immunohistochemical description of the endogenous … · 2008. 10. 2. · immunohistochemical...

Immunohistochemical Description of theEndogenous Cannabinoid System in the

Rat Cerebellum and Functionally RelatedNuclei

JUAN SUAREZ,1* FRANCISCO JAVIER BERMUDEZ-SILVA,1 KEN MACKIE,2

CATHERINE LEDENT,3 ANDREAS ZIMMER,4 BENJAMIN F. CRAVATT,5

AND FERNANDO RODRIGUEZ DE FONSECA1*1Laboratorio de Medicina Regenerativa, Fundacion IMABIS, 29010 Malaga, Spain

2Departments of Psychological and Brain Sciences, Indiana University, Bloomington, IN 474013Institut de Recherche Interdisciplinaire en Biologie Humaine et Moleculaire, Universite Libre

de Bruxelles, B-1050 Bruxelles, Belgium4Institute of Molecular Psychiatry, University of Bonn, 53115 Bonn, Germany

5Chemical Biology and Cell Biology, The Scripps Research Institute,La Jolla, California 92037

ABSTRACTWe 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) andthe two functionally related nuclei, the vestibular nuclei and the inferior olive. These proteinsinclude the two main cannabinoid receptors (CB1 and CB2), the enzymes involved in canna-binoid biosynthesis (DAGL�, DAGL�, and NAPE-PLD), and the endocannabinoid-degradating enzymes (FAAH and MAGL). With regard to the cerebellar cortex, these dataconfirm several published reports on the distribution of cannabinoid CB1 receptors, DAGL�,MAGL, and FAAH, which suggests a role of endocannabinoids as retrograde messengers inthe synapses of the Purkinje cells by either parallel fibers of granule cells or climbing fibersfrom the inferior olive or GABAergic interneuron. Additionally, we describe the presence ofCB2 receptors in fibers related to Purkinje somata (Pinceau formations) and dendrites(parallel fibers), suggesting a potential role of this receptor in the retrograde cannabinoidsignaling. A remarkable finding of the present study is the description of the differentelements of the endogenous cannabinoid system in both the main afferent nuclei to thecerebellar cortex (the inferior olive) and the efferent cerebellar pathway (the deep cerebellarnuclei). The presence of the endogenous cannabinoid system at this level establishes the basisfor 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 disordersaffecting 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 Internetat http://www.interscience.wiley.com/jpages/0021-9967/suppmat.

Grant sponsor: Consejerıa de Salud (Junta de Andalucıa); Grant num-ber: PI-0220; MEC; Grant number: SAF 2004/07762; Grant sponsor: Insti-tuto de Salud Carlos III; Grant number: 07/1226; Grant number: 07/0880;Grant sponsor: Plan Nacional Sobre Drogas; Grant sponsor: Consejeria deInnovacion Ciencia y Empresa (Junta de Andalucıa); Grant number: RE-DES RTA RD06/001; Grant sponsor: 5th Framework Programme; Grantnumber: TARGALC QLRT-2001-01048.

*Correspondence to: Juan Suarez and Fernando Rodrıguez de Fonseca,Laboratorio de Medicina Regenerativa, Fundacion IMABIS, Avenida CarlosHaya 82, 29010 Malaga, Spain. E-mail: [email protected];[email protected]

Received 21 February 2007; Revised 14 August 2007; Accepted 28 April2008

DOI 10.1002/cne.21774Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 509:400–421 (2008)

© 2008 WILEY-LISS, INC.

Analysis of the CB1 receptor expression in the rat brainby in situ hybridization histochemistry and immunocyto-chemistry has provided important insights into the func-tional neuroanatomy of the endocannabinoid system (Mat-suda et al., 1993; Pettit et al., 1998; Egertova and Elphick,2000; Van Sickle et al., 2005; Gong et al., 2006). Electro-physiological studies and the finding of CB1 receptor inthe cerebellar synapses suggest that endocannabinoidsact as retrograde messengers in the cerebellum. This roleof the endocannabinoid system is confirmed by the findingof different types of endocannabinoid-mediated synapticplasticity, including both short-term [depolarization-induced suppression of inhibition (DSI) and excitation(DSE)] and the more permanent long-term depression(LTD; for review see Wilson and Nicoll, 2002; Diana andMarty, 2004; Safo and Regehr, 2005). The presence of theendocannabinoid CB1 receptor in the cerebellum impliesthat the endocannabinoid system plays a central role inreal-time regulation of movement and neuroadaptationsunderlying motor control and motor learning. In fact, rel-evant pharmacological actions of exogenously adminis-tered cannabinoids are ataxia and catalepsy (Rodrıguez deFonseca et al., 1998) and modulation of eye blink condi-tioning (Kishimoto and Kano, 2006; Skosnik et al., 2007).

CB1 receptors are located in axon terminals of parallelfibers of cerebellar granular cells and climbing fibers ofinferior olive neurons that provide excitatory input onPurkinje cells. In addition, CB1 receptors are located inaxon terminals of cerebellar basket and stellate cells pro-viding inhibitory input on Purkinje cells (Mailleux andVanderhaeghen, 1992; Matsuda et al., 1993; Tsou et al.,1998; Egertova and Elphick, 2000; Cristino et al., 2006;Kawamura et al., 2006). Modulation of excitatory andinhibitory input on Purkinje cells by the endocannabinoidsystem allows Purkinje cells to refine the output of motorresponses from cerebellum.

However, so far, little information is available on thepresence 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; Egertova et al., 2003; Okamoto et al.,2004). Recently, immunohistochemical studies revealedthe distribution of CB2 receptor in the rat brain, particu-larly in cerebellum and hippocampus (Van Sickle et al.,2005; Gong et al., 2006). The finding of CB2 receptor in thecerebellum suggests the need to reevaluate the effects ofexogenous and endogenous cannabinoids on neurotrans-mission.

There are some studies demonstrating the presence ofthe cannabinoid degradation enzymes FAAH (Cravatt etal., 1995, 1996; Egertova et al., 1998; Goparaju et al.,1998; Tsou et al., 1998) and MAGL (Dihn et al., 2002) inthe brain. Other studies report a general analysis ofFAAH expression in specific neuronal population of mouseand human brain, including cerebellar Purkinje cells, neu-rons of cerebellar nuclei and inferior olive, neocortical andhippocampal pyramidal neurons, and striatal projectingneurons. These localizations of FAAH suggest a comple-mentary distribution with CB1 expression in these brainregions (Romero et al., 2002; Egertova et al., 2003). North-ern blot and in situ hybridization analyses reveal thatMAGL is heterogeneously expressed in some brain areas,including hippocampus, cortex, cerebellum, and anteriorthalamus, where CB1 receptor is also expressed, indicat-ing a presynaptic localization of the enzyme (Dihn et al.,2002).

Indeed, the recent identification of 2-AG and AEA bio-synthesis and release enzymes, DAGL� and DAGL�(Bisogno et al., 2003), and NAPE-PLD (Okamoto et al.,2004) has provided new insights on the endocannabinoidsignaling system in the brain. Pharmacological studiessuggest that DAGL and NAPE-PLD activity is requiredfor inhibition of �-aminobutyric acid (GABA)-ergic trans-mission by glutamatergic input (Chevaleyre and Castillo,2003). Additionally, DAGL activity is related to axonalgrowth and guidance during development (Brittis et al.,1996; Williams et al., 2003). The expression of DAGLisozymes (� and � forms) changes during development ofthe brain; that is, they are expressed in axonal tracts ofthe embryo and then in dendritic fields of the adult mousebrain (Bisogno et al., 2003). In the adult mouse cerebel-

Abbreviations

b basket cellc collateralsCbCx cerebellar cortexCbN cerebellar nucleicf climbing fibersDEn dorsal endopiriform nucleusDG dentate gyrusg granular cellG golgi cellGrL granular layerHi hippocampusicp inferior cerebellar peduncleIntA interposed cerebellar nucleus, part anteriorIntDL interposed cerebellar nucleus, dorsolateral humpIntDM interposed cerebellar nucleus, dorsomedial crestIntP interposed cerebellar nucleus, part posteriorIntPPC interposed cerebellar nucleus, posterior parvicellular partIO inferior oliveIOD inferior olive, dorsal nucleusIODM inferior olive, dorsomedial cell groupIOM inferior olive, medial nucleusIOPr inferior olive, principal nucleus

LA lateral amygdaloid nucleusLat lateral (dentate) cerebellar nucleusLatPC lateral cerebellar nucleus, parvicellular partLVe lateral vestibular nucleusmf mossy fibersMed medial (fastigial) cerebellar nucleusMedDL medial cerebellar nucleus, dorsolateral protuberanceML molecular layerMVe medial vestibular nucleusMVeMC medial vestibular nucleus, magnocellular partMVePC medial vestibular nucleus, parvicellular partP purkinje cellpf parallel fiberspif pinceau formationpy pyramidal tracts superficial stellate cellscp superior cerebellar pedunclesp5 spinal trigeminal tractSpVe spinal vestibular nucleusSuVe superior vestibular nucleusVeCb vestibulocerebellar nucleusVeN vestibular nuclei

The Journal of Comparative Neurology

401ENDOCANNABINOID AND CEREBELLUM

lum, the substantial down-regulation of DAGL� contrastswith the strong staining of DAGL� in the Purkinje celldendritic field (Bisogno et al., 2003).

For this study, we selected NAPE-PLD as theanandamide-synthesizing enzyme. A recent paper de-scribed the molecular characterization of NAPE-PLD; itsauthors noted the presence of NAPE-PLD activity inmouse brain, including the cerebellum (Okamoto et al.,2004). We have also detected NAPE-PLD expression in ratcerebellum (Ferrer et al., 2007). However, its immunohis-tochemical localization in the brain has not been analyzedso far. It is important to note that NAPE-PLD is not theonly source for anandamide in the brain (Leung et al.,2006) but is the first of a series of enzymes capable ofgenerating anandamide from the membrane precursorN-arachidonyl-phosphatidyl ethanolamide (NAPE), suchas �/� hydrolase 4, lyso-PLD, lyso-PLC, and phosphatasessuch as PTPN22 (Leung et al., 2006; Simon and Cravatt,2006; Liu et al., 2007). However, despite discrepancies insubstrate specificity and the lack of specific test for theactivation of NAPE-PLD in neural circuits (Liu et al.,2007), NAPE-PLD remains an important source of anan-damide in the brain. Therefore, the molecular character-ization of new synthesis pathways for anandamide in thebrain will determine this important aspect of endocan-nabinoid physiology.

The aim of this study was to determine the distributionof the endocannabinoid receptors CB1 and CB2 and theendocannabinoid biosynthesis and degradation enzymesDAGL�, DAGL�, NAPE-PLD, FAAH, and MAGL by im-munohistochemistry in the rat cerebellum (cerebellar cor-tex and cerebellar nuclei) and other functionally relatedbrain areas, such as vestibular nuclei and inferior olive.The exact localization of these cannabinoid enzymes andreceptors in the rat cerebellum may facilitate a neuroana-tomical framework for the analysis of the physiologicalroles of the endocannabinoid signaling system.

MATERIAL AND METHODS

Generation of NAPE-PLD-, DAGL�-, DAGL�-,and MAGL-specific antibodies

We have generated polyclonal rabbit antibodies againstproteins of the cannabinoid machinery. Immunizing pep-tides were 1) a 13-amino-acid (aa) peptide comprising partof both the C-terminal and the N-terminal regions ofNAPE-PLD (MDENSCDKAFEET); 2) a 16-aa peptidefrom the C-terminal region of DAGL� (CGASPTKQDDL-VISAR); 3) a 16-aa peptide from an internal sequence ofDAGL� (SSDSPLDSPTKYPTLC); 4) a 15-aa peptide fromthe N-terminal region of MAGL (SSPRRTPQNVPYQDL);5) a 73-aa peptide (401–473) from the C-terminal region ofCB1 receptor; and 6) a 14-aa peptide (328–342) from theC-terminal region of CB2 receptor. We employed a chi-meric sequence peptide as immunogen for NAPE-PLDantibody generation. The aim of this chimeric constructionwas to contain two distant epitopes exposed in the nativeprotein because one of them belongs to the N-terminal andthe other to the C-terminal region of the protein, bothregions having random coil structures. NAPE-PLD,DAGL�, and DAGL� peptides were synthesized and cou-pled to keyhole limpet hemocyanin (KLH; JPT PeptideTechnologies, Berlin, Germany). The three peptides wereinjected into rabbits (two animals per peptide), according

to standard protocols for generation of antisera, with theIgG fraction subsequently purified by means of a protein Acolumn (Sigma, St. Louis, MO). MAGL antibody was pro-duced in the laboratory of Dr. D. Piomelli (Dihn et al.,2002). MAGL peptide was synthesized and coupled toKLH by addition of a cysteine at the peptide N terminus(United Biochemical, Seattle, WA). The conjugated pep-tide was injected into two rabbits to generate antisera(Strategic Biosolutions, Ramona, CA). The peptide wasthen conjugated to an agarose column and the antiserumpurified according to the manufacturer’s instructions(AminoLink; Pierce Endogen, Rockford, IL). For CB1 andCB2 antibody generation, we injected rabbits with a fusionprotein composed of glutathione-S transferase (GST) andCB1 residues 401–473 or CB2 residues 328–342, usingconventional techniques. Polyclonal antisera and purifiedantibodies specific for CB1 and CB2 were collected byaffinity chromatography against both GST and immuniz-ing fusion protein.

Immunohistochemistry

We have evaluated the presence of CB1 and CB2 recep-tors, FAAH, MAGL, DAGL�, DAGL�, and NAPE-PLD inthe adult rat cerebellum by immunohistochemistry. Ma-nipulation of animals was in accordance with the Euro-pean Communities Council Directives (86/609/EEC) onthe treatment of experimental animals.

Adult Wistar rats (n � 6; 300 g) were deeply anesthe-tized with 2,2,2-tribromoethanol (300 mg/kg i.p.) andbriefly transcardially perfused with 0.1 M phosphate-buffered saline (PBS; pH 7.4), followed by 4% paraformal-dehyde in PBS at 4°C for 30 minutes. Brains were dis-sected, postfixed overnight in buffered paraformaldehydeat 4°C, equilibrated with 30% sucrose in PBS at 4°C,frozen, and cut into 40-�m-thick-transverse or sagittalsections using a sliding microtome. We then collected 19alternate series of sections from each rat brain to processthe seven antibodies and Nissl staining.

Free-floating sections were first incubated in H2O dis-tilled containing 50 mM sodium citrate (pH 9) for 30minutes at 80°C, followed by several washes in PBS.Then, we incubated sections with 3% hydrogen peroxide inPBS for 20 minutes at room temperature to inhibit endog-enous peroxidase, followed by three times in PBS.

We have used seven primary antibodies. The anti-CB1was developed in rabbits by using a fusion protein asimmunogen containing 73 amino acid residues (401–473)of the rat CB1 receptor (Wager-Miller et al., 2002); theanti-CB2 was produced in rabbits by using a fusion proteincontaining 14 amino acid residues (328–342) from rat CB2receptor; the anti-FAAH was developed in rabbits by us-ing a synthetic peptide corresponding to 561–579 aminoacid fragment of rat fatty acid amine hydrolase conjugatedto KLH as immunogen (Cayman Chemical; catalog No.101600, lot. No. 157878). The anti-NAPE-PLD, anti-DAGL�, anti-DAGL�, and anti-MAGL were developed inrabbits as described above. Sections were incubated in thediluted primary antibody (anti-CB1, diluted 1:500; anti-rat CB2, 1:500; anti-rat FAAH, 1:200; anti-MAGL, 1:200;anti-DAGL�, 1:500; anti-DAGL�, 1:200; and anti-NAPE-PLD, 1:400) overnight at room temperature. After threewashes in PBS, the sections were incubated in a biotinyl-ated donkey anti-rabbit immunoglobulin (Amersham, Lit-tle Chalfont, England) diluted 1:500 for 1 hour, washedagain in PBS, and incubated in ExtrAvidin peroxidase


402 J. SUAREZ ET AL.

(Sigma) diluted 1:2,000 for 1 hour. We revealed immuno-labeling 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 roomtemperature. After the sections had been washed in PBS,they were mounted on gelatinized slides, air dried, dehy-drated in ethanol, cleared in xylene, and coverslipped withEukitt mounting medium (Kindler GmBH and Co.,Freiburg, Germany).

Digital photographs were taken on an Olympus BX41microscope equipped with an Olympus DP70 digital cam-era. Digital images were adjusted for brightness/contrastin Adobe Photoshop (Adobe, San Jose, CA), and the figureswere mounted and labelled in Adobe PageMaker.

Antibody specificity and controls

We performed Western blot analyses to demonstratethat CB1, CB2, FAAH, MAGL, DAGL�, DAGL�, andNAPE-PLD antibodies recognized the corresponding anti-gen in the rat cerebellum. To perform Western blot anal-ysis, we used fresh tissue from Wistar male rats. Animalswere 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 untiluse. Membrane extracts of rat cerebellum were preparedin HEPES 50 mM (pH 8)-sucrose 0.32 M buffer by using ahomogenizer. The homogenate was centrifuged at 800g for10 minutes at 4°C and the supernatant centrifuged at40,000g for 30 minutes. We resuspended the pellets inHEPES 50 mM buffer and potterized using a homoge-nizer.

For immunoblotting, equivalent amounts of membraneproteins (45 �g) from rat cerebellum were separated by10% sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE), electroblotted onto nitrocellulosemembranes, and controlled by Ponceau red staining. ForDAGL� immunoblotting, we used stringent conditionswith the addition of dithiothreitol 0.15 mM in the samplebuffer. We preincubated blots with a blocking buffer con-taining PBS, 0.1% Tween 20, and 2% albumin fraction Vfrom bovine serum (Merck) at room temperature for 1hour. For protein detection, each blotted membrane lanewas 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) antibodiesand diluted in PBS containing 0.1% Tween 20 and 2%albumin fraction V from bovine serum at room tempera-ture overnight. After extensive washing in PBS containing1% Tween 20 (PBS-T), a peroxydase-conjugated goat anti-rabbit antibody (Promega, Madison, WI) was added (1:2,500) for 1 hour at room temperature. Biotinylatedmarker proteins with defined molecular weights wereused for molecular weight determination in Western blots(ECL Western Blotting Molecular Weight Markers; Am-ersham). We incubated the corresponding markers lanewith ExtrAvidin peroxidase (Sigma). Membranes werethen subjected to repeated washing in PBS-T and thespecific protein bands visualized by using the enhancedchemiluminiscence technique (ECL; Amersham) andAuto-Biochemi Imaging System (LTF LabortechnikGmbH, Wasserburg/Bodensee, Germany). Western Blotsshowed that each primary antibody detected a protein ofthe expected molecular size (see Fig. 1A).

As additional controls, cerebellar and hippocampal sec-tions from CB1 receptor knockout mice (Ledent et al.,

1999), CB2 receptor knockout mice (Buckley et al., 2000),NAPE-PLD knockout mice (Cravatt et al., 2001), and wild-type controls (n � 2 pairs) were also analyzed. Immuno-histochemical protocol was carried out as described above(anti-CB1, diluted 1:500; anti-rat CB2, 1:500; anti-ratNAPE-PLD, 1:400). We observed that immunostainingwas almost completely absent in CB1 knockout mousebrain, but weak staining was found in the spinal trigem-inal tract at cerebellar levels and in the cerebral peduncleat hippocampal levels (Suppl. Fig. 1). With the exceptionof these features, all of the staining in wild-type brain isspecifically attributable to CB1 expression. We did notobserved labeling in the CB2 receptor knockout mouse orNAPE-PLD receptor knockout mouse, whereas the wild-type mouse showed labelling similar to that of the ratbrain (Suppl. Figs. 2, 3).

We also incubated blotted membrane lanes with theprimary antibody preadsorbed with the immunizing pep-tide CB1 (10 �g/ml), CB2 (10 �g/ml), FAAH (20 �g/ml;Cayman, Ann Harbor, MI), MAGL (13 �g/ml; kindly do-nated by Dr. D. Piomelli), DAGL� (175 �g/ml; JPT; seeabove), DAGL� (32 �g/ml; JPT; see above), and NAPE-PLD (16 �g/ml; JPT; see above) or incubated by replacingthe primary antiserum by 2% albumin fraction V frombovine serum (see Fig. 1A). In addition, we incubatedbrain sections with the primary antibody preadsorbedwith the immunizing peptide under the same conditionsas described above. We did not detect staining under theseconditions (Suppl. Figs. 4, 5).

RESULTS

In the present study, we mapped the expression of thecannabinoid receptors CB1 and CB2; the degradation en-zymes FAAH and MAGL; and the synthesis enzymesDAGL�, DAGL�, and NAPE-PLD in the rat cerebellumand two main cerebellar-related areas, vestibular nucleiand inferior olive. The analysis of the immunostainingpatterns was carried out at transverse and sagittal planesof the adult rat brain by comparing it with the cytoarchi-tecture and with published data on its neurochemistryand connections. The intensity of the immunoreactivityfor each antibody was very similar in all the six rat brainsused in this study. Numerous brain regions such as hip-pocampus, basal ganglia, substantia nigra, and cerebel-lum intensely expressed CB1 receptors, FAAH, MAGL,and DAGL�, in comparison with CB2 receptors, DAGL�,and NAPE-PLD expression. The nomenclature for nucleiand subdivisions referred to in the present study is widelyaccepted and used in the rat brain atlas by Paxinos andWatson (1998). Results for this study are described in thetext and summarized in a rating scale (Table 1). Gray-scale values measured in single cerebellar, vestibular, andolive nuclei are represented on an arbitrary scale of fourlabelling intensities, from “�,” meaning “very low”(slightly above the density measured in sections incubatedin preadsorbed antibody; see Suppl. Figs. 4, 5) to “��,”meaning “very high” (according to the highest signal den-sity in the specimen, i.e., parvicellular part of the medialvestibular nucleus). Previously, we analyzed controls andWestern blot results to demonstrate that CB1, CB2,FAAH, MAGL, DAGL�, DAGL�, and NAPE-PLD antibod-ies recognize the corresponding antigen in the rat cerebel-lum.



Western blot analysis

Western blot analyses of membrane extracts from ratcerebellum revealed CB1 immunostaining as a prominentband at about 60 kD (Fig. 1A, lane 1). CB2 immunostain-ing also showed a prominent band at about 55 kD (Fig. 1A,lane 7). Immunoblots for FAAH and NAPE-PLD revealeda single band with a molecular mass of 63 and 46 kD,respectively (Fig. 1A, lanes 3 and 5, respectively). In ad-dition, under more stringent conditions, DAGL� immuno-blotting showed a prominent band of 76 kD and another,less intense one of 97 kD (Fig. 1A, lane 11). DAGL� im-munoblotting showed an expected band of 120 kD (Fig. 1A,lane 13). Analysis of MAGL immunoreactivity confirmedtwo bands of 37 and 35 kD, but we also observed anadditional band at about 62 kD (Fig. 1A, lane 9). In allcases, the immunoreactive bands were abolished afterabsorption with the immunizing peptides (Fig. 1A, lanes2, 4, 6, 8, 10, 12, 14).

CB1 immunoreactivity

We observed a prominent fiber CB1 labelling surround-ing Purkinje somata and proximal axons that may consistof clustered basket cell axons, called Pinceau formations(Fig. 2B, inset). Numerous immunostained fibers are alsowidespread throughout the branches of the cerebellarwhite 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 representaxons from inferior olive (climbing fibers). We clearly dis-tinguished the molecular layer of cerebellar cortex by anintense CB1 immunoreactivity consisting of a dense net-work of immunostaining fibers and puncta, which mayprincipally correspond to climbing fiber terminals but alsoparallel fibers from granular cells. The climbing fibersrepresented the main external Purkinje afferent innerva-tions originating from the inferior olive (see Fig. 10B). Wedid not observed stained cell bodies in the cerebellar cor-tex.

Cerebellar and vestibular nuclei present a very low CB1immunoreactivity (Fig. 2A,C–F). In general, our results

showed that all cerebellar nuclei (medial, lateral, andinterposed nuclei) contained very weak neuropil. How-ever, it should be noted that numerous fiber tracts crossedthe cerebellar nuclei, which could be distinguished bytheir position and orientation. Some of these fiber bundlescrossed unstained peduncles and formed part of the supe-rior cerebellar peduncle (Fig. 2C,D). Therefore, part ofthese fibers may represent axons from cerebellar nucleithat projected to red nuclei, dorsal thalamus, and motorcortex. Other peduncles of CB1

� fibers coursed dorsallyfrom the inferior cerebellar peduncle to the cerebellarcortex. Most of these fibers constituted axons from theinferior olive, in agreement with the presence of climbingfibers in the molecular layer (Fig. 2E). An intense CB1immunoreactivity consisting of a network of neuropil andpuncta characterized the dorsal portion of the principalnucleus of the inferior olive (IOPr; see Fig. 9A). The pres-ence of numerous CB1

� fibers in the rubroolivary tract(data not shown) suggested that most of the fiber termi-nals in the IOPr originated from the red nucleus. The restof the inferior olivary complex showed very low immuno-reactivity.

Most vestibular nuclei also presented very weak CB1immunoreactivity (Fig. 2C). We distinguished spinal ves-tibular nucleus (SpVe) and the magnocellular part of me-dial vestibular nucleus (MVeMC) from its parvocellularpart (MVePC) by a stronger CB1

� neuropil (Fig. 2C). It isworth noticing that typical giant neurons of the lateralvestibular nuclei (LVe) were moderately CB1 immuno-stained, whereas their dendritic initial segments could beclearly distinguished (Fig. 2E).

CB2 immunoreactivity

The distribution of CB2 immunoreactivity in the cere-bellar cortex was similar to that of CB1 immunoreactivity.As occurred with CB1 immunoreactivity, we observedCB2-immunoreactive (CB2

�) fiber terminals surroundingPurkinje somata and proximal axons (Pinceau forma-tions). Purkinje cells were not CB2 immunoreactive. The

TABLE 1. Immunoreactivity in Cerebellum, Vestibular Nuclei, and Inferior Olive1

CB1 CB2 FAAH MAGL DAGL� DAGL� NAPE-PLD

c f c f c f c f c f c f c f

Cerebellar cortexMolecular layer – �� – �� – �� – �� – �� Granular layer – �� – �� – �� Purkinje cells – – �� Cerebellar nucleiIntA – � – �� – �� IntP – � – �� – �� IntPPC – � – �� – �� – �� IntDL – � – �� – �� IntDM – � – �� – �� Lat – � – �� – �� LatPC – � – �� – �� – �� Med – � – �� – �� MedDL – � – �� – �� Vestibular nucleiLVe �� – �� – �� MVeMC – �� – �� – �� MVePC – � – �� – �� SpVe – �� – �� – �� SuVe – � – �� – �� VeCb – � – �� – �� – �� Inferior olive – �� – �� – ��

1Rating scale of the immunoreactivity of each structure in cells (c) and fibers (f). Symbols are as follows: very high (��), high (��), low (��), very low (�), and withoutimmunoreactivity (–).



molecular layer also contained a dense network of immu-nostained fibers and neuropil, extending from the granu-lar layer to the pial surface of the molecular layer. MostCB2

� fibers of the molecular layer were disposed in par-allel and presented numerous varicosities along their sur-face. Therefore, these fibers may principally representparallel fibers originating from granular cells (Fig. 3B,inset). In contrast to CB1 immunoreactivity, the granularlayer showed moderate neuropil CB2 immunoreactivity.These fibers showed a mossy aspect (mossy fibers) and soprobably may represent axons from pontine nuclei, vestib-ular nuclei, and spinal cord (Fig. 3B). In addition, allsubdivisions of the inferior olive showed a strong CB2immunoreactivity that consisted of a dense network offibers (Fig. 9B). The main olivary afferent innervations

originated from the spinal cord, so these fiber terminals inthe IO may constitute collaterals of the mossy fibers in thecerebellar cortex from the spinal cord.

The CB2 immunoreactivity in the cerebellar and vestib-ular nuclei was different from the CB1 immunoreactivity,especially the prominent neuropil immunoreactivity (Fig.3C–F). Most cerebellar nuclei are characterized by a densenetwork of CB2

� neuropil and fiber terminals that definenumerous unstained fiber bundles and cell profiles (Fig.3D,F). These CB2

� fibers may have the same origin as theCB2

� mossy fibers in the granular layer of the vestibularnuclei, pontine nuclei, and spinal cord, but they may alsoconstitute projections from Purkinje cells. A stronger neu-ropil immunostaining also characterized vestibular nucleisuch as MVePC and SpVe (Fig. 3C,E). Most fiber termi-

Fig. 1. A: Western blots of membrane extracts from rat cerebellumshow prominent immunoreactive bands of expected molecular massesof 60 kD for CB1; 63 kD for FAAH; 46 kD for NAPE-PLD; 55 kD forCB2; 62, 37, and 35 kD for MAGL; 97 and 76 kD for DAGL�; and 120kD for DAGL�. Positions of molecular markers (MW) are indicated atleft. B: Major biochemical pathways for endogenous cannabinoid sig-nalling system. Anandamide (AEA) is released from a membrane lipidprecursor, N-arachidonoyl-phosphatidylethanolamine (NAPE), by theaction of a specific phospholipase D (PLD) activated by depolarization,postsynaptic calcium increases, or G-protein-coupled receptor stimu-lation. The membrane enzyme N-acyltransferase (NAT) catalyses

NAPE biosynthesis, which transfers arachidonic acid from phosphati-dylcholine (PhChol) to the head group of phosphatidylethanolamine(PhEth). Postsynaptic calcium influx and the activation of metabo-tropic receptors coupled to phosphatidyl-inositol-specific phospho-lipase C (PLC) and diacylglycerol (DAG) lipase pathway lead to in-creases in 2-arachidonoylglycerol (2-AG) production.Endocannabinoid signalling includes uptake into cells mediated by atransporter (AT) and hydrolysis by two specific enzymatic systems,the fatty acid amide hydrolase (FAAH) and the monoacylglyceridelipase (MAGL).



Fig. 2. CB1 immunoreactivity in the rat cerebellum and vestibularnuclei. A: General view of a coronal section through the cerebellumand vestibular nuclei. B: High-magnification photomicrographs of thecerebellar cortex showing clustered basket cell axons (pinceau forma-tions) surrounding unstained Purkinje somata (inset). Numerousfibers fill the branches of the cerebellar white matter and dispersefrom the granular layer into the molecular layer (arrowhead in inset).

C: Low-magnification photomicrograph showing CB1 immunoreactiv-ity throughout the cerebellar and vestibular nuclei. Note fiber tractsthat cross the cerebellar nuclei and can be distinguished by theirposition and orientation into the superior (D) and inferior (E) cere-bellar peduncle and the giant neurons in LVe (F). For abbreviationssee list. Scale bars � 1 mm in A; 200 �m in B,C; 100 �m in D–F; 20�m in inset.



Fig. 3. CB2 immunoreactivity in the rat cerebellum and vestibularnuclei. A: General view of a coronal section through the cerebellumand vestibular nuclei. B: Detail of a transverse section of the cerebel-lar cortex showing mossy fibers in the granular layer, parallel fibers inthe molecular layer (arrows), and “pinceau” formations in the Pur-kinje layer (arrowheads). Note the unstained Purkinje somata (in-

set). C: Low-magnification photomicrograph showing CB2 immunore-activity throughout the cerebellar and vestibular nuclei. Note cellprofiles immersed in a dense fiber network in all cerebellar nuclei(D,E) and the intense neuropil immunoreactivity in the MVe (F). Forabbreviations 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.



nals in the vestibular nuclei may constitute projectionsfrom Purkinje cells.

FAAH immunoreactivity

We clearly observed FAAH immunoreactivity in Pur-kinje neurons, which showed intensely stained cell bodiesand a dense network of fibers in the molecular layer that,in sagittal sections, characterized the dendrite tree of thePurkinje cells (Fig. 4B, inset). On the other hand, weobserved an evident FAAH immunoreactivity in a sub-population of granular cells (Fig. 4B) and in numerousneurons of IO (Fig. 9C). Therefore, we can expect thatsome of the FAAH-immunoreactive (FAAH�) puncta ofthe molecular layer probably represent climbing and par-allel fiber terminals. Poor neuropil immunoreactivity inthe granular layer was similar to that of fibers in thecerebellar white matter.

The strong FAAH immunoreactivity observed in all cer-ebellar nuclei was related mainly to the presence of adense meshwork of fibers, consisting of FAAH� punctatelabelling that contained immunoreactive cell bodies (Fig.4C,D). Most likely, these fibers represented dendritic fi-bers. Note the moderate density of FAAH� neurons in theIntDL, MeDL, and Med (Fig. 4C,D) and the stronglyFAAH� fibers in all cerebellar nuclei (Fig. 4C).

However, we observed that most of the vestibular nucleipresented a low immunoreactivity for FAAH (Fig. 4C,E,F),with the exception of LVe, which showed immunoreactiv-ity similar to that of cerebellar nuclei. LVe staining con-sisted of a dense network of fibers disposed between tractsof the juxtarestiform body along with a number of giantsFAAH� neurons spreading from LVe to SpVe (Fig. 4E).Both parts (parvicellular and magnocellular) of the medialvestibular nucleus showed a very low FAAH immuno-staining consisting of a number of FAAH� cells embeddedin a very poorly stained neuropil (Fig. 4F).

MAGL immunoreactivity

MAGL immunostaining of the cortex cerebellumshowed a distinct pattern in the cerebellar layers (Fig.5A). The molecular layer was distinguished by its moder-ate MAGL immunoreactivity consisting of a number ofsmall cells that likely represented basket cells and super-ficial stellate cells, clearly discernable within a moder-ately MAGL-immunoreactive (MAGL�) neuropil (Fig. 5B).Most MAGL� neuropil in the molecular layer seemed torepresent parallel fiber terminals from the granular cells(Fig. 5B) and also probably climbing fibers from IO. Pur-kinje cell bodies were moderately stained, whereas Pur-kinje dendrites were not immunoreactive (Fig. 5B, arrow-heads). A remarkable feature was the MAGLimmunoreactivity of the granular layer, related mainly tothe presence of densely packed, well-stained granularcells and possibly others cell types embedded in a moder-ate neuropil labelling (Fig. 5B). Part of this neuropil likelyconsisted of mossy fibers from spinal, pontine, and vestib-ular nuclei. The outer one-third of the granular layer (withrespect to its radial dimension to the pial surface) con-tained dispersed cells with a stronger staining that likelyrepresented Golgi cells according to their unique locationunder the Purkinje layer (Fig. 5B, inset).

Cerebellar and vestibular nuclei presented numerousstrongly stained MAGL� neurons, showing a perikaryaland dendritic Golgi-like labelling (Fig. 5C–F). Medium-sized 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 giantneurons (more dispersed in SpVe) showing a prominentGolgi-like labelling (Fig. 5E). MVePC showed numeroussmall MAGL� neurons embedded in a moderately immu-noreactive neuropil, in contrast to the large neurons inMVeMC (Fig. 5F). All IO subdivisions presented numer-ous small MAGL� neurons showing less staining thanthose of the Golgi-like labelling of surrounding areas (Fig.9D).

DAGL� immunoreactivity

In contrast to MAGL immunoreactivity, the expressionof DAGL� was particularly prominent in the molecularlayer that clearly corresponded to the dendritic field of thePurkinje cells (Fig. 6A,B). Note the weak DAGL� immu-noreactivity Purkinje cell bodies and the numerous vari-cosities along the dendritic fibers in transverse and sagit-tal views (Fig. 6B, insets b�,b��, respectively). However,the lack of DAGL�� cells in the granular layer and in allsubdivisions of IO (see Fig. 9E) suggests that parallel andclimbing fibers did not present DAGL� immunoreactivity.We also observed typically DAGL�� mossy fibers thatcoursed along the branches of the cerebellar white matterand spread into the granular layer, showing a moderatelyimmunoreactive neuropil (Fig. 6B).

However, we did not observe DAGL� immunoreactivityin cell bodies of cerebellar and vestibular nuclei (Fig.6C–F) or in any other region with mossy fiber projectionsin the granular layer such as the pontine nuclei or thespinal cord (data not shown). Cerebellar and vestibularnuclei presented moderate DAGL� immunoreactivity thatconsisted of a conspicuous network of neuropil and puncta(Fig. 6C–F). In some cerebellar and vestibular regions, thedense neuropil defined numerous profiles of unstained cellbodies (Fig. 6D,F). Of relevance, IntPPC, LatPC, andMVePC were characterized by a prominent neuropilDAGL� immunoreactivity (Fig. 6D,F). Additionally, IOwas also characterized by the presence of intense neuropilimmunoreactivity in contrast to that of surrounding areas(Fig. 9E).

DAGL� immunoreactivity

DAGL� immunostaining in the cerebellar cortex wasconsiderably less pronounced than that of DAGL� (Fig.7A,B). We also observed stained DAGL�� Purkinje cellbodies and a moderately DAGL� immunoreactivity in themolecular layer that may be consistent with the immuno-histochemical description by Bisogno and collaborators(2003) for mouse cerebellum (Fig. 7B). In the granularlayer, DAGL� immunoreactivity was associated mainlywith the presence of immunoreactive neuropil but wasweaker than that of the molecular layer and some dis-persed granular cells (Fig. 7B). Additionally, IO containedabundant DAGL�� neurons (Fig. 9F), so DAGL�� neuro-pil of the molecular layer probably contained parallel andclimbing fibers from granular cells and IO neurons, re-spectively, in contrast to the DAGL� immunoreactivity.

DAGL� immunoreactivity in the cerebellar and vestib-ular nuclei was associated mainly with cell bodies embed-ded in a network of fibers, in contrast to DAGL� immu-noreactivity (Fig. 7C). Most cerebellar nuclei presented aDAGL�� neuronal distribution similar to that of MAGL�

neurons, which is in medium-sized cell bodies homog-



Fig. 4. FAAH immunoreactivity in the rat cerebellum and vestib-ular nuclei. A: General view of a coronal section through the cerebel-lum and vestibular nuclei. B: High-magnification photomicrographsof the cerebellar cortex showing intensely stained Purkinje cell bodiesand the dense network of Purkinje dendritic fibers (inset in B) anddispersed granular cells (arrowheads). C: Low-magnification photomi-

crograph showing FAAH immunoreactivity throughout the cerebellarand vestibular nuclei. Note the low density of FAAH� neurons in theMeDL (D) and the weak immunostaining of the giant neurons in theLVe (E) and the small neurons of the MVePC (F). For abbreviationssee list. Scale bars � 1 mm in A; 100 �m in B,D–F; 200 �m in C; 20�m in inset.



Fig. 5. MAGL immunoreactivity in the rat cerebellum and vestib-ular nuclei. A: General view of a coronal section through the cerebel-lum and vestibular nuclei. B: High-magnification photomicrographsof the cerebellar cortex showing the high density of granular cells,Purkinje somata, and basket and stellate cells homogeneously distrib-uted in the molecular layer. Purkinje dendrites are not immuno-stained (arrowheads). Note the prominent immunoreactivity of a neu-ronal subpopulation into the granular layer that, by its position under

the Purkinje layer, may correspond to Golgi cells (inset in B). C: Low-magnification photomicrograph showing MAGL immunoreactivitythroughout the cerebellar and vestibular nuclei. Note the Golgi-likelabelling of the medium-sized neurons in IntP, IntA, and Lat (D) andthe small neurons in IntPPC (D) and MVePC (F). Giant neurons of theLVe and large neurons of the MVeMC are also intensely immunore-active (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.



Fig. 6. DAGL� immunoreactivity in the rat cerebellum and ves-tibular nuclei. A: General view of a coronal section through the cere-bellum and vestibular nuclei. B: High-magnification photomicro-graphs of the cerebellar cortex showing the Purkinje somata and thedense network of Purkinje dendritic fibers in the molecular layer.Note the numerous varicosities along the dendritic fibers in trans-

verse (b�) and sagittal (b��) views. C: Low-magnification photomicro-graph showing an intense DAGL� immunoreactivity in a network offibers throughout the cerebellar and vestibular nuclei. Note thehigher density of fibers in the IntPPC, LatPC, and MVePC. Scalebars � 1 mm in A; 100 �m in B,D–F; 200 �m in C; 20 �m in b�; 10 �min b��.



Fig. 7. DAGL� immunoreactivity in the rat cerebellum and ves-tibular nuclei. A: General view of a coronal section through the cere-bellum and vestibular nuclei. Stained DAGL�� Purkinje somata andscattered DAGL�� granular cells are observed in the cerebellar cortex(B, arrowheads). C: Low-magnification photomicrograph showing

DAGL� immunoreactivity throughout the cerebellar and vestibularnuclei. Note the small neurons in the IntDM (D) and MVePC (F), incomparison with the larger neurons in the IntA (D) and MVeMC (F),and the giant neurons in LVe (E). For abbreviations see list. Scalebars � 1 mm in A; 100 �m in B,D–F; 200 �m in C.



enously distributed (Fig. 7C,D). However, fewer smallDAGL�� neurons were observed in IntPPC and parts ofLatPC and IntDM compared with the remaining parts ofcerebellar nuclei (Fig. 7C,D). Giant DAGL�� neuronscharacterized LVe (Fig. 7E), whereas small DAGL�� neu-rons immersed in a dense immunoreactive neuropil char-acterized MVePC (Fig. 7F).

NAPE-PLD immunoreactivity

The distribution of the NAPE-PLD immunoreactivitywas quite different from that described above for CB1 inthe cerebellar cortex (Fig. 8A,B). The molecular layer wascharacterized by a moderately immunoreactive neuropil,in contrast to the considerably weaker immunostaining ofthe granular layer, which was partially related to den-dritic fiber from the strongly stained Purkinje cell bodies(Fig. 8B, inset) but also possibly to parallel fibers from anumber of granular cells showing NAPE-PLD immunore-activity (Fig. 8B). In addition, most IO subdivisions pre-sented a number of weakly NAPE-PLD-immunoreactive(NAPE-PLD�) neurons (Fig. 9G), so the molecular layer ofthe cerebellar cortex could also contain NAPE-PLD�

climbing fibers. Note the weakly stained NAPE-PLD�

cells in the molecular layer, which may correspond tobasket and stellate cells (Fig. 8B, inset).

The distribution of NAPE-PLD immunoreactivity in thecerebellar and vestibular nuclei was quite similar to thatof DAGL� immunoreactivity (Figs. 7C, 8C). Most cerebel-lar nuclei consisted of intense neuropil immunoreactivityand a number of moderately labelled NAPE-PLD� neu-rons (Fig. 8C,D). As with DAGL� immunoreactivity, In-tPPC and LatPC showed considerably lower number ofNAPE-PLD� neurons and a less intense neuropil immu-noreactivity than in the remaining cerebellar regions (Fig.8C).

Vestibular nuclei showed well-stained neurons consist-ing of abundant giant NAPE-PLD� neurons in LVe andSpVe (Fig. 8E), and large neurons in MVeMC (Fig. 8F).MVePC also showed numerous stained cell bodies im-mersed in moderately NAPE-PLD� neuropil (Fig. 8F).

DISCUSSION

Here we report the first detailed analysis of the pres-ence and the comparative distribution of functionallyrelevant proteins of the endogenous cannabinoid sys-tem, namely, the two main cannabinoid receptors (CB1and CB2), the enzymes involved in cannabinoid biosyn-thesis (DAGL�, DAGL�, and NAPE-PLD), and twoendocannabinoid-degradating enzymes (FAAH andMAGL) in the rat cerebellum (cerebellar cortex andcerebellar nuclei) and two functionally related nuclei,the vestibular nuclei and the inferior olive. It is impor-tant to note that additional putative endocannabinoidreceptors (i.e., orphan receptor GPR55, vanilloid VR1receptor) and enzymes for biosynthesis and degradationhave been proposed (Leung et al., 2006; Simon andCravatt, 2006; Liu et al., 2007). However, their molec-ular characterization and their contribution to endocan-nabinoid physiology are still under active investigation.

Our results confirm data from previous studies on thepresence and localization of CB1 in the cerebellar cortex.Our study also provides new insight in relation to thelocalization of CB1 and CB2 receptors and FAAH, butprincipally in relation to the presence of MAGL, DAGL�,

DAGL�, and NAPE-PLD in cerebellum and functionallyrelated nuclei that have not been described previously.Additionally, the segregated localization of CB1 and CB2in the cerebellum suggests a complementary distributionof the two receptors associated with the specific distribu-tion of the cannabinoid degradation and biosynthesis en-zymes in the cerebellum.

Because of the described variability of NAPE-PLD, CB1,and CB2 distribution with regard to the different antibod-ies used, we carried out careful control experiments forspecificity. Thus, we have used the NAPE-PLD knockoutmouse, CB1 knockout mouse, CB2 knockout mouse, andWestern blot analyses as additional controls for immuno-histochemistry to characterize the NAPE-PLD, CB1, andCB2 antibodies and demonstrate their antibody specific-ity. We observed that immunostaining was almost com-pletely absent in CB1 knockout mouse brain. However,weak staining was found in the spinal trigeminal tract atcerebellar levels and in the cerebral peduncle at hip-pocampal levels (Suppl. Fig. 1). With the exception ofthese features, all of the staining in wild-type brain isspecifically attributable to CB1 expression. We did notobserved labeling in the CB2 receptor knockout mouse orNAPE-PLD receptor knockout mouse, whereas the wild-type mouse showed labelling similar to that of the ratbrain (Suppl. Figs. 2, 3). CB1-immunostained bands (60kD) were similar to those described by Egertova and El-phick (2000). CB2 immunoblotting also showed a band (55kD) similar to that described in recent reports for rat brain(Van Sickle et al., 2005; Gong et al., 2006). The singlebands observed for FAAH (63 kD) and NAPE-PLD (46 kD)were identical to those described previously (Giang andCravatt, 1997; Okamoto et al., 2004). The DAGL� molec-ular mass (120 kD) is identical to that described for COScells by Bisogno and collaborators (2003). Carrying outmore stringent conditions for DAGL� immunoblotting, wedetected a prominent DAGL� band (76 kD) that was sim-ilar to that described by Bisogno and collaborators (2005;70 kD). The weaker band at 97 kD may be explained bythe presence of a glycosylated form of DAGL� that has yetto be defined. Analysis of MAGL immunoreactivity con-firmed two bands of 37 and 35 kD, but we also observed anadditional band at about 62 kD, similar to the weak bandobserved in Figure 2 of Dihn et al. (2002). This band canrepresent a post-translationally modified form of MAGL inthe rat cerebellum that still has to be characterized. Wecarried out Western blots and immunohistochemistry inthe presence of specific immunizing peptides to confirmthe specificity of the labelling. The absence of labellingunder these conditions indicated that the seven antibodiesutilized in the present study were selective for the histo-logical identification and discrimination of their proteinexpression.

Distribution of CB1 and CB2 in cerebellum

Our data indicated that CB1 and CB2 immunoreactivi-ties show partial complementary localization in the cere-bellar cortex. Characterization and localization of CB1-immunopositive staining throughout all the cerebellarlobules was consistent with previous studies on the ex-pression of CB1 mRNA, the detection of [3H]CP-55,940binding sites, and the immunocytochemical mapping ofCB1 in rodent brain (Herkenham et al., 1991b; Matsuda etal., 1993; Pettit et al., 1998; Egertova and Elphick, 2000;Cristino et al., 2006). We have located the majority of CB1



Fig. 8. NAPE-PLD immunoreactivity in the rat cerebellum andvestibular nuclei. A: General view of a coronal section through thecerebellum and vestibular nuclei. NAPE-PLD-immunoreactive(NAPE-PLD�) Purkinje somata and granular cell (arrows) are ob-served in the cerebellar cortex (B,D). Note the weakly stained NAPE-PLD� cells in the molecular layer that may correspond to basket and

stellate cells (arrowheads). C: Low-magnification photomicrographsshowing NAPE-PLD immunoreactivity throughout the cerebellar andvestibular nuclei. Note the well-labeled somata of the giant neurons inthe LVe (E) and the small neurons in the MVePC (F). For abbrevia-tions 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 infe-rior 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, ex-cept for CB1, CB2, and DAGL� immunoreactivity, whereas IO shows

a denser neuropil than the surrounding areas (A,B,E). Note the re-stricted location of CB1

� neuropil in the dorsal part of the IOPr (A).H: Schematic representation of the IO subdivisions at Bregma –12.72mm, described in the rat brain atlas of Paxinos and Watson (1998).For abbreviations see list. Scale bars � 100 �m.

immunoreactivity in the molecular layer of the rat cere-bellar cortex, largely associated with climbing and parallelfibers that extended on the Purkinje dendrites and inclustered basket cell axons surrounding Purkinje somata,especially on their basal areas, which correspond to theinitial axonal segment (Herkenham, 1995; Egertova andElphick, 2000; Egertova et al., 2003; Cristino et al., 2006;Kawamura et al., 2006). Previous studies have reporteddense [3H]CP-55,940 labelling in the molecular layer andsparse binding in the granular layer, including mutantmice deficient in Purkinje cell expression, suggesting thatPurkinje cells were not the source of CB1 expression in themolecular layer (Herkenham et al., 1991a,b; Herkenham,1995). As expected, cell bodies of the molecular layer (bas-ket and stellate cells) expressed CB1 mRNA, but Purkinjecells did not (Mailleux and Vanderhaeghen, 1992; Mat-suda et al., 1993). In agreement with these studies, we didnot detect CB1 immunoreactivity in Purkinje somata andtheir dendritic processes (Egertova and Elphick, 2000).The present study showed an intense CB1 immunoreac-tivity in fibers of the molecular layer, which agrees withprevious studies describing the expression of CB1 mRNAin fibers of the molecular layer and neurons of IO (Mail-leux and Vanderhaeghen, 1992), which suggests the pres-ence of CB1 immunoreactivity in climbing fibers (Pettit etal., 1998). However, by silver-enhanced immunogold,Kawamura et al. (2006) detected occasionally weak CB1labelling in climbing fibers that terminated on the proxi-mal Purkinje dendrites. On the other hand, our resultsalso indicated an absence of CB1 immunoreactivity in thegranular layer (as in mossy fibers), in contrast to thedetection of CB1 mRNA labelling in the deep cerebellarnuclei (Mailleux and Vanderhaeghen, 1992; Matsuda etal., 1993). However, we have detected CB1

� immunoreac-tivity 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 nucleiof the dorsal thalamus and motor cortex (data not shown).

Our results revealed a distribution of CB2 immunoreac-tivity in part similar to that of CB1 immunoreactivity. Incontrast to recent immunocytochemical studies (Ashton etal., 2006; Gong et al., 2006; Onaivi et al., 2006), CB2immunoreactivity was not associated with Purkinje cellbodies and their dendritic processes. We have observedstrong CB2 immunostaining in a number of varicose fibers(parallel fibers) in the molecular layer; most of them maybe associated with granular cells, but they could also beassociated with mossy fibers in the granular layer. Thesedata match the detection of granular layer cells and neu-rons in brainstem and spinal cord by in situ hybridizationin previous reports (Skaper et al., 1996; Van Sickle et al.,2005).

The results obtained for CB1 and CB2 immunostainingindicate that CB1 and CB2 receptors are in part located inthe same presynaptic structures of the cerebellar cortex,such as clustered basket cell axons (Pinceau formation),but they are also present in complementary presynapticstructures. Therefore, CB1 receptors are preferably lo-cated in climbing fibers (olivary projections), whereas CB2receptors are preferably located in mossy fibers (spinal,pontine, and vestibular projections) and parallel fibers(cerebellar granular cells). The presynaptic localization ofboth cannabinoid receptors in the cerebellar cortex sup-ports the hypothesis of endocannabinoids as retrogrademessengers proposed for different brain areas, including

cerebellum, amygdala, basal ganglia, and hippocampus(Stella et al., 1997; Rodrıguez de Fonseca et al., 1998,2005; Giufrida et al., 1999; Wilson and Nicoll, 2001; Wil-son et al., 2001; Diana et al., 2002; Gerdeman et al., 2002;Robbe et al., 2002; Chevaleyre and Castillo, 2003). At thismoment, we cannot exclude that CB2 receptors mightserve as a retrograde signalling gate controlling neuronaldepolarization or trophic maintenance of the synapses. Inany case, the finding of CB2 receptor in the cerebellumsuggests the need for reevaluating the effects of exogenousand endogenous cannabinoids on neurotransmission.

Presence and distribution of cannabinoiddegradation enzymes in cerebellum in

relation to CB1 and CB2 receptors

FAAH and MAGL are two hydrolytic enzymes that me-diate the degradation of different endocannabinoids.FAAH mediates endocannabinoid degradation, includingAEA, but also 2-AG, whereas MAGL was found to mediate85% of total brain membrane 2-AG hydrolase activity (Fig.1B; Piomelli et al., 2000; Blankman et al., 2007). As de-scribed for the synthesis of endocannabinoids, additionalhydrolytic enzymes degradating anandamide and 2-AGhave been recently proposed (Wei et al., 2006; Blankmanet al., 2007; Muccioli et al., 2007), but their role in neuralcircuits is still unknown. Thus we will limit the discussionto both FAAH and MAGL. The presence of both endocan-nabinoid degradation enzymes in the cerebellum givessupport for the existence of multiple regulatory mecha-nisms terminating endocannabinoid signaling. As occurswith CB1 and CB2 receptors, the specific localization ofFAAH and MAGL also suggests a complementary distri-bution of the two enzymes in the cerebellar cortex. Con-sistent with previous studies of the rodent and humancerebellar cortex (Egertova et al., 1998, 2003; Romero etal., 2002; Gulyas et al., 2004), FAAH immunoreactivitywas present in Purkinje somata. Additionally, we haveclearly detected in sagittal cerebellar slides the character-istic dendritic tree of the Purkinje cell, including the tini-est branches, which contained intense FAAH immuno-staining. These data disagree with data from Egertova etal. (2003) but agree with the dendritic staining describedin the molecular layer of human and rat cerebellum (Ro-mero et al., 2002; Gulyas et al., 2004) and observed inhuman cerebellar samples in our laboratory (Suarez et al.,unpublished). However, we have not detected stained cellsin rat cerebellar molecular layer. In contrast to previousstudies in rat cerebellum (Egertova et al., 1998; Tsou etal., 1998; Gulyas et al., 2004), FAAH immunoreactivitywas also evident in a small population of granular cellsthat was consistent with the detection of granular cells inrat cerebellum by in situ hybridization (Thomas et al.,1997) and in human and mouse cerebellum by immuno-histochemistry (Romero et al., 2002; Egertova et al., 2003).All these data indicate that the localization of FAAH isquite complementary to that of CB1 and CB2 receptors inthe cerebellar cortex, so Purkinje somata and their den-dritic processes and a specific population of granular cellsexpressed FAAH, whereas theirs presynaptic structures,climbing fibers, expressed CB1 receptor, and parallel fi-bers 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), wefound that not only the molecular layer neuropil (possibly



consisting of axon terminals) but also densely packed cellsof the granular layer showed MAGL immunoreactivity.We have clearly distinguished two cell types according tothe intensity of MAGL immunoreactivity: a dense popula-tion of moderately immunoreactive cells disposedthroughout the granular layer, which may be granularcells, and lower numbers of strongly immunoreactive cellsdisposed near Purkinje layer, which probably are Golgicells. Additionally, cells of the molecular layer, as well asPurkinje somata, showed moderate MAGL staining.These molecular layer cells probably represented basketcells and stellate cells.

The localization of MAGL in the cerebellar cortex couldpartially overlap that of CB1 and CB2 receptors. Despitethe difficulty in defining the specific localization of MAGLexpression because of the immunoreactivity in the granu-lar layer, the stained fibers and puncta in the molecularand granular layers could be related to mossy and climb-ing and/or parallel fibers; although negative Purkinje celldendrites could be clearly seen (Gulyas et al., 2004). Thelocation of MAGL as a presynaptic enzyme, which agreedwith CB1 and CB2 receptor distribution in presynapticstructures of the cerebellar cortex, may be related to theretrograde messenger role of 2-AG, as described recentlyfor the hippocampus, which determines basal endocan-nabinoid tone (Hashimotodani et al., 2007).

For the present study, we have optimized the immuno-histochemical protocol for MAGL antibody (kindly do-nated by Dr. D. Piomelli) by using a combination of im-munological methods. We tested different incubations,pretreatments, and antibody dilutions in comparison witha commercial MAGL antibody (Cayman; catalog No.100035; Suppl. Fig. 6). Both MAGL antibodies recognizedthe same N-terminal aa sequence and revealed the samemolecular masses. Only the distinct dilutions tested forboth antibodies resulted in differences in the general in-tensity of the MAGL immunoreactivity. In contrast, bothMAGL antibodies resulted in the same molecular weightand the same immunohistochemical distribution in the ratcerebellum. Therefore, the immunohistochemical differ-ences observed in this study and that of Gulyas et al.(2004) can only be explained by the use of different MAGLlots.

Additional explanations might be considered in order toclarify the nature of this potentially nonspecific signal.Although MAGL is thought to be the major 2-AG-hydrolyzing enzyme (Blankman et al., 2007), it also actsas an inactivator of other monoacylglycerol and prosta-glandin glycerol esters (Dihn et al., 2002, 2004; Vila et al.,2007). Thus, MAGL is also a relevant enzyme controllingthe acyl glycerol metabolism that might not be orientedonly to synaptic transmission. Furthermore, a recentstudy has provided evidence of MAGL activity that con-trols 2-AG levels in microglia, not described previously(Muccioli et al., 2007). The novel MAGL activity is espe-cially rich in mitochondrial and nuclei. The findings ofMucciolli and collaborators suggest that the clonedMAGL, which is responsible for the majority of 2-AG hy-drolysis in healthy brains (Hohmann et al., 2005), does notplay a major role in primary microglia. Possible explana-tions for this include cell-specific regulation of MAGLtranslation and, furthermore, the differential regulationthat cytokine activation produces on the expression ofMAGL and the novel MAGL in the brain. The authorsobserved an inverse regulation of both enzymes by

interferon-� (Witting et al., 2006). All these possibilitiesrequire further clarification but may support the evidenceof a wider distribution of the enzyme in the brain.

Presence and distribution of cannabinoidbiosynthesis enzymes in the cerebellum in

relation to CB1 and CB2 receptors

We have reported here the first analysis of the presenceof DAGL�, DAGL�, and NAPE-PLD in the cerebellum.The expression of these biosynthesis enzymes will deter-mine where endocannabinoids are made and released inthe cerebellum. DAGL� and DAGL� constitute two re-cently identified isoforms of closely related genes corre-lated with 2-AG biosynthesis and release (Fig. 1B;Mechoulam et al., 1995; Sugiura et al., 1995; Piomelli etal., 2000; Bisogno et al., 2003). Additionally, Bisogno andcollaborators (2003) have found coexpression of DAGL�and DAGL� in a similar staining pattern in mouse brain.They also indicated that the expression of both isozymeschanged in the developing brain from axonal tracts inthe embryonic stages to dendritic fields in the adult-hood. It is important to note that, even though 2-AG isconsidered a full cannabinoid receptor agonist, it is alsoan important intermediate in triacyl/diacylglycerol me-tabolism as well as a prominent molecule linking thecannabinoid signaling with lysophospholipids and dia-cycilglycerol-PKC signaling systems. Therefore, we can-not strictly consider both DAGL� and DAGL� as pureendocannabinoid-synthesizing enzymes. Besides theirobvious role on the endocannabinoid system, it is verypossible that they also play other, additional physiolog-ical roles. For instance, pharmacological studies havesuggested that DAGL activity is required for axonalgrowth and guidance in developing brain (Brittis et al.,1996; Williams et al., 2003). However, we will focus onlyon 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 con-tained numerous varicosities on their surface. In addi-tion, Purkinje cells expressed the highest levels ofDAGL� mRNA but not cerebellar granular cells (Yo-shida et al., 2006). However, our results indicated thatthe staining of DAGL� was quite different from that ofDAGL� in rat cerebellum; that is, whereas Purkinje celldendrites strongly expressed DAGL�, Purkinje cell bod-ies specifically expressed DAGL�. Additionally, we havedetected a small population of granular cells that wasDAGL� immunoreactive. The distribution of DAGL�mRNA and DAGL� mRNA also differed in the cerebel-lar cortex. DAGL� mRNA was highly expressed in thecerebellar granular layer (Yoshida et al., 2006). Thedifferent localizations of DAGL� and DAGL� suggestthat at least three different postsynaptic locations pref-erably make and release 2-AG in the cerebellar cortex:Purkinje cell dendrites postsynaptically release 2-AG byDAGL� and Purkinje somata and granular somata/dendrites by DAGL�. Indeed, the postsynaptic localiza-tion of DAGL� in Purkinje cell dendrites correlates withthe presynaptic localization of CB1 receptor in climbingfibers that terminate on distal and proximal Purkinjecell dendrites and with the presynaptic location of CB2receptor in mossy and parallel fibers that terminate on



granular cells and Purkinje dendrites, respectively. Inagreement with our results, a previous report indicatedthat DAGL� was essentially targeted by postsynapticspines in cerebellar Purkinje cells and suggested closeproximity between production sites of endocannabinoidsand their receptors (Yoshida et al., 2006). The postsyn-aptic localization of DAGL� in Purkinje somata andgranular somata also agrees with the presynaptic local-ization of CB1 and CB2 receptors in clustered basket cellaxons (Pinceau formation) and with the location of CB2receptor in mossy fibers that surround granular somata.

On the other hand, NAPE-PLD is another recently char-acterized cannabinoid biosynthesis enzyme that mediatesthe release of N-acyl ethanolamines (including AEA) from aphospholipid precursor [N-acyl-phosphatidylethanolamide(NAPE); Fig. 1B; Piomelli et al., 2000; Okamoto et al., 2004].Again, N-acyl ethanolamides are not only endocannabinoidmediators; some of them (oleoylethanolamide, palmithyleth-anolamide) are also activators of other receptor types, in-cluding nuclear receptors of the peroxisome proliferators-activated receptor family (Fu et al., 2003). However, c-fosmapping did not reveal a substantial change in the patternof cellular activity in the cerebellum after exogenousoleoylethanolamide administration, underscoring its contri-bution to cerebellar physiology (Rodriguez de Fonseca et al.,2001).

Our results indicated that the staining of NAPE-PLDwas quite similar to that of DAGL� in the cerebellarcortex, as indicated by the fact that Purkinje somata anda small population of granular somata presented promi-nent NAPE-PLD immunoreactivity. Here, we have alsoobserved weak NAPE-PLD expression in cells and fibers ofthe molecular layer. The appearance of these fibers in themolecular layer possibly represents the dendritic field ofthe Purkinje cells. So, NAPE-PLD and DAGL� may becoexpressed in the Purkinje cell dendritic field. Similarlyto DAGL� and DAGL�, the postsynaptic localization ofNAPE-PLD in Purkinje somata and granular somata cor-relates with the presynaptic localization of CB1 and CB2receptor in clustered basket cell axons (Pinceau forma-tion) and the location of CB2 receptor in mossy fibers,whereas the postsynaptic localization of NAPE-PLD inPurkinje dendrites and molecular layer cells correlateswith the presynaptic localization of CB1 receptor in climb-ing fibers and the location of CB2 receptor in parallelfibers. The induction of retrograde signals by the biosyn-thesis of endocannabinoids in Purkinje dendrites(DAGL�� and NAPE-PLD�) is enhanced when parallelfiber (CB2

�) stimulation is combined with climbing fiber(CB1

�) stimulation (Brenowitz and Regehr, 2005).

Presence of the endocannabinoid system inthe cerebellar nuclei, vestibular nuclei, and

inferior olive

The importance should be noted of the CB2 receptor infiber terminals, which homogeneously filled all cerebellarand vestibular nuclei and suggests a Purkinje origin. Inaddition, IO also showed a dense network of CB2

� fibersthat may originate principally from spinal projections. Onthe other hand, the presence of the CB1 receptor in fibersthat coursed into the inferior cerebellar peduncles couldrelate to the olivary-cerebellar projections, which also cor-relates with the presence of climbing fibers in the cerebel-lar cortex (Fig. 10).

These data were not consistent with previous studiesshowing an intense CB1 and CB2 immunoreactivities as-sociated with neuronal somata of the cerebellar nuclei (seeFig. 7 in Pettit et al., 1998; Gong et al., 2006) and vestib-

Fig. 10. A: Schematic parasagittal section of a mammalian cere-bellum redrawn from Cajal (1911). B: Schematic representation of theinferior olive-cerebellar and vestibulocerebellar connections and in-trinsic cerebellar circuits in mammals. For abbreviations see list.



ular nuclei (Ashton et al., 2004; see Figs. 5F and 13A inGong et al., 2006). However, our study agrees with the lowlevel of [3H]CP-55,940 binding and the low intensity ofCB1 mRNA signal found in the deep cerebellar nuclei,vestibular nuclei, and IO (Herkenham et al., 1991b; Mat-suda et al., 1993) but also with the low levels of CB1radiographic labelling in the human medial and lateralvestibular nucleus (Glass et al., 1997). In contrast, thepresence of cannabinoid degradation and biosynthesis en-zymes was prominent in cerebellar nuclei, vestibular nu-clei, and IO complex. Although FAAH, MAGL, DAGL�,and NAPE-PLD immunoreactivities were associatedmainly with cell bodies, DAGL� immunoreactivity wasassociated exclusively with a conspicuous network of fi-bers and puncta in these three functionally related re-gions. In agreement with Bisogno and collaborators(2003), DAGL� expression became restricted to synapticfields in the adult brain, possibly in postsynaptic den-drites, correlating with the postsynaptic requirement forthe synthesis of endocannabinoids as a retrograde mes-senger (Kreitzer and Regehr, 2001; Diana et al., 2002;Bisogno et al., 2003). The presence of the proteins of theendogenous cannabinoid system in the input and outputrelays of the cerebellar cortex suggests a potential role forendocannabinoid-mediated plasticity in motor timing andlearning mediated by these circuits (Raymond et al.,1996).

Role for the endocannabinoid system in thecerebellum: new targets for the study of

motor learning and the ataxias

Early studies using natural and synthetic cannabinoidsreported ataxia as one of the key features of the pharma-cological profile of these compounds (Dewey, 1986; Pateland Hillard, 2001). The effects on ataxia are mediated bycannabinoid CB1 receptors, and apparently there are noCB2-mediated gait alterations in experimental animals(Patel and Hillard, 2001; Valenzano et al., 2005). Theseresults indicated that, at least, motor timing and coordi-nation require an intact endogenous cannabinoid receptorsignalling system that, despite the activation of CB1 re-ceptors, may regulate sensorimotor integration. However,the cerebellum is an additional motor learning deviceneeded for many different forms of motor learning (Ray-mond et al., 1996). It has been suggested that the endog-enous cannabinoid system modulates motor learning (i.e.,eye blink conditioning) in the cerebellum (Kishimoto andKano, 2006; Skosnik et al., 2007). The truth is that thereis a lack of information on the role of CB1 and CB2 recep-tors in these learning processes involving cerebellar cir-cuits. Most of the work so far has been performed in thecerebellar cortex, using synaptic plasticity paradigms. Re-cent studies have clearly established a role for the endog-enous cannabinoid system in short-term and long-termforms of synaptic plasticity in the cerebellar cortex. Long-term depression, depolarization-induced suppression ofinhibition, or depolarization-induced suppression of exci-tation is modulated by endogenous cannabinoid release(Diana and Marty, 2004; Kreitzer and Regehr, 2001; Safoand Regehr, 2005). These effects are mediated throughcannabinoid CB1 receptors. However, little is known onthe role of cannabinoid CB2 receptors, which, as describedhere, are present in clustered basket cell axons (Pinceauformations) and other relevant relays of the cerebellar

circuitry. Whether they affect postsynaptic endocannabi-noid production, downstream intracellular signalling, orsurvival/remodelling processes still requires further in-vestigation. This is relevant not only for motor learningbut also for the neuroadaptions associated with cerebellarinsults or chronic degenerative disorders such as the atax-ias.

Moreover, we lack crucial information on the role of theendogenous cannabinoid system in deep cerebellar nucleiand the IO complex. Because of the crucial role of thesestructures in motor timing and learning, especially insensorymotor integration and conditioning, it is possibleto anticipate a role for cannabinoid receptors in modulat-ing the input and output information streams of the cer-ebellar cortex, beyond the Purkinje cell (as an example, itis worth remembering that the IO is the sole source for anentire afferent system to the cerebellum, the climbingfibers). More research is thus needed to establish thishypothesis by analyzing the effects of cannabinoid CB1and CB2 receptors as well as FAAH and MAGL inhibitorsin simple learning paradigms, such as the vestibuloocularreflex and the eyelid conditioning responses. Such studiesmay also help to establish the potential pharmacologicalutility of cannabinoids in cerebellar disorders.

ACKNOWLEDGMENTS

The authors are indebted to Dr. Daniele Piomellli forkindly providing MAGL antibody.

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