V. Di Marzo

Immunohistochemical localization of cannabinoid type 1 and vanilloid transient receptor potential vanilloid type 1 receptors in the mouse brain

Cannabinoid type 1 receptors and transient receptor potential vanilloid type 1 channels have been proposed to act as metabotropic and ionotropic receptors, respectively, for two classes of endogenous polyunsaturated fatty acid amides, the acylethanolamides and the acyldopamides. Furthermore, we and others have shown that functional crosstalk occurs between these two receptors when they are expressed in the same cell. Although demonstrated in sensory neurons of the dorsal root ganglia, spinal cord and myenteric neurons, co-expression of cannabinoid type 1 and transient receptor potential vanilloid type 1 has not yet been studied in the brain. In the present study, we addressed this issue by using commercially available specific antibodies whose specificity was confirmed by data obtained with brains from cannabinoid type 1(-/-) and transient receptor potential vanilloid type 1(-/-) mice. Double cannabinoid type 1/transient receptor potential vanilloid type 1 immunofluorescence and single cannabinoid type 1 or transient receptor potential vanilloid type 1 avidin-biotin complex immunohistochemistry techniques were performed and both methods used point to the same results. Cannabinoid type 1/transient receptor potential vanilloid type 1 expression was observed in the hippocampus, basal ganglia, thalamus, hypothalamus, cerebral peduncle, pontine nuclei, periaqueductal gray matter, cerebellar cortex and dentate cerebellar nucleus. In particular, in the hippocampus, cannabinoid type 1/transient receptor potential vanilloid type 1 expression was detected on cell bodies of many pyramidal neurons throughout the CA1-CA3 subfields and in the molecular layer of dentate gyrus. In the cerebellar cortex, expression of cannabinoid type 1/transient receptor potential vanilloid type 1 receptors was found surrounding soma and axons of the vast majority of Purkinje cell bodies, whose cytoplasm was found unstained for both receptors. Cannabinoid type 1 and transient receptor potential vanilloid type 1 immunoreactivity was also detected in: a) the globus pallidus and substantia nigra, in which some intensely transient receptor potential vanilloid type 1 immunopositive cell bodies were found in dense and fine cannabinoid type 1/transient receptor potential vanilloid type 1 positive and cannabinoid type 1 positive nerve fiber meshworks, respectively; b) the cytoplasm of thalamic and hypothalamic neurons; and c) some neurons of the ventral periaqueductal gray. These data support the hypothesis of a functional relationship between the two receptor types in the CNS.

Endocannabinoids control spasticity in a multiple sclerosis model

Spasticity is a complicating sign in multiple sclerosis that also develops in a model of chronic relapsing experimental autoimmune encephalomyelitis (CREAE) in mice. In areas associated with nerve damage, increased levels of the endocannabinoids, anandamide (arachidonoylethanolamide, AEA) and 2‐arachidonoyl glycerol (2‐AG), and of the AEA congener, palmitoylethanolamide (PEA), were detected here, whereas comparable levels of these compounds were found in normal and non‐spastic CREAE mice. While exogenously administered endocannabinoids and PEA ameliorate spasticity, selective inhibitors of endocannabinoid re‐uptake and hydrolysis—probably through the enhancement of endogenous levels of AEA, and, possibly, 2‐arachidonoyl glycerol—significantly ameliorated spasticity to an extent comparable with that observed previously with potent cannabinoid receptor agonists. These studies provide definitive evidence for the tonic control of spasticity by the endocannabinoid system and open new horizons to therapy of multiple sclerosis, and other neuromuscular diseases, based on agents modulating endocannabinoid levels and action, which exhibit little psychotropic activity.

IMMUNOHISTOCHEMICAL LOCALIZATION OF ANABOLIC AND CATABOLIC ENZYMES FOR ANANDAMIDE AND OTHER PUTATIVE ENDOVANILLOIDS IN THE HIPPOCAMPUS AND CEREBELLAR CORTEX OF THE MOUSE BRAIN

An increasing body of evidence indicates that: 1) the endocannabinoid anandamide (AEA) and other unsaturated N-acylethanolamines (NAEs), 2) 12-(S)-lipoxygenase (12-LOX) products of arachidonic acid, and 3) unsaturated N-acyldopamines (NADAs), act as endogenous ligands of transient receptor potential vanilloid type 1 (TRPV1) channels at intracellular binding sites. AEA is synthesized and released on demand in neurons from its membrane precursor, N-arachidonoyl-phosphatidylethanolamine, by an N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), and is inactivated by intracellular hydrolysis by fatty acid amide hydrolase (FAAH), whereas catechol-O-methyl-transferase (COMT) was suggested to inactivate NADAs. However, it is not known whether these enzymes or 12-LOX co-localize to any extent with TRPV1 receptors in the brain. In this study we used immunohistochemical techniques (single peroxidase and double immunofluorescence staining), and analyzed the localization of the TRPV1 channel in mouse hippocampal and cerebellar neurons with respect to NAPE-PLD, FAAH, 12-LOX and COMT. Cycloxygenase-2 (COX-2), another putative AEA-degrading enzyme, was also studied. Co-localization between TRPV1 and either NAPE-PLD or FAAH, COX-2, 12-LOX and COMT was found in Ammons horn (CA3) hippocampal pyramidal neurons and (with the exception of 12-LOX) in some Purkinje cells. At the cellular level, both anabolic and catabolic enzymes appeared as fine grains with immunoperoxidase labeling and were observed in the somatodendritic compartment of CA3 pyramidal cells as well as (with the exception of 12-LOX) in the cytoplasm of Purkinje neurons, in which FAAH and COX-2 immunoreactivities were, however, preferentially localized in the large extension of the dendritic arbor. Our data agree with the hypothesis that, in potential endovanillergic neurons, endogenous TRPV1 agonists, and AEA in particular, act as intracellular mediators by being produced from and/or degraded by the same mouse brain cells that express TRPV1 receptors.

Protective role of palmitoylethanolamide in contact allergic dermatitis

To cite this article: Petrosino S, Cristino L, Karsak M, Gaffal E, Ueda N, Tüting T, Bisogno T, De Filippis D, D’Amico A, Saturnino C, Orlando P, Zimmer A, Iuvone T, Di Marzo V. Protective role of palmitoylethanolamide in contact allergic dermatitis. Allergy 2010; 65: 698–711.

Effect of maternal under-nutrition on pup body weight and hypothalamic endocannabinoid levels.

Abstract. Dietary long-chain polyunsaturated fatty acids are known to influence brain levels of the endocannabinoid anandamide in newborn pigs and mice. Furthermore, endocannabinoids were shown to control pup suckling and body weight in mice, and food intake in adult rodents. Here we determined the effect of maternal under-nutrition during gestation, lactation, or both, on body weight, and on the levels of endocannabinoids and expression of cannabinoid CB1 receptors and fatty acid amide hydrolase in the hypothalamus of rat pups at weaning (21 days old) or adult rats (4 months old). Maternal under-nutrition resulted in a striking decrease in body weight of weaning rats, paralleled by a decrease in the hypothalamic levels of the endocannabinoid anandamide, but not of 2-arachidonoylglycerol. No significant change in the hypothalamic expression of either cannabinoid CB1 receptors or fatty acid amide hydrolase mRNA was detected in any of the three groups of weaned pups. The decrease in pup body weight and hypothalamic anandamide levels was not observable in 4-month-old rats from any of the three groups. These data suggest that maternal under-nutrition causes a decrease in hypothalamic anandamide levels and loss of body weight, and confirm a crucial role for endocannabinoid signalling in neonatal development.

Control of experimental spasticity by targeting the degradation of endocannabinoids using selective fatty acid amide hydrolase inhibitors

Background: It has been previously shown that CB1 cannabinoid receptor agonism using cannabis extracts alleviates spasticity in both a mouse experimental autoimmune encephalomyelitis (EAE) model and multiple sclerosis (MS) in humans. However, this action can be associated with dose-limiting side effects. Objective: We hypothesised that blockade of anandamide (endocannabinoid) degradation would inhibit spasticity, whilst avoiding overt cannabimimetic effects. Methods: Spasticity eventually developed following the induction of EAE in either wild-type or congenic fatty acid amide hydrolase (FAAH)-deficient Biozzi ABH mice. These animals were treated with a variety of different FAAH inhibitors and the effect on the degree of limb stiffness was assessed using a strain gauge. Results: Control of spasticity was achieved using FAAH inhibitors CAY100400, CAY100402 and URB597, which was sustained following repeated administrations. Therapeutic activity occurred in the absence of overt cannabimimetic effects. Importantly, the therapeutic value of the target could be definitively validated as the treatment activity was lost in FAAH-deficient mice. Spasticity was also controlled by a selective monoacyl glycerol lipase inhibitor, JZL184. Conclusions: This study demonstrates definitively that FAAH inhibitors provide a new class of anti-spastic agents that may have utility in treating spasticity in MS and avoid the dose-limiting side effects associated with cannabis use.

Lipids in neural function: modulation of behavior by oral administration of endocannabinoids found in foods.

Although uf7319(–)-tetrahydrocannabinol (THC) is not naturally found in the body, receptors for this compound have been described in brain since 1988 [1]. These receptors were later named CB1 and a receptor subtype (CB2) has been found in immune tissues [2]. An endogenous ligand for these receptors has more recently been discovered: it is the ethanolamide of arachidonic acid and was named ‘anandamide’ from the Sanskrit word ‘ananda’ which means ‘bliss’. When anandamide was injected into animals, hypomobility, hypothermia, analgesia and catalepsy were observed [3]. These are the classical behavioral effects of THC administration. Since this discovery, other endogenous metabolites which have been shown to be functional agonists of these receptors have been several polyunsaturated fatty acid derivatives, some of which include uf7314,7,10,13,16,19docosahexaenoylethanolamide, uf7318,11,14,17-docosatetraenoylethanolamide, uf7318,11,14-di-homo γ-linolenoylethanolamide [4], 2-arachidonylglycerol (2-AG) [5, 6] and uf7315,8,11-eicosatrienoyl (Mead acid) ethanolamide [7] (Fig. 1). These compounds are collectively termed endocannabinoids and have been shown to bind to brain CB receptors with an IC50 value equal to or 1.5-fold that of anandamide [8, 9].

Hydra Tentacle Regeneration: A Model for the Study of the Involvement of Protein Kinase C and Phospholipase A2 in Cell Differentiation

The involvement of protein kinase C (PKC) and phospholipase A2 (PLA2) activation in the cel differentiation processes accompanying Hydra tentacle regeneration was studied using respectively a novel non-linear diacylglycerol, verrucosin B, and the potent site-specific PLA2 inhibitor oleyl-oxyethyl-phosphorylcholine (OOPC). Verrucosin B was found to induce tentacle regeneration at doses 10000 times lower than 1,2-sn-dioctanoylglycerol (diC8), and this effect was blocked by PKC inhibitors. OOPC potently inhibited tentacle regeneration and the inhibition was reversed by arachidonic acid (AA). A role as transmembrane signalling effectors for a Ca2+-independent PKC and for PLA2-derived AA and its metabolites in Hydra regenerative processes is hypothesized.

Alteration of the endocannabinoid system in mouse brain during prion disease

Prion diseases are neurodegenerative disorders characterized by deposition of the pathological prion protein (PrPsc) within the brain of affected humans and animals. Microglial cell activation is a common feature of prion diseases; alterations of various neurotransmitter systems and neurotransmission have been also reported. Owing to its ability to modulate both neuroimmune responses and neurotransmission, it was of interest to study the brain endocannabinoid system in a prion-infected mouse model. The production of the endocannabinoid, 2-arachidonoyglycerol (2-AG), was enhanced 10 weeks post-infection, without alteration of the other endocannabinoid, anandamide. The CB2 receptor expression was up-regulated in brains of prion-infected mice as early as 10 weeks and up to 32 weeks post-infection whereas the mRNAs of other cannabinoid receptors (CBRs) remain unchanged. The observed alterations of the endocannabinoid system were specific for prion infection since no significant changes were observed in the brain of prion-resistant mice, that is, mice devoid of the Prnp gene. Our study highlights important alterations of the endocannabinoid system during early stages of the disease long before the clinical signs of the disease.