJ. 1993. Localization of cannabinoid
receptor mRNA in rat brain. J Comp Neurol 327:535–550.
Mechoulam R, Ben-Shabat S, Hanus L, Ligumsky M, Kaminski NE, Schatz
AR, Gopher A, Almog S, Martin BR, Compton DR, Pertwee RG, Griffin
G, Bayewitch M, Barg J, Vogel Z. 1995. Identification of an endogenous
2-monoglyceride, present in canine gut, that binds to cannabinoid
receptors. Biochem Pharmacol 50:83–90.
Muccioli GG, Xu C, Odah E, Cudaback E, Cisneros JA, Lambert DM, Lopez
Rodriguez ML, Bajjalieh S, Stella N. 2007. Identification of a novel
endocannabinoid-hydrolyzing enzyme expressed by microglial cells.
J Neurosci 2007 27:2883–2889.
Okamoto Y, Morishita J, Tsuboi K, Tonai T, Ueda N. 2004. Molecular
characterization of a phospholipase D generating anandamide and its
congeners. J Biol Chem 279:5298 –305.
Onaivi ES, Ishiguro H, Gong JP, Patel S, Perchuk A, Meozzi PA, Myers L,
Mora Z, Tagliaferro P, Gardner E, Brusco A, Akinshola BE, Liu QR,
Hope B, Iwasaki S, Arinami T, Teasenfitz L, Uhl GR. 2006. Discovery
of the presence and functional expression of cannabinoid CB2 receptors
in brain. Ann N Y Acad Sci 1074:514 –536.
Patel S, Hillard CJ. 2001. Cannabinoid CB(1) receptor agonists produce
cerebellar dysfunction in mice. J Pharmacol Exp Ther 297:629 – 637.
Paxinos G, Watson C. 1998. The rat brain in stereotaxic coordinates, 24th
ed. London: Academic Press.
Pazos MR, Nunez E, Benito C, Tolon RM, Romero J. 2004. Role of the
endocannabinoid system in Alzheimer’s disease: new perspectives. Life
Sci 75:1907–1915.
Pettit DAD, Harrison MP, Olson JM, Spencer RF, Cabral GA. 1998. Immunohistochemical localization of the neural cannabinoid receptor in
rat brain. J Neurosci Methods 51:391– 402.
Piomelli D, Giuffrida A, Calignano A, Rodrı́guez de Fonseca F. 2000. The
endocannabinoid system as a target for therapeutic drugs. Trends
Pharmacol Sci 21:218 –224.
Raymond JL, Lisberger SG, Mauk MD. 1996. The cerebellum: a neuronal
learning machine? Science 272:1126 –1131.
The Journal of Comparative Neurology
ENDOCANNABINOID AND CEREBELLUM
Robbe D, Kopf M, Remaury A, Bockaert J, Manzoni OJ. 2002. Endogenous
cannabinoids mediate long-term synaptic depression in the nucleus
accumbens. Proc Natl Acad Sci U S A 99:8384 – 8348.
Rodrı́guez de Fonseca F, Del Arco I, Martin-Calderon JL, Gorriti MA,
Navarro M. 1998. Role of the endogenous cannabinoid system in the
regulation of motor activity. Neurobiol Dis 5:483–501.
Rodriguez de Fonseca F, Navarro M, Gomez R, Escuredo L, Nava F, Fu J,
Murillo-Rodriguez E, Giuffrida A, LoVerme J, Gaetani S, Kathuria S,
Gall C, Piomelli D. 2001. An anorexic lipid mediator regulated by
feeding. Nature 414:209 –212.
Rodrı́guez de Fonseca F, Del Arco I, Bermúdez-Silva FJ, Bilbao A, Cippitelli A, Navarro M. 2005. The endocannabinoid system: physiology and
pharmacology. Alcohol Alcoholism 40:2–14.
Romero J, Hillard CJ, Calero M, Rábano A. 2002. Fatty acid amide hydrolase localization in the human central nervous system: an immunohistochemical study. Brain Res Mol Brain Res 100:85–93.
Safo PK, Regehr WG. 2005. Endocannabinoids control the induction of
cerebellar LTD. Neuron 48:647– 659.
Sheng WS, Hu S, Min X, Cabral GY, Lokensgard JR, Peterson PK. 2005.
Synthetic cannabinoid WIN55212-2 inhibits generation of inflammatory mediators by IL-IB-stimulated human astrocytes. Glia 49:211–
219.
Skaper SD, Buriani A, Dal Toso R, Petrelli L, Romanello S, Facci L, Leon
A. 1996. The ALIAmide palmitoylethanolamide and cannabinoids, but
not anandamide, are protective in a delayed postglutamate paradigm of
excitotoxic death in cerebral granule neurons. Proc Natl Acad Sci U S
A 93:3984 –3989.
Skosnik PD, Edwards CR, O’Donnell BF, Steffen A, Steinmetz JE, Hetrick
WP. 2007. Cannabis use disrupts eyeblink conditioning: evidence for
cannabinoid modulation of cerebellar-dependent learning. Neuropsychopharmacology [July 18 E-pub ahead of print].
Simon GM, Cravatt BF. 2006. Endocannabinoid biosynthesis proceeding
through glycerophospho-N-acyl ethanolamine and a role for alpha/
beta-hydrolase 4 in this pathway. J Biol Chem 281:26465–26472.
Stella N, Schweitzer P, Piomelli D. 1997. A second endogenous cannabinoid
that modulates long-term potentiation. Nature 388:773–778.
Sugiura T, Kondo S, Sukagawa A, Nakane S, Shinoda A, Itoh K, Yamashita A, Waku K. 1995. 2-Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain. Biochem Biophys Res Commun 215:89 –97.
Thomas EA, Cravatt BF, Danielson PE, Gilula NB, Sutcliffe JG. 1997.
Fatty acid amide hydrolase, the degradative enzyme for anandamide
and oleamide, has selective distribution in neurons within the rat
central nervous system. J Neurosci Res 50:1047–1052.
Tsou K, Brown S, Sañudo-Peña MC, Mackie K, Walker JM. 1998. Immu-
421
nohistochemical distribution of cannabinoid CB1 receptors in the rat
central nervous system. Neuroscience 83:393– 411.
Valenzano KJ, Tafesse L, Lee G, Harrison JE, Boulet JM, Gottshall SL,
Mark L, Pearson MS, Miller W, Shan S, Rabadi L, Rotshteyn Y, Chaffer
SM, Turchin PI, Elsemore DA, Toth M, Koetzner L, Whiteside GT.
2005. Pharmacological and pharmacokinetic characterization of the
cannabinoid receptor 2 agonist, GW405833, utilizing rodent models of
acute and chronic pain, anxiety, ataxia and catalepsy. Neuropharmacology 48:658 – 672.
Van Sickle MD, Duncan M, Kingsley PJ, Mouihate A, Urbani P, Mackie K,
Stella N, Makriyannis A, Piomelli D, Davison JS, Marnett LJ, Di
Marzo V, Pittman QJ, Patel KD, Sharkey KA. 2005. Identification and
functional characterization of brainstem cannabinoid CB2 receptors.
Science 310:329 –332.
Vila A, Rosengarth A, Piomelli D, Cravatt B, Marnett LJ. 2007. Hydrolysis
of prostaglandin glycerol esters by the endocannabinoid-hydrolyzing
enzymes, monoacylglycerol lipase and fatty acid amide hydrolase. Biochemistry 46:9578 –9585.
Wager-Miller J, Westenbroek R, Mackie K. 2002. Dimerization of G
protein-coupled receptors: CB1 cannabinoid receptors as an example.
Chem Phys Lipids 121:83– 89.
Wei BQ, Mikkelsen TS, McKinney MK, Lander ES, Cravatt BF. 2006. A
second fatty acid amide hydrolase with variable distribution among
placental mammals. J Biol Chem. 281:36569 –36578.
Williams EJ, Walsh FS, Doherty P. 2003. The FGF receptor uses the
endocannabinoid signaling system to couple to an axonal growth response. J Cell Biol 160:481– 486.
Wilson RI, Nicoll RA. 2001. Endogenous cannabinoids mediate retrograde
signalling at hippocampal synapses. Nature 410:588 –592.
Wilson RI, Nicoll RA. 2002. Endocannabinoid signaling in the brain. Science 296:678 – 682.
Wilson RI, Kunos G, Nicoll RA. 2001. Presynaptic specificity of endocannabinoid signaling in the hippocampus. Neuron 31:453– 462.
Witting A, Chen L, Cudaback E, Straiker A, Walter L, Rickman B, Moller
T, Brosnan C, Stella N. 2006. Experimental autoimmune
encephalomyeli-tis disrupt endocannabinoid-mediated neuroprotection. Proc Natl Acad Sci U S A 103:6362– 6367.
Yoshida T, Fukaya M, Uchigashima M, Miura E, Kamiya H, Kano M,
Watanabe M. 2006. Localization of diacylglycerol lipase-␣ around
postsynaptic spine suggests close proximity between production site of
an endocannabinoid, 2-arachidonoyl-glycerol, and presynaptic cannabinoid CB1 receptor. J Neurosci 26:4740 – 4751.
Zhang J, Hoffert C, Vu HK, Groblewski T, Ahmad S, O’Donnell D. 2003.
Induction of CB2 receptor expression in the rat spinal cord of neuropathic but not inflammatory chronic pain models. Eur J Neurosci
17:2750 –2754.