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 SUA´ REZ,1* FRANCISCO JAVIER BERMU´ DEZ-SILVA,1 KEN MACKIE,2 CATHERINE LEDENT,3 ANDREAS ZIMMER,4 BENJAMIN F. CRAVATT,5 AND FERNANDO RODRI´GUEZ DE FONSECA1* 1Laboratorio de Medicina Regenerativa, Fundacio´n IMABIS, 29010 Ma´laga, Spain 2Departments of Psychological and Brain Sciences, Indiana University, Bloomington, IN 47401 3Institut de Recherche Interdisciplinaire en Biologie Humaine et Mole´culaire, Universite´ Libre de Bruxelles, B-1050 Bruxelles, Belgium 4Institute of Molecular Psychiatry, University of Bonn, 53115 Bonn, Germany 5Chemical 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 canna- binoid biosynthesis (DAGL␣, DAGL␤, and NAPE-PLD), and the endocannabinoid- degradating 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 *Correspondence to: Juan Sua´rez and Fernando Rodrı´guez de Fonseca, at http://www.interscience.wiley.com/jpages/0021-9967/suppmat. Laboratorio de Medicina Regenerativa, Fundacio´n IMABIS, Avenida Carlos Grant sponsor: Consejerı´a de Salud (Junta de Andalucı´a); Grant num- Haya 82, 29010 Ma´laga, Spain. E-mail: [email protected]; ber: PI-0220; MEC; Grant number: SAF 2004/07762; Grant sponsor: Insti- [email protected] tuto de Salud Carlos III; Grant number: 07/1226; Grant number: 07/0880; Received 21 February 2007; Revised 14 August 2007; Accepted 28 April Grant sponsor: Plan Nacional Sobre Drogas; Grant sponsor: Consejeria de 2008 Innovacio´n Ciencia y Empresa (Junta de Andalucı´a); Grant number: RE- DES RTA RD06/001; Grant sponsor: 5th Framework Programme; Grant DOI 10.1002/cne.21774 number: TARGALC QLRT-2001-01048. Published online in Wiley InterScience (www.interscience.wiley.com). © 2008 WILEY-LISS, INC. The Journal of Comparative Neurology ENDOCANNABINOID AND CEREBELLUM 401 Analysis of the CB1 receptor expression in the rat brain zymes, in the brain (Dihn et al., 2002; Romero et al., 2002; by in situ hybridization histochemistry and immunocyto- Bisogno et al., 2003; Egertova´ et al., 2003; Okamoto et al., chemistry has provided important insights into the func- 2004). Recently, immunohistochemical studies revealed tional neuroanatomy of the endocannabinoid system (Mat- the distribution of CB2 receptor in the rat brain, particu- suda et al., 1993; Pettit et al., 1998; Egertova´ and Elphick, larly in cerebellum and hippocampus (Van Sickle et al., 2000; Van Sickle et al., 2005; Gong et al., 2006). Electro- 2005; Gong et al., 2006). The finding of CB2 receptor in the physiological studies and the finding of CB1 receptor in cerebellum suggests the need to reevaluate the effects of the cerebellar synapses suggest that endocannabinoids exogenous and endogenous cannabinoids on neurotrans- act as retrograde messengers in the cerebellum. This role mission. of the endocannabinoid system is confirmed by the finding There are some studies demonstrating the presence of of different types of endocannabinoid-mediated synaptic the cannabinoid degradation enzymes FAAH (Cravatt et plasticity, including both short-term [depolarization- al., 1995, 1996; Egertova´ et al., 1998; Goparaju et al., induced suppression of inhibition (DSI) and excitation 1998; Tsou et al., 1998) and MAGL (Dihn et al., 2002) in (DSE)] and the more permanent long-term depression the brain. Other studies report a general analysis of (LTD; for review see Wilson and Nicoll, 2002; Diana and FAAH expression in specific neuronal population of mouse Marty, 2004; Safo and Regehr, 2005). The presence of the and human brain, including cerebellar Purkinje cells, neu- endocannabinoid CB1 receptor in the cerebellum implies rons of cerebellar nuclei and inferior olive, neocortical and that the endocannabinoid system plays a central role in hippocampal pyramidal neurons, and striatal projecting real-time regulation of movement and neuroadaptations neurons. These localizations of FAAH suggest a comple- underlying motor control and motor learning. In fact, rel- mentary distribution with CB1 expression in these brain evant pharmacological actions of exogenously adminis- regions (Romero et al., 2002; Egertova´ et al., 2003). North- tered cannabinoids are ataxia and catalepsy (Rodrı´guez de ern blot and in situ hybridization analyses reveal that Fonseca et al., 1998) and modulation of eye blink condi- MAGL is heterogeneously expressed in some brain areas, tioning (Kishimoto and Kano, 2006; Skosnik et al., 2007). including hippocampus, cortex, cerebellum, and anterior CB1 receptors are located in axon terminals of parallel thalamus, where CB1 receptor is also expressed, indicat- fibers of cerebellar granular cells and climbing fibers of ing a presynaptic localization of the enzyme (Dihn et al., inferior olive neurons that provide excitatory input on 2002). Purkinje cells. In addition, CB1 receptors are located in Indeed, the recent identification of 2-AG and AEA bio- axon terminals of cerebellar basket and stellate cells pro- synthesis and release enzymes, DAGL␣ and DAGL␤ viding inhibitory input on Purkinje cells (Mailleux and (Bisogno et al., 2003), and NAPE-PLD (Okamoto et al., Vanderhaeghen, 1992; Matsuda et al., 1993; Tsou et al., 2004) has provided new insights on the endocannabinoid 1998; Egertova´ and Elphick, 2000; Cristino et al., 2006; signaling system in the brain. Pharmacological studies Kawamura et al., 2006). Modulation of excitatory and suggest that DAGL and NAPE-PLD activity is required inhibitory input on Purkinje cells by the endocannabinoid for inhibition of ␥-aminobutyric acid (GABA)-ergic trans- system allows Purkinje cells to refine the output of motor mission by glutamatergic input (Chevaleyre and Castillo, responses from cerebellum. 2003). Additionally, DAGL activity is related to axonal However, so far, little information is available on the growth and guidance during development (Brittis et al., presence and function of the cannabinoid CB2 receptor 1996; Williams et al., 2003). The expression of DAGL (Skaper et al., 1996; Lu et al., 2000; Zhang et al., 2003; isozymes (␣ and ␤ forms) changes during development of Pazos et al., 2004; Benito et al., 2005; Sheng et al., 2005) the brain; that is, they are expressed in axonal tracts of and other components of the endocannabinoid system, the embryo and then in dendritic fields of the adult mouse such as cannabinoid biosynthesis and degradation en- brain (Bisogno et al., 2003). In the adult mouse cerebel- Abbreviations b basket cell LA lateral amygdaloid nucleus c collaterals Lat lateral (dentate) cerebellar nucleus CbCx cerebellar cortex LatPC lateral cerebellar nucleus, parvicellular part CbN cerebellar nuclei LVe lateral vestibular nucleus cf climbing fibers mf mossy fibers DEn dorsal endopiriform nucleus Med medial (fastigial) cerebellar nucleus DG dentate gyrus MedDL medial cerebellar nucleus, dorsolateral protuberance g granular cell ML molecular layer G golgi cell MVe medial vestibular nucleus GrL granular layer MVeMC medial vestibular nucleus, magnocellular part Hi hippocampus MVePC medial vestibular nucleus, parvicellular part icp inferior cerebellar peduncle P purkinje cell IntA interposed cerebellar nucleus, part anterior pf parallel fibers IntDL interposed cerebellar nucleus, dorsolateral hump pif pinceau formation IntDM interposed cerebellar nucleus, dorsomedial crest py pyramidal tract IntP interposed cerebellar nucleus, part posterior s superficial stellate cell IntPPC interposed cerebellar nucleus, posterior parvicellular part scp superior cerebellar peduncle IO inferior olive sp5 spinal trigeminal tract IOD inferior olive, dorsal nucleus SpVe spinal vestibular